Detection of some virulence factors in Staphylococcus aureus ...

African Journals Online (AJOL)

Detection of some virulence factors in Staphylococcus aureus isolated from clinical and subclinical bovine mastitis in Iran. ... Mastitis is one of the common diseases of dairy cattle and an inflammatory response of the mammary glands tissue. ... and B genes, 10 samples contained agrI gene, 42 samples contained agrII gene, ...

Optimization of a Laboratory-Developed Test Utilizing Roche Analyte-Specific Reagents for Detection of Staphylococcus aureus, Methicillin-Resistant S. aureus, and Vancomycin-Resistant Enterococcus Species▿

OpenAIRE

Mehta, Maitry S.; Paule, Suzanne M.; Hacek, Donna M.; Thomson, Richard B.; Kaul, Karen L.; Peterson, Lance R.

2008-01-01

Nasal and perianal swab specimens were tested for detection of Staphylococcus aureus and vancomycin-resistant Enterococcus species (VRE) using a laboratory-developed real-time PCR test and microbiological cultures. The real-time PCR and culture results for S. aureus were similar. PCR had adequate sensitivity, but culture was more specific for the detection of VRE.

Effect of Promoter Region Mutations and mgrA Overexpression on Transcription of norA, Which Encodes a Staphylococcus aureus Multidrug Efflux Transporter

OpenAIRE

Kaatz, Glenn W.; Thyagarajan, Rama V.; Seo, Susan M.

2005-01-01

NorA is a Staphylococcus aureus multidrug transporter that confers resistance to structurally distinct compounds. The MgrA global regulatory protein is reported to augment norA expression when mgrA is overexpressed from an undefined plasmid-based promoter. Further details about norA regulatory mechanisms are scant. A chromosomal norA::lacZ transcriptional fusion was constructed in different S. aureus strains, and allele replacement was used to define the relevance of promoter region sequences...

A new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of Staphylococcus aureus.

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Lian, Yan; He, Fengjiao; Wang, Huan; Tong, Feifei

2015-03-15

A novel aptamer/graphene interdigitated gold electrode piezoelectric sensor was developed for the rapid and specific detection of Staphylococcus aureus (S. aureus) by employing S. aureus aptamer as a biological recognition element. 4-Mercaptobenzene-diazonium tetrafluoroborate (MBDT) salt was used as a molecular cross-linking agent to chemically bind graphene to interdigital gold electrodes (IDE) that are connected to a series electrode piezoelectric quartz crystal (SPQC). S. aureus aptamers were assembly immobilized onto graphene via the π-π stacking of DNA bases. Due to the specific binding between S. aureus and aptamer, when S. aureus is present, the DNA bases interacted with the aptamer, thereby dropping the aptamer from the surface of the graphene. The electric parameters of the electrode surface was changed and resulted in the change of oscillator frequency of the SPQC. This detection was completed within 60min. The constructed sensor demonstrated a linear relationship between resonance frequency shifts with bacterial concentrations ranging from 4.1×10(1)-4.1×10(5)cfu/mL with a detection limit of 41cfu/mL. The developed strategy can detect S. aureus rapidly and specifically for clinical diagnosis and food testing. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of An Impedimetric Aptasensor for the Detection of Staphylococcus aureus

OpenAIRE

Reich, Peggy; Stoltenburg, Regina; Strehlitz, Beate; Frense, Dieter; Beckmann, Dieter

2017-01-01

In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-...

Metabolic profiling for detection of Staphylococcus aureus infection and antibiotic resistance.

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Henrik Antti

Full Text Available Due to slow diagnostics, physicians must optimize antibiotic therapies based on clinical evaluation of patients without specific information on causative bacteria. We have investigated metabolomic analysis of blood for the detection of acute bacterial infection and early differentiation between ineffective and effective antibiotic treatment. A vital and timely therapeutic difficulty was thereby addressed: the ability to rapidly detect treatment failures because of antibiotic-resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA and methicillin-sensitive S. aureus (MSSA were used in vitro and for infecting mice, while natural MSSA infection was studied in humans. Samples of bacterial growth media, the blood of infected mice and of humans were analyzed with combined Gas Chromatography/Mass Spectrometry. Multivariate data analysis was used to reveal the metabolic profiles of infection and the responses to different antibiotic treatments. In vitro experiments resulted in the detection of 256 putative metabolites and mice infection experiments resulted in the detection of 474 putative metabolites. Importantly, ineffective and effective antibiotic treatments were differentiated already two hours after treatment start in both experimental systems. That is, the ineffective treatment of MRSA using cloxacillin and untreated controls produced one metabolic profile while all effective treatment combinations using cloxacillin or vancomycin for MSSA or MRSA produced another profile. For further evaluation of the concept, blood samples of humans admitted to intensive care with severe sepsis were analyzed. One hundred thirty-three putative metabolites differentiated severe MSSA sepsis (n = 6 from severe Escherichia coli sepsis (n = 10 and identified treatment responses over time. Combined analysis of human, in vitro, and mice samples identified 25 metabolites indicative of effective treatment of S. aureus sepsis. Taken together, this

Beta-lactamase detection in Staphylococcus aureus and coagulase-negative Staphylococcus isolated from bovine mastitis

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Bruno F. Robles

2014-04-01

Full Text Available The objectives of the study were to evaluate the presence/production of beta-lactamases by both phenotypic and genotypic methods, verify whether results are dependent of bacteria type (Staphylococcus aureus versus coagulase-negative Staphylococcus - CNS and verify the agreement between tests. A total of 200 bacteria samples from 21 different herds were enrolled, being 100 CNS and 100 S. aureus. Beta-lactamase presence/detection was performed by different tests (PCR, clover leaf test - CLT, Nitrocefin disk, and in vitro resistance to penicillin. Results of all tests were not dependent of bacteria type (CNS or S. aureus. Several S. aureus beta-lactamase producing isolates were from the same herd. Phenotypic tests excluding in vitro resistance to penicillin showed a strong association measured by the kappa coefficient for both bacteria species. Nitrocefin and CLT are more reliable tests for detecting beta-lactamase production in staphylococci.

Detecting Staphylococcus aureus in milk from dairy cows using sniffer dogs.

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Fischer-Tenhagen, C; Theby, V; Krömker, V; Heuwieser, W

2018-05-01

Fast and accurate identification of disease-causing pathogens is essential for specific antimicrobial therapy in human and veterinary medicine. In these experiments, dogs were trained to identify Staphylococcus aureus and differentiate it from other common mastitis-causing pathogens by smell. Headspaces from agar plates, inoculated raw milk samples, or field samples collected from cows with Staphylococcus aureus and other mastitis-causing pathogens were used for training and testing. The ability to learn the specific odor of Staphylococcus aureus in milk depended on the concentration of the pathogens in the training samples. Sensitivity and specificity for identifying Staphylococcus aureus were 91.3 and 97.9%, respectively, for pathogens grown on agar plates; 83.8 and 98.0% for pathogens inoculated in raw milk; and 59.0 and 93.2% for milk samples from mastitic cows. The results of these experiments underline the potential of odor detection as a diagnostic tool for pathogen diagnosis. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Evaluation of different methods to detect methicillin resistance in Staphylococcus aureus (MRSA).

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Alipour, Farzad; Ahmadi, Malahat; Javadi, Shahram

2014-01-01

The studies suggest that dogs living with human are potential risk of becoming MRSA carrier and increased risk of infections caused by MRSA. Phenotypic methods to detect methicillin resistance in Staphylococcus aureus (MRSA) are inadequate. The objective of the present study was to determine methicillin resistance in S. aureus by phenotypic susceptibility test (oxacillin disk diffusion, cefoxitin disk diffusion, oxacillin screen agar) and molecular methods (PCR as a gold standard) and the latex agglutination test for the detection of PBP2a and to evaluate the results of these tests for its sensitivity and specificity. A total of 100 swab samples were taken from muzzle site, in more contact with human, of dogs and MRSA were isolated. Oxacillin (1 μg), cefoxitin (30 μg) disk diffusion and oxacillin screen agar method were used. The isolates were also subjected to latex agglutination test for detection of PBP2a and PCR to detect mecA gene. By PCR 37% of isolates show the presence of mecA. Latex agglutination was found to be the most sensitive (97.29%) and cefoxitin disk diffusion to be the most specific (96.82%) tests for detection of MRSA. Our finding showed that combining oxacillin screen agar or cefoxitin disk diffusion with latex agglutination improves sensitivity and specificity to detect methicillin resistance S. aureus (MRSA) isolates. Copyright © 2014 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved.

Gold nanoparticles enhanced SERS aptasensor for the simultaneous detection of Salmonella typhimurium and Staphylococcus aureus.

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Zhang, Hui; Ma, Xiaoyuan; Liu, Ying; Duan, Nuo; Wu, Shijia; Wang, Zhouping; Xu, Baocai

2015-12-15

Salmonella typhimurium and Staphylococcus aureus are most common causes of food-associated disease. A Raman based biosensor was developed for S. typhimurium and S. aureus detection simultaneously. The biosensor was based on nanoparticles enhanced Raman intensity and the specific recognition of aptamer. The Raman signal probe and the capture probe are built. Gold nanoparticles (GNPs) modified with Raman molecules (Mercaptobenzoic acid and 5,5'-Dithiobis(2-nitrobenzoic acid)) and aptamer are used as the signal probe for S. typhimurium and S. aureus, respectively. Fe3O4 magnetic gold nanoparticles (MGNPs) immobilized with both aptamer of S. typhimurium and S. aureus are used as the capture probe. When S. typhimurium and S. aureus are added in the reaction system, the capture probe will capture the target bacteria through the specific binding effect of aptamer. And then the signal probe will be connected to the bacteria also by the effect of aptamer to form the sandwich like detection structure. The Raman intensified spectrum was measured to quantify S. typhimurium and S. aureus. Under optimal conditions, the SERS intensity of MBA at 1582 cm(-1) are used to measure S. typhimurium (y=186.4762+704.8571x, R(2)=0.9921) and the SERS intensity of DNTB at 1333 cm(-1) are used to measure S. aureus (y=135.2381+211.4286x, R(2)=0.9946) in the range of 10(2)-10(7) cfu mL(-1). The LOD is 35 cfu mL(-1) for S. aureus and 15 cfu mL(-1) for S. typhimurium. This method is simple and rapid, results in high sensitivity and specificity, and can be used to detect actual samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Staphylococcus aureus shifts towards commensalism in response to Corynebacterium species

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Matthew M Ramsey

2016-08-01

Full Text Available Staphylococcus aureus–human interactions result in a continuum of outcomes from commensalism to pathogenesis. S. aureus is a clinically important pathogen that asymptomatically colonizes ~25% of humans as a member of the nostril and skin microbiota, where it resides with other bacteria including commensal Corynebacterium species. Commensal Corynebacterium spp. are also positively correlated with S. aureus in chronic polymicrobial diabetic foot infections, distinct from acute monomicrobial S. aureus infections. Recent work by our lab and others indicates that microbe-microbe interactions between S. aureus and human skin/nasal commensals, including Corynebacterium species, affect S. aureus behavior and fitness. Thus, we hypothesized that S. aureus interactions with Corynebacterium spp. diminish S. aureus virulence. We tested this by assaying for changes in S. aureus gene expression during in vitro mono- versus coculture with Corynebacterium striatum, a common skin and nasal commensal. We observed a broad shift in S. aureus gene transcription during in vitro growth with C. striatum, including increased transcription of genes known to exhibit increased expression during human nasal colonization and decreased transcription of virulence genes. S. aureus uses several regulatory pathways to transition between commensal and pathogenic states. One of these, the quorum signal accessory gene regulator (agr system, was strongly inhibited in response to Corynebacterium spp. Phenotypically, S. aureus exposed to C. striatum exhibited increased adhesion to epithelial cells, reflecting a commensal state, and decreased hemolysin activity, reflecting an attenuation of virulence. Consistent with this, S. aureus displayed diminished fitness in experimental in vivo coinfection with C. striatum when compared to monoinfection. These data support a model in which S. aureus shifts from virulence towards a commensal state when exposed to commensal Corynebacterium species.

Detection of Antibiotic Resistant Staphylococcus aureus from Milk: A Public Health Implication

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Akindolire, Muyiwa Ajoke; Babalola, Olubukola Oluranti; Ateba, Collins Njie

2015-01-01

The aim of this study was to investigate the occurrence, antibiotic susceptibility profiles, and virulence genes determinants of S. aureus isolated from milk obtained from retail outlets of the North-West Province, South Africa. To achieve this, 200 samples of raw, bulk and pasteurised milk were obtained randomly from supermarkets, shops and some farms in the North-West Province between May 2012 and April 2013. S. aureus was isolated and positively identified using morphological (Gram staining), biochemical (DNase, catalase, haemolysis and rapid slide agglutination) tests, protein profile analysis (MALDI-TOF mass spectrometry) and molecular (nuc specific PCR) methods. The antimicrobial resistance profiles of the isolates were determined using the phenotypic agar diffusion method. Genes encoding enterotoxins, exfoliative toxins and collagen adhesins were also screened using PCR. Among all the samples examined, 30 of 40 raw milk samples (75%), 25 of 85 bulk milk samples (29%) and 10 of 75 pasteurised milk samples (13%) were positive for S. aureus. One hundred and fifty-six PCR-confirmed S. aureus isolates were obtained from 75 contaminated milk samples. A large proportion (60%–100%) of the isolates was resistant to penicillin G, ampicillin, oxacillin, vancomycin, teicoplanin and erythromycin. On the contrary, low level resistance (8.3%–40%) was observed for gentamicin, kanamycin and sulphamethoxazole. Methicillin resistance was detected in 59% of the multidrug resistant isolates and this was a cause for concern. However, only a small proportion (20.6%) of these isolates possessed PBP2a which codes for Methicillin resistance in S. aureus. In addition, 32.7% of isolates possessed the sec gene whereas the sea, seb sed, see, cna, eta, etb genes were not detected. The findings of this study showed that raw, bulk and pasteurised milk in the North-West Province is contaminated with toxigenic and multi-drug resistant S. aureus strains. There is a need to implement

Aptamer-fluorescent silica nanoparticles bioconjugates based dual-color flow cytometry for specific detection of Staphylococcus aureus.

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He, Xiaoxiao; Li, Yuhong; He, Dinggen; Wang, Kemin; Shangguan, Jingfang; Shi, Hui

2014-07-01

This paper describes a sensitive and specific determination strategy for Staphylococcus aureus (S. aureus) detection using aptamer recognition and fluorescent silica nanoparticles (FSiNPs) label based dual-color flow cytometry assay (Aptamer/FSiNPs-DCFCM). In the protocol, an aptamer, having high affinity to S. aureus, was first covalently immobilized onto chloropropyl functionalized FSiNPs through a click chemistry approach to generate aptamer-nanoparticles bioconjugates (Aptamer/FSiNPs). Next, S. aureus was incubated with Aptamer/FSiNPs, and then stained with SYBR Green I (a special staining material for the duplex DNA). Upon target binding and nucleic acid staining with SYBR Green I, the S. aureus was determined using two-color flow cytometry. The method took advantage of the specificity of aptamer, signal amplification of FSiNPs label and decreased false positives of two-color flow cytometry assay. It was demonstrated that these Aptamer/FSiNPs could efficiently recognize and fluorescently label target S. aureus. Through multiparameter determination with flow cytometry, this assay allowed for detection of as low as 1.5 x 10(2) and 7.6 x 10(2) cells mL(-1) S. aureus in buffer and spiked milk, respectively, with higher sensitivity than the Aptamer/FITC based flow cytometry.

Loop-mediated isothermal amplification for detection of Staphylococcus aureus in dairy cow suffering from mastitis.

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Tie, Zhang; Chunguang, Wang; Xiaoyuan, Wei; Xinghua, Zhao; Xiuhui, Zhong

2012-01-01

To develop a rapid detection method of Staphylococcus aureus using loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of the nuc gene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was 1 × 10² CFU/mL and that of PCR was 1 × 10⁴ CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection of Staphylococcus aureus has many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection of Staphylococcus aureus.

A combination of positive dielectrophoresis driven on-line enrichment and aptamer-fluorescent silica nanoparticle label for rapid and sensitive detection of Staphylococcus aureus.

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Shangguan, Jingfang; Li, Yuhong; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Zou, Zhen; Shi, Hui

2015-07-07

Staphylococcus aureus (S. aureus) is an important human pathogen that causes several diseases ranging from superficial skin infections to life-threatening diseases. Here, a method combining positive dielectrophoresis (pDEP) driven on-line enrichment and aptamer-fluorescent silica nanoparticle label has been developed for the rapid and sensitive detection of S. aureus in microfluidic channels. An aptamer, having high affinity to S. aureus, is used as the molecular recognition tool and immobilized onto chloropropyl functionalized fluorescent silica nanoparticles through a click chemistry approach to obtain S. aureus aptamer-nanoparticle bioconjugates (Apt(S.aureus)/FNPs). The pDEP driven on-line enrichment technology was used for accumulating the Apt(S.aureus)/FNP labeled S. aureus. After incubating with S. aureus, the mixture of Apt(S.aureus)/FNP labeled S. aureus and Apt(S.aureus)/FNPs was directly introduced into the pDEP-based microfluidic system. By applying an AC voltage in a pDEP frequency region, the Apt(S.aureus)/FNP labelled S. aureus moved to the electrodes and accumulated in the electrode gap, while the free Apt(S.aureus)/FNPs flowed away. The signal that came from the Apt(S.aureus)/FNP labelled S. aureus in the focused detection areas was then detected. Profiting from the specificity of aptamer, signal amplification of FNP label and pDEP on-line enrichment, this assay can detect as low as 93 and 270 cfu mL(-1)S. aureus in deionized water and spiked water samples, respectively, with higher sensitivities than our previously reported Apt(S.aureus)/FNP based flow cytometry. Moreover, without the need for separation and washing steps usually required for FNP label involved bioassays, the total assay time including sample pretreatment was within 2 h.

Detection of Staphylococcus aureus by functional gold nanoparticle-based affinity surface-assisted laser desorption/ionization mass spectrometry.

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Lai, Hong-Zheng; Wang, Sin-Ge; Wu, Ching-Yi; Chen, Yu-Chie

2015-02-17

Staphylococcus aureus is one of the common pathogenic bacteria responsible for bacterial infectious diseases and food poisoning. This study presents an analytical method based on the affinity nanoprobe-based mass spectrometry that enables detection of S. aureus in aqueous samples. A peptide aptamer DVFLGDVFLGDEC (DD) that can recognize S. aureus and methicillin-resistant S. aureus (MRSA) was used as the reducing agent and protective group to generate DD-immobilized gold nanoparticles (AuNPs@DD) from one-pot reactions. The thiol group from cysteine in the peptide aptamer, i.e., DD, can interact with gold ions to generate DD-immobilized AuNPs in an alkaline solution. The generated AuNPs@DD has an absorption maximum at ∼518 nm. The average particle size is 7.6 ± 1.2 nm. Furthermore, the generated AuNPs@DD can selectively bind with S. aureus and MRSA. The conjugates of the target bacteria with AuNPs were directly analyzed by surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The gold ions generated from the AuNPs@DD anchored on the target bacteria were monitored. Gold ions (m/z 197 and 394) were only generated from the conjugates of the target bacterium-AuNP@DD in the SALDI process. Thus, the gold ions could be used as the indicators for the presence of the target bacteria. The detection limit of S. aureus using this method is in the order of a few tens of cells. The low detection limit is due to the ease of generation of gold cluster ion derived from AuNPs under irradiation with a 355 nm laser beam. Apple juice mixed with S. aureus was used as the sample to demonstrate the suitability of the method for real-world application. Because of its low detection limit, this approach can potentially be used to screen the presence of S. aureus in complex samples.

Detection of Staphylococcus aureus among coagulase positive staphylococci from animal origin based on conventional and molecular methods

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Nikolina Velizarova Rusenova

2017-03-01

Full Text Available The present study aimed to detect Staphylococcus aureus (S. aureus among other coagulase positive staphylococci from animal origin by using conventional methods (biochemical tests and latex agglutination and a molecular method, based on the nuc gene, as the gold standard and to assess the usefulness of these methods. For this purpose, total of 344 staphylococcal isolates were collected and analysed. A total of 156 isolates suspicious for S. aureus were detected by a conventional biochemical method – 88 from cows, 18 from goats, 7 from pigs, 17 from poultry, 7 from rabbits and 19 from dogs. The majority of S. aureus strains gave typical biochemical reactions with the exception of 30 (19.2% and 25 (16% that were VP negative and weak positive in fermenting mannitol, respectively. Twelve strains were found to be non-haemolytic (7.7% and four strains did not ferment trehalose (2.6%. Other staphylococci were identified as S. pseudintermedius (n = 103, S. hyicus (n = 23 and the rest were coagulase-negative staphylococci. Latex agglutination test resulted in rapid positive reactions with S. aureus with exception of 5 strains (3.2% from cow mastitis milk. Positive agglutination reactions were also established with S. pseudintermedius, and S. hyicus. PCR confirmed all strains that were preliminary identified as S. aureus by amplification of 270 bp fragment of nuc gene specific for this species. The atypical reactions in certain strains established in this study have shown that the precise detection of S. aureus from animal origin should be done by combination of conventional and molecular methods.

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles.

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Chang, Yi-Chung; Yang, Chia-Ying; Sun, Ruei-Lin; Cheng, Yi-Feng; Kao, Wei-Chen; Yang, Pan-Chyr

2013-01-01

Staphylococcus aureus is one of the most important human pathogens, causing more than 500,000 infections in the United States each year. Traditional methods for bacterial culture and identification take several days, wasting precious time for patients who are suffering severe bacterial infections. Numerous nucleic acid-based detection methods have been introduced to address this deficiency; however, the costs and requirement for expensive equipment may limit the widespread use of such technologies. Thus, there is an unmet demand of new platform technology to improve the bacterial detection and identification in clinical practice. In this study, we developed a rapid, ultra-sensitive, low cost, and non-polymerase chain reaction (PCR)-based method for bacterial identification. Using this method, which measures the resonance light-scattering signal of aptamer-conjugated gold nanoparticles, we successfully detected single S. aureus cell within 1.5 hours. This new platform technology may have potential to develop a rapid and sensitive bacterial testing at point-of-care.

Detection of Genes for Superantigen Toxins in Methicillin-Resistant Staphylococcus aureus Clinical Isolates in Karachi

International Nuclear Information System (INIS)

Taj, Y.; Fatima, I.; Ali, S. W.; Kazmi, S. U.

2014-01-01

Objective: To detect genes for enterotoxins, exfoliative and toxic shock syndrome toxins in Staphylococcus aureus (S. aureus) strains isolated from clinical specimens. Study Design: Cross-sectional observational study. Place and Duration of Study: Department of Molecular Genetics, Dr. Ziauddin Hospital, Karachi, from January to December 2010. Methodology: Two hundred and ninety eight S. aureus clinical isolates were obtained from various clinical samples received at Dr. Ziauddin Hospital, Karachi. Out of these, 115 were detected as methicillin resistant (MRSA) by cefoxitin disk diffusion test showing a prevalence rate of 38.6%. Detection of individual toxin genes was performed by Polymerase Chain Reaction (PCR) by using only one primer pair for each tube. Uniplex primers were preferred as multiplex primers are longer in base pairs and have the potential for cross reaction due to non-specific binding and increase in optimization time. Results: The possession of a single gene or more than a single gene in MRSA isolates was found in 61.73% of clinical samples; the highest number was found in pus swab, followed by sputum, blood, urethral swab, and urine. The prevalence of toxin genes was higher in MRSA as compared to methicillin sensitive (MSSA) isolates (19.12%). Conclusion: PCR detects strains possessing toxin genes independent of their expression. The possession of genes for super-antigens seems to be a frequent and habitual trait of S. aureus more so in MRSA. (author)

Molecular detection of the carriers of Staphylococcus aureus golden in referred to the Imam Ali Clinic in Shahrekord, Iran

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Maryam Reisi

2014-12-01

Full Text Available Objective: To conduct for the molecular detection of Staphylococcus aureus (S. aureus (Golden staph carriers among patients referred to the Imam Ali Clinic, Shahrekord, Iran. Methods: This sectional-descriptive study was conducted with 200 persons with suspected upper respiratory tract infections, who were referred to the Imam Ali Clinic in Shahrekord, Iran, in 2012. After culturing the nasal swab samples in mannitol salt agar and blood agar, S. aureus colonies were confirmed by biochemical methods. To determine the susceptibility of S. aureus strains isolated, molecular methods were used. Results: Among the 200 investigated samples, 60 cases (30%, comprising 25 men (41.66% and 35 women (58.33%, were found to be S. aureus carriers. Conclusions: The results of the present study showed that the frequency of the S. aureus strain isolated from the nasal swabs of patients with respiratory tract infections admitted to the Imam Ali Clinic in Shahrekord, Iran, was remarkable. Thus, knowing detection of S. aureus carriers, who are at a risk of spreading nosocomial infection among the staff, is vital to control and prevent nosocomial infections.

Detection of methicillin resistant Staphylococcus aureus (MRSA) from recreational beach using the mecA gene

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Zulkifli, Aisya; Ahmad, Asmat

2015-09-01

Water samples were collected in triplicates from three different locations choosen from the recreational beach of Teluk Kemang, Port Dickson as sampling station including main area of recreation activity for the public. Bacteria were isolated from the water and cultured. Out of 286 presumptive Staphylococcus aureus enumerated by using culture method, only 4 (1.4 %) confirmed as Meticillin Resistant S. aureus (MRSA) based on PCR detection of mecA gene. Interestingly, all of MRSA detections were found at the main area of recreational activity. Our results suggested that public beaches may be reservoir for transmission of MRSA to beach visitors and PCR using the mecA gene is the fastest way to detect this pathogenic bacteria.

Visual detection and microplate assay for Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification

International Nuclear Information System (INIS)

Yuan, Jinglei; Li, Can; Ma, Xiaoyuan; Xia, Yu; Chen, Jie; Wang, Zhouping; Yu, Ye

2014-01-01

We have developed a specific method for the visual detection of Staphylococcus aureus based on aptamer recognition coupled to tyramine signal amplification technology. A biotinylated aptamer specific for S. aureus was immobilized on the surface of the wells of a microplate via biotin-avidin binding. Then, the target bacteria (S. aureus), the biotinylated-aptamer-streptavidin-HRP conjugates, biotinylated tyramine, hydrogen peroxide and streptavidin-HRP were successively placed in the wells of the microplate. After adding TMB reagent and stop solution, the intensity of the yellow reaction product can be visually inspected or measured with a plate reader. Under optimized conditions, there is a linear relationship between absorbance at 450 nm and the concentration of S. aureus in the 10 to 107 cfu mL −1 concentration range (with an R 2 of 0.9976). The limit of detection is 8 cfu mL −1 . (author)

Polymerase chain reaction assay for detection of Staphylococcus aureus in buffalo milk

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V.K. Jain

2010-02-01

Full Text Available In India, Haryana has the world’s best dairy type buffalo, the Murrah capable of milk yields as high as 35 kg a day. Clinical and Sub clinical mastitis exerts a negative impact on milk quality, quantity and animal health and profits. In India, Staphylococci are the main causative agents responsible for mastitis of economic importance. Therefore, a suitable and specific test is required for the rapid diagnosis of Staphylococcus aureus. For definitive diagnosis of Staphylococcus aureus in mastitic milk, a polymerase chain reaction assay was developed using target sequence of 16S to 23S rRNA spacer region. This test can be performed within hours and avoids cumbersome and lengthy steps involved in microbiological culture of milk and biochemical tests. Polymerase chain reaction assay can be used as a screening test for a large herd to detect Staphylococcus aureus in milk.

Capture and detection of Staphylococcus aureus with dual labeled aptamers to cell surface components.

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Ramlal, Shylaja; Mondal, Bhairab; Lavu, Padma Sudharani; N, Bhavanashri; Kingston, Joseph

2018-01-16

In the present study, a high throughput whole cell SELEX method has been applied successfully in selecting specific aptamers against whole cells of Staphylococcus aureus, a potent food poisoning bacterium. A total ten rounds of SELEX and three rounds of intermittent counter SELEX, was performed to obtain specific aptamers. Obtained oligonucleotide pool were cloned, sequenced and then grouped into different families based on their primary sequence homology and secondary structure similarity. FITC labeled sequences from different families were selected for further characterization via flow cytometry analysis. The dissociation constant (K d ) values of specific and higher binders ranged from 34 to 128nM. Binding assays to assess the selectivity of aptamer RAB10, RAB 20, RAB 28 and RAB 35 demonstrated high affinity against S. aureus and low binding affinity for other bacteria. To demonstrate the potential use of the aptamer a sensitive dual labeled sandwich detection system was developed using aptamer RAB10 and RAB 35 with a detection limit of 10 2 CFU/mL. Furthermore detection from mixed cell population and spiked sample emphasized the robustness as well as applicability of the developed method. Altogether, the established assay could be a reliable detection tool for the routine investigation of Staphylococcus aureus in samples from food and clinical sources. Copyright © 2017. Published by Elsevier B.V.

Development of An Impedimetric Aptasensor for the Detection of Staphylococcus aureus.

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Reich, Peggy; Stoltenburg, Regina; Strehlitz, Beate; Frense, Dieter; Beckmann, Dieter

2017-11-21

In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus . The used aptamer targets protein A, a surface bound virulence factor of S. aureus . The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1.01 ± 0.44 × 10 13 aptamer molecules per cm². As shown with quartz crystal microbalance experiments, the immobilized aptamer retained its functionality to bind recombinant protein A. Our impedimetric biosensor is based on the principle that binding of target molecules to the immobilized aptamer decreases the electron transfer between electrode and ferri-/ferrocyanide in solution, which is measured as an increase of impedance. Microscale thermophoresis measurements showed that addition of the redox probe ferri-/ferrocyanide has no influence on the binding of aptamer and its target. We demonstrated that upon incubation with various concentrations of S. aureus , the charge-transfer resistance increased proportionally. The developed biosensor showed a limit of detection of 10 CFU·mL -1 and results were available within 10 minutes. The biosensor is highly selective, distinguishing non-target bacteria such as Escherichia coli and Staphylococcus epidermidis . This work highlights the immense potential of impedimetric aptasensors for future biosensing applications.

Development of An Impedimetric Aptasensor for the Detection of Staphylococcus aureus

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Peggy Reich

2017-11-01

Full Text Available In combination with electrochemical impedance spectroscopy, aptamer-based biosensors are a powerful tool for fast analytical devices. Herein, we present an impedimetric aptasensor for the detection of the human pathogen Staphylococcus aureus. The used aptamer targets protein A, a surface bound virulence factor of S. aureus. The thiol-modified protein A-binding aptamer was co-immobilized with 6-mercapto-1-hexanol onto gold electrodes by self-assembly. Optimization of the ratio of aptamer to 6-mercapto-1-hexanol resulted in an average density of 1.01 ± 0.44 × 1013 aptamer molecules per cm2. As shown with quartz crystal microbalance experiments, the immobilized aptamer retained its functionality to bind recombinant protein A. Our impedimetric biosensor is based on the principle that binding of target molecules to the immobilized aptamer decreases the electron transfer between electrode and ferri-/ferrocyanide in solution, which is measured as an increase of impedance. Microscale thermophoresis measurements showed that addition of the redox probe ferri-/ferrocyanide has no influence on the binding of aptamer and its target. We demonstrated that upon incubation with various concentrations of S. aureus, the charge-transfer resistance increased proportionally. The developed biosensor showed a limit of detection of 10 CFU·mL−1 and results were available within 10 minutes. The biosensor is highly selective, distinguishing non-target bacteria such as Escherichia coli and Staphylococcus epidermidis. This work highlights the immense potential of impedimetric aptasensors for future biosensing applications.

The detection and differentiation of methicillin-resistant and methicillin-susceptible Staphylococcus aureus endocarditis by using the BD GeneOhm StaphSR Assay.

Science.gov (United States)

Frey, Amy B; Wilson, Deborah A; LaSalvia, Margaret M; Tan, Carmela D; Rodriguez, E Rene; Shrestha, Nabin K; Hall, Gerri S; Procop, Gary W

2011-11-01

We use the BD GeneOhm StaphSR Assay (BD Diagnostics, Oakville, Canada) to screen for Staphylococcus aureus nasal colonization and sought to evaluate this assay for the assessment of valve specimens from patients with endocarditis. We examined 23 paired fresh and formalin-fixed, paraffin-embedded cardiac valve tissue samples, 12 of which had S aureus endocarditis, using the BD GeneOhm StaphSR Assay for the detection and differentiation of methicillin-susceptible and methicillin-resistant S aureus. This assay appropriately characterized all specimens with respect to the presence or absence of S aureus. There was an 87.5% correlation between the presence or absence of the mecA gene and the oxacillin susceptibility results for the S aureus isolates studied. The GeneOhm StaphSR assay accurately detected S aureus in cardiac valve tissue samples. Rare discordances were observed between oxacillin susceptibility status and mecA gene detection by this assay.

Staphylococcus aureus Transcriptome Architecture

DEFF Research Database (Denmark)

Mäder, Ulrike; Nicolas, Pierre; Depke, Maren

2016-01-01

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of...

Molecular basis of virulence in Staphylococcus aureus mastitis.

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Caroline Le Maréchal

Full Text Available S. aureus is one of the main pathogens involved in ruminant mastitis worldwide. The severity of staphylococcal infection is highly variable, ranging from subclinical to gangrenous mastitis. This work represents an in-depth characterization of S. aureus mastitis isolates to identify bacterial factors involved in severity of mastitis infection.We employed genomic, transcriptomic and proteomic approaches to comprehensively compare two clonally related S. aureus strains that reproducibly induce severe (strain O11 and milder (strain O46 mastitis in ewes. Variation in the content of mobile genetic elements, iron acquisition and metabolism, transcriptional regulation and exoprotein production was observed. In particular, O11 produced relatively high levels of exoproteins, including toxins and proteases known to be important in virulence. A characteristic we observed in other S. aureus strains isolated from clinical mastitis cases.Our data are consistent with a dose-dependant role of some staphylococcal factors in the hypervirulence of strains isolated from severe mastitis. Mobile genetic elements, transcriptional regulators, exoproteins and iron acquisition pathways constitute good targets for further research to define the underlying mechanisms of mastitis severity.

Eugenol reduces the expression of virulence-related exoproteins in Staphylococcus aureus.

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Qiu, Jiazhang; Feng, Haihua; Lu, Jing; Xiang, Hua; Wang, Dacheng; Dong, Jing; Wang, Jianfeng; Wang, Xiaoliang; Liu, Juxiong; Deng, Xuming

2010-09-01

Eugenol, an essential oil component in plants, has been demonstrated to possess activity against both gram-positive and gram-negative bacteria. This study examined the influence that subinhibitory concentrations of eugenol may have on the expression of the major exotoxins produced by Staphylococcus aureus. The results from a tumor necrosis factor (TNF) release assay and a hemolysin assay indicated that S. aureus cultured with graded subinhibitory concentrations of eugenol (16 to 128 microg/ml) dose dependently decreased the TNF-inducing and hemolytic activities of culture supernatants. Western blot analysis showed that eugenol significantly reduced the production of staphylococcal enterotoxin A (SEA), SEB, and toxic shock syndrome toxin 1 (the key exotoxins to induce TNF release), as well as the expression of alpha-hemolysin (the major hemolysin to cause hemolysis). In addition, this suppression was also evaluated at the transcriptional level via real-time reverse transcription (RT)-PCR analysis. The transcriptional analysis indicated that 128 microg/ml of eugenol remarkably repressed the transcription of the S. aureus sea, seb, tst, and hla genes. According to these results, eugenol has the potential to be rationally applied on food products as a novel food antimicrobial agent both to inhibit the growth of bacteria and to suppress the production of exotoxins by S. aureus.

Detection of Small Colony Variants Among Methicillin-Resistant Staphylococcus aureus Blood Isolates.

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Yagci, Server; Sancak, Banu; Hascelik, Gulsen

2016-12-01

Staphylococcus aureus small colony variants (SCVs) are associated with chronic and persistent infections. Methicillin-resistant S. aureus (MRSA) SCVs cause more severe infections and mortality rates are higher in comparison with infections caused by MRSA. Our objective was to document the prevalence and phenotypical characteristics of SCVs among MRSA blood isolates. MRSA strains isolated from blood during 1999-2009 were evaluated retrospectively. Among 299 MRSA isolates, suspected colonies were inoculated onto Columbia blood agar and Schaedler agar. Columbia blood agar was incubated in normal atmosphere and Schaedler agar in 5-10% CO 2 , both at 35°C. If the small, nonpigmented, nonhemolytic colonies on Columbia blood agar were seen as normal-sized, hemolytic, and pigmented colonies on Schaedler agar, they were considered as MRSA SCVs. Six MRSA SCVs were detected. When subcultures were made, four of them reversed to phenotypically normal S. aureus, but two isolates were stable as SCV phenotype. The prevalence of SCVs among MRSA blood isolates was found as 6/299 (2%) with 2 (0.67%) stable. The detection of SCVs among MRSA blood isolates was reported from Turkey for the first time in this study. As the clinical significance of MRSA infections is well documented, evaluation of MRSA SCVs in clinical samples, especially from intensive care patients and those who have chronic and persistent infections are important to consider.

Detection of Methicillin Resistance and Various Virulence Factors in Staphylococcus aureus Strains Isolated from Nasal Carriers

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Hatice Türk Dağı

2015-06-01

Full Text Available Background: Staphylococus aureus can be found as a commensal on skin and nasal flora or it may cause local and invasive infections. S. aureus has a large number of virulence factors. Aims: To investigate the methicillin resistance and frequency of various virulence factors in S. aureus nasal isolates. Study Design: Descriptive study. Methods: Nasal samples collected from university students were cultured in media. S. aureus was identified by conventional methods and the Staphyloslide latex test (Becton Dickinson, Sparks, USA. Antibiotic susceptibility tests were conducted, and the methicillin resistance was determined. The mecA, nuc, pvl and staphylococcal toxin genes were examined by polymerase chain reaction (PCR. Results: S. aureus was isolated in 104 of 600 (17.3% nasal samples. In total, 101 (97.1% S. aureus isolates were methicillin-sensitive and the remaining 3 (2.9% were methicillin-resistant. Furthermore, all but five isolates carried at least one staphylococcal enterotoxin gene, with seg being predominant. The tst and eta genes were determined in 29 (27.9%, and 3 (2.9% isolates, respectively. None of the S. aureus isolates harbored see, etb, and pvl genes. Conclusion: A moderate rate of S. aureus carriage and low frequency of MRSA were detected in healthy students. S. aureus isolates had a high prevalence of staphylococcal enterotoxin genes and the tst gene. In this study, a large number of virulence factors were examined in S. aureus nasal isolates, and the data obtained from this study can be used for monitoring the prevalence of virulence genes in S. aureus strains isolated from nasal carriers.

Specific capture and detection of Staphylococcus aureus with high-affinity modified aptamers to cell surface components.

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Baumstummler, A; Lehmann, D; Janjic, N; Ochsner, U A

2014-10-01

Slow off-rate modified aptamer (SOMAmer) reagents were generated to several Staphylococcus aureus cell surface-associated proteins via SELEX with multiple modified DNA libraries using purified recombinant or native proteins. High-affinity binding agents with sub-nanomolar Kd 's were obtained for staphylococcal protein A (SpA), clumping factors (ClfA, ClfB), fibronectin-binding proteins (FnbA, FnbB) and iron-regulated surface determinants (Isd). Further screening revealed several SOMAmers that specifically bound to Staph. aureus cells from all strains that were tested, but not to other staphylococci or other bacteria. SpA and ClfA SOMAmers proved useful for the selective capture and enrichment of Staph. aureus cells, as shown by culture and PCR, leading to improved limits of detection and efficient removal of PCR inhibitors. Detection of Staph. aureus cells was enhanced by several orders of magnitude when the bacterial cell surface was coated with SOMAmers followed by qPCR of the SOMAmers. Furthermore, fluorescence-labelled SpA SOMAmers demonstrated their utility as direct detection agents in flow cytometry. Significance and impact of the study: Monitoring for microbial contamination of food, water, nonsterile products or the environment is typically based on culture, PCR or antibodies. Aptamers that bind with high specificity and affinity to well-conserved cell surface epitopes represent a promising novel type of reagents to detect bacterial cells without the need for culture or cell lysis, including for the capture and enrichment of bacteria present at low cell densities and for the direct detection via qPCR or fluorescent staining. © 2014 Soma Logic, Inc. published by John Wiley & Sons Ltd On behalf of the society for Applied Microbiology.

Detection of toxic shock toxin (tst gene in Staphylococcus aureus isolated from bovine milk samples

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S. Baniardalan

2017-09-01

Full Text Available Staphylococcus aureus is a major causative pathogen of clinical and subclinical mastitis in dairy cattle all over the world. This agent produces a variety of extracellular toxins and virulence factors in-cluding toxic shock syndrome toxin-1 (TSST-1 which is the major cause of toxic shock syndrome (TSS. In the present study, 76 S. aureus isolates have been obtained from milk samples collected from 7 dairy herds in Hamedan province of Iran. The isolates were identified based on the biochemical and molecular methods using PCR amplification of the femA gene. The staphylococcal isolates were also examined for the presence of TSST-1 (tst encoding gene. This gene was detected in only one S. aureus isolate (1.3%. The results revealed that S. aureus strains causing bovine mastitis may potentially produce staphylococcal toxic shock syndrome toxin-1, indicating that it is very important to follow the presence of TSST-1 producing S. aureus isolates in foodstuffs to protect consumers against the risk of toxic shock syndrome

Detection of Staphylococcus aureus enterotoxins in sheep cheese and dairy desserts by multiplex PCR technique.

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Ertas, Nurhan; Gonulalan, Zafer; Yildirim, Yeliz; Kum, Erhan

2010-08-15

The aim of this study was to investigate the presence of Staphylococcus aureus (S. aureus) and staphylococcal enterotoxins (SEs) genes in sheep cheese and dairy dessert samples by multiplex PCR (mPCR) technique. A total of 150 samples were analyzed consisting of 50 dairy dessert samples and 100 sheep cheese. Coagulase positive staphylococci (CPS) were found in 86 (57.3%) out of 150 analyzed samples. S. aureus were isolated from 60 (60%), 26 (52%) of sheep cheese and from of dairy desserts, respectively. Five suspected colonies were tested from each sheep cheese and dairy dessert samples for phenotypic and genotypic characterizations. A total of 430 isolates from the 86 positive samples were investigated in this study. Eighty (18.6%) isolates were characterized as S. aureus. The enterotoxin genes (sea, seb, sec, sed) were found in 13 (3.02%) out of 80 isolates. From cheese isolates, sea, seb and sed were detected in 5 (1.6%), 2 (0.6%), 1 (0.3%), respectively. From dairy dessert isolates, sea, sec and sed were detected in 3 (2.3%), 1 (0.76%), 1 (0.76%), respectively. The presence of SEs was identified in 12 (2.8%) out of 80 isolates by using ELISA technique. It was determined that these SEs had a distribution of 7 (1.6%) SEA, 2 (0.46%) SEB, 1 (0.23%) SEC, and 2 (0.46%) SED. SEs were found in 7 (2.3%) cheese and 5 (3.8%) dairy dessert isolates. In conclusion, S.aureus and their SEs were found to be present in sheep cheese and dairy desserts in this study. It is emphasized that the presence of S. aureus and their SEs genes in sheep cheese and dairy desserts may be regarded as a potential risk for human health. Copyright 2010 Elsevier B.V. All rights reserved.

Ultra-sensitive chemiluminescent detection of Staphylococcus aureus based on competitive binding of Staphylococcus protein A-modified magnetic beads to immunoglobulin G

International Nuclear Information System (INIS)

Xiong, Jie; Wang, Wenwen; Zhou, Yali; Kong, Weijun; Wang, Zhenxing; Fu, Zhifeng

2016-01-01

Staphylococcus protein A (SPA) is a surface protein only expressed naturally in the cell walls of Staphylococcus aureus (S. aureus) and binds specifically to the Fc region of immunoglobulin G (IgG). This fact can be utilized for the detection of S. aureus. Specifically, SPA-modified magnetic beads, compete with S. aureus pathogens for binding to rabbit IgG that previously was labeled with horseradish peroxidase (HRP). The beads were then magnetically separated, and chemiluminescence (CL) was generated by adding the reagents luminol and H_2O_2. Under optimal conditions, the intensity of CL decreases with increasing concentration of S. aureus over a very wide linear range (10 to 1.0 × 10"9 cfu·mL"−"1), with a limit of detection of 6.0 cfu·mL"−"1 at an S/N ratio of 3. The assay (including binding reaction, magnetic separation, washing of beads and detection) is completed within 50 min which is faster than many reported methods. It can well distinguish S. aureus from other Gram-positive and Gram-negative bacteria. The magnetic beads have the beneficial effect of eliminating undesired matrix effects and of concentrating the sample. The method was applied to the analysis of urine, apple juice and glucose injection samples spiked with S. aureus, and recoveries ranged from 85 to 107 %. (author)

Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry.

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Julie Gagnaire

Full Text Available The aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF mass spectrometry (MS, correlate delta-toxin expression with accessory gene regulator (agr status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively. In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies.

Rapid detection of methicillin resistance in Staphylococcus aureus isolates by the MRSA-screen latex agglutination test

NARCIS (Netherlands)

W.B. van Leeuwen (Willem); C. van Pelt (Cindy); A. Luijendijk (Ad); H.A. Verbrugh (Henri); W.H.F. Goessens (Wil)

1999-01-01

textabstractThe slide agglutination test MRSA-Screen (Denka Seiken Co., Niigata, Japan) was compared with the mecA PCR ("gold standard") for the detection of methicillin resistance in Staphylococcus aureus. The MRSA-Screen test detected the penicillin-binding protein 2a

Detection of some virulence factors in Staphylococcus aureus ...

African Journals Online (AJOL)

USER

2010-06-21

Jun 21, 2010 ... Mastitis is one of the common diseases of dairy cattle and an inflammatory ... Key words: Bovine mastitis, Staphylococcus aureus, virulence factors, ... frequent cause of subclinical intramammary infections in ... genotypes has not been investigated. ... genes in S. aureus, we were particularly interested in the.

Detection of meca gene from methicillin resistant staphylococcus aureus isolates of north sumatera

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Septiani Nasution, Gabriella; Suryanto, Dwi; Lia Kusumawati, R.

2018-03-01

Methicillin Resistant Staphylococcus aureus (MRSA) is a major pathogen associated with hospital-acquired infections (nosocomial infections). MRSA is a type of S. aureus resistant to the sub-group of beta-lactam antibiotics such as penicillin, cephalosporin, monobactam, and carbapenem. MRSA is resistant because of genetic changes caused by exposure to irrational antibiotic therapy. This study aimed to detect mecA gene in North Sumatra isolates of MRSA and to determine the pattern of antibiotic resistance in S.aureus isolates classified as MRSA by Vitek 2 Compact in the Central Public Hospital Haji Adam Malik, Medan. Samples were 40 isolates of S. aureus classified as MRSA obtained from clinical microbiology specimens. DNA isolation of the isolates was conducted by a method of freeze-thaw cycling. Amplification of mecA gene was done by PCR technique using specific primer for the gene. PCR products were visualized using mini-gel electrophoresis. The results showed that all MRSA isolates showed to have 533 bp band of mecA. Antibiotics test of Vitek 2 Compact showed that despite all isolates were resistant to beta-lactam antibiotics groups; the isolates showed multidrug resistant to other common antibiotics, such as aminoglycosides, macrolides, and fluoroquinolones. However, they were still sensitive to vancomycin (82.5% isolates), linezolid (97.5% isolates), and tigecycline (100% isolates).

Detection of enterotoxins and genotyping of Staphylococcus aureus strains isolated from Isfahan Educational Hospital, Iran

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Seyed Asghar Havaei

2017-10-01

Full Text Available Background and aims: Staphylococcus aureus is known as one of the most important nosocomial pathogens, which may lead to several infections. The aim of this study was determining the enterotoxins A, C, and TSST-1 and molecular characterization of S. aureus strains with PFGE and MLST typing methods. Materials and methods: In the present study during the sixmonths sampling, fifty S. aureus strains were isolated from patients admitted to Al-Zahra university hospital. Antimicrobial susceptibility testing, Multiplex PCR for detection of enterotoxin A, C and TSST-1, pulse field gel electrophoresis (PFGE and multilocus sequence typing (MLST were used for molecular typing. Results: In antibiogram the highest and lowest percentage of resistance was belonged to tetracycline and rifampin respectively. Multiplex PCR indicated that 30% of the strains harbored sea and 34% harbored sec genes. However, only 4% of our collected isolates had tsst gene. In PFGE method analysis on all S. aureus strains, a total of 19 different patterns were identified. Nine various sequence types in 27 selected S. aureus isolates were identified by MLST. Conclusions: Present study indicates a possible higher variability among our S. aureus strains by two different molecular typing methods; nevertheless four main common types (CT1, CT7, CT9, and CT11 with at least one toxin genes were determined.

Multiplex Real-Time PCR for Detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL) Genes from Selective Enrichments from Animals and Retail Meat

Science.gov (United States)

Velasco, Valeria; Sherwood, Julie S.; Rojas-García, Pedro P.; Logue, Catherine M.

2014-01-01

The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL) genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat). The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL (virulence factor), and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68–0.88 (from substantial to almost perfect agreement) and 0.29–0.77 (from fair to substantial agreement) for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0–0.49 (from no agreement beyond that expected by chance to moderate agreement) for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA) using

Multiplex real-time PCR for detection of Staphylococcus aureus, mecA and Panton-Valentine Leukocidin (PVL genes from selective enrichments from animals and retail meat.

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Valeria Velasco

Full Text Available The aim of this study was to compare a real-time PCR assay, with a conventional culture/PCR method, to detect S. aureus, mecA and Panton-Valentine Leukocidin (PVL genes in animals and retail meat, using a two-step selective enrichment protocol. A total of 234 samples were examined (77 animal nasal swabs, 112 retail raw meat, and 45 deli meat. The multiplex real-time PCR targeted the genes: nuc (identification of S. aureus, mecA (associated with methicillin resistance and PVL (virulence factor, and the primary and secondary enrichment samples were assessed. The conventional culture/PCR method included the two-step selective enrichment, selective plating, biochemical testing, and multiplex PCR for confirmation. The conventional culture/PCR method recovered 95/234 positive S. aureus samples. Application of real-time PCR on samples following primary and secondary enrichment detected S. aureus in 111/234 and 120/234 samples respectively. For detection of S. aureus, the kappa statistic was 0.68-0.88 (from substantial to almost perfect agreement and 0.29-0.77 (from fair to substantial agreement for primary and secondary enrichments, using real-time PCR. For detection of mecA gene, the kappa statistic was 0-0.49 (from no agreement beyond that expected by chance to moderate agreement for primary and secondary enrichment samples. Two pork samples were mecA gene positive by all methods. The real-time PCR assay detected the mecA gene in samples that were negative for S. aureus, but positive for Staphylococcus spp. The PVL gene was not detected in any sample by the conventional culture/PCR method or the real-time PCR assay. Among S. aureus isolated by conventional culture/PCR method, the sequence type ST398, and multi-drug resistant strains were found in animals and raw meat samples. The real-time PCR assay may be recommended as a rapid method for detection of S. aureus and the mecA gene, with further confirmation of methicillin-resistant S. aureus (MRSA

Lab on a chip sensor for rapid detection and antibiotic resistance determination of Staphylococcus aureus.

Science.gov (United States)

Abeyrathne, Chathurika D; Huynh, Duc H; Mcintire, Thomas W; Nguyen, Thanh C; Nasr, Babak; Zantomio, Daniela; Chana, Gursharan; Abbott, Iain; Choong, Peter; Catton, Mike; Skafidas, Efstratios

2016-03-21

The Gram-positive bacterium, Staphylococcus aureus (S. aureus), is a major pathogen responsible for a variety of infectious diseases ranging from cellulitis to more serious conditions such as septic arthritis and septicaemia. Timely treatment with appropriate antibiotic therapy is essential to ensure clinical defervescence and to prevent further complications such as infective endocarditis or organ impairment due to septic shock. To date, initial antibiotic choice is empirical, using a "best guess" of likely organism and sensitivity- an approach adopted due to the lack of rapid identification methods for bacteria. Current culture based methods take up to 5 days to identify the causative bacterial pathogen and its antibiotic sensitivity. This paper provides proof of concept for a biosensor, based on interdigitated electrodes, to detect the presence of S. aureus and ascertain its sensitivity to flucloxacillin rapidly (within 2 hours) in a cost effective manner. The proposed method is label-free and uses non-faradic measurements. This is the first study to successfully employ interdigitated electrodes for the rapid detection of antibiotic resistance. The method described has important potential outcomes of faster definitive antibiotic treatment and more rapid clinical response to treatment.

Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

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Megan R Kiedrowski

Full Text Available Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2 that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity.

Science.gov (United States)

Henke, Sarah K; Cronan, John E

2016-11-01

Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins. However, the S. aureus BirA was reported to dimerize and bind DNA in the absence of biotin or biotinoyl-AMP (Soares da Costa et al. (2014) Mol Microbiol 91: 110-120). These in vitro results argued that the protein would be unable to respond to the levels of biotin or acceptor proteins and thus would lack the regulatory properties of the other characterized BirA proteins. We tested the regulatory function of the protein using an in vivo model system and examined its DNA binding properties in vitro using electrophoretic mobility shift and fluorescence anisotropy analyses. We report that the S. aureus BirA is an effective regulator of biotin operon transcription and that the prior data can be attributed to artifacts of mobility shift analyses. We also report that deletion of the DNA binding domain of the S. aureus BirA results in loss of virtually all of its ligation activity. © 2016 John Wiley & Sons Ltd.

Copper-Based Metal-Organic Framework Nanoparticles with Peroxidase-Like Activity for Sensitive Colorimetric Detection of Staphylococcus aureus.

Science.gov (United States)

Wang, Shuqin; Deng, Wenfang; Yang, Lu; Tan, Yueming; Xie, Qingji; Yao, Shouzhuo

2017-07-26

Cu-MOF nanoparticles with an average diameter of 550 nm were synthesized from 2-aminoterephthalic acid and Cu(NO 3 ) 2 by a mixed solvothermal method. The Cu-MOF nanoparticles can show peroxidase-like activity that can catalyze 3,3',5,5'-tetramethylbenzidine to produce a yellow chromogenic reaction in the presence of H 2 O 2 . The presence of abundant amine groups on the surfaces of Cu-MOF nanoparticles enables facile modification of Staphylococcus aureus (S. aureus) aptamer on Cu-MOF nanoparticles. By combining Cu-MOF-catalyzed chromogenic reaction with aptamer recognition and magnetic separation, a simple, sensitive, and selective colorimetric method for the detection of S. aureus was developed.

Detection of Alpha-Toxin and Other Virulence Factors in Biofilms of Staphylococcus aureus on Polystyrene and a Human Epidermal Model.

Science.gov (United States)

den Reijer, P M; Haisma, E M; Lemmens-den Toom, N A; Willemse, J; Koning, R I; Koning, R A; Demmers, J A A; Dekkers, D H W; Rijkers, E; El Ghalbzouri, A; Nibbering, P H; van Wamel, W

2016-01-01

The ability of Staphylococcus aureus to successfully colonize (a)biotic surfaces may be explained by biofilm formation and the actions of virulence factors. The aim of the present study was to establish the presence of 52 proteins, including virulence factors such as alpha-toxin, during biofilm formation of five different (methicillin resistant) S. aureus strains on Leiden human epidermal models (LEMs) and polystyrene surfaces (PS) using a competitive Luminex-based assay. All five S. aureus strains formed biofilms on PS, whereas only three out of five strains formed biofilms on LEMs. Out of the 52 tested proteins, six functionally diverse proteins (ClfB, glucosaminidase, IsdA, IsaA, SACOL0688 and nuclease) were detected in biofilms of all strains on both PS and LEMs. At the same time, four toxins (alpha-toxin, gamma-hemolysin B and leukocidins D and E), two immune modulators (formyl peptide receptor-like inhibitory protein and Staphylococcal superantigen-like protein 1), and two other proteins (lipase and LytM) were detectable in biofilms by all five S. aureus strains on LEMs, but not on PS. In contrast, fibronectin-binding protein B (FnbpB) was detectable in biofilms by all S. aureus biofilms on PS, but not on LEMs. These data were largely confirmed by the results from proteomic and transcriptomic analyses and in case of alpha-toxin additionally by GFP-reporter technology. Functionally diverse virulence factors of (methicillin-resistant) S. aureus are present during biofilm formation on LEMs and PS. These results could aid in identifying novel targets for future treatment strategies against biofilm-associated infections.

Virulence Factors and Antibiotic Susceptibility of Staphylococcus aureus Isolates in Ready-to-Eat Foods: Detection of S. aureus Contamination and a High Prevalence of Virulence Genes

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Suat Moi Puah

2016-02-01

Full Text Available Staphylococcus aureus is one of the leading causes of food poisoning. Its pathogenicity results from the possession of virulence genes that produce different toxins which result in self-limiting to severe illness often requiring hospitalization. In this study of 200 sushi and sashimi samples, S. aureus contamination was confirmed in 26% of the food samples. The S. aureus isolates were further characterized for virulence genes and antibiotic susceptibility. A high incidence of virulence genes was identified in 96.2% of the isolates and 20 different virulence gene profiles were confirmed. DNA amplification showed that 30.8% (16/52 of the S. aureus carried at least one SE gene which causes staphylococcal food poisoning. The most common enterotoxin gene was seg (11.5% and the egc cluster was detected in 5.8% of the isolates. A combination of hla and hld was the most prevalent coexistence virulence genes and accounted for 59.6% of all isolates. Antibiotic resistance studies showed tetracycline resistance to be the most common at 28.8% while multi-drug resistance was found to be low at 3.8%. In conclusion, the high rate of S. aureus in the sampled sushi and sashimi indicates the need for food safety guidelines.

Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A

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Lama, Amrita; Pané-Farré, Jan; Chon, Tai; Wiersma, Anna M.; Sit, Clarissa S.; Vederas, John C.; Hecker, Michael; Nakano, Michiko M.

2012-01-01

Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiho...

Direct detection of methicillin-resistant Staphylococcus aureus from blood cultures using an immunochromatographic immunoassay-based MRSA rapid kit for the detection of penicillin-binding protein 2a.

Science.gov (United States)

Shin, Kyeong Seob; Song, Hyung Geun; Kim, Haejung; Yoon, Sangsun; Hong, Seung Bok; Koo, Sun Hoe; Kim, Jimyung; Kim, Jongwan; Roh, Kyoung Ho

2010-07-01

Using an EZ-Step MRSA rapid kit, a novel screening test for methicillin-resistant Staphylococcus aureus (MRSA) that detects penicillin-binding protein 2a, 34 of 36 MRSA-positive clinical blood culture samples were positive on direct testing (sensitivity, 94.4%), whereas 21 of 21 methicillin-susceptible S. aureus-positive samples were negative (specificity, 100%).

Flow cytometry as a rapid test for detection of penicillin resistance directly in bacterial cells in Enterococcus faecalis and Staphylococcus aureus.

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Jarzembowski, T; Wiśniewska, K; Józwik, A; Bryl, E; Witkowski, J

2008-08-01

We studied the usefulness of flow cytometry for detection of penicillin resistance in E. faecalis and S. aureus by direct binding of commercially available fluorescent penicillin, Bocillin FL, to cells obtained from culture. There were significantly lower percentages of fluorescent cells and median and mean fluorescence values per particle in penicillin-resistant than in penicillin-sensitive strains of both species observed. The method allows rapid detection of penicillin resistance in S. aureus and E. faecalis. The results encourage further investigations on the detection of antibiotic resistance in bacteria using flow cytometry.

Colorimetric DNAzyme Biosensor for Convenience Detection of Enterotoxin B Harboring Staphylococcus aureus from Food Samples.

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Mondal, Bhairab; N, Bhavanashri; Ramlal, Shylaja; Kingston, Joseph

2018-02-14

In the present study, a colorimetric DNAzymes biosensor strategy was devised in combination with immunomagnetic separation for rapid and easy detection of enterotoxin B harboring Staphylococcus aureus from food and clinical samples. The method employs immunocapture of S. aureus and amplification of seb gene by DNAzyme complementary sequence integrated forward primer and with specific reverse primer. The DNAzyme sequence integrated dsDNA PCR products when treated with hemin and TMB (3,3',5,5'-tetramethylbenzidine) in the presence of H 2 O 2 produce colorimetric signal. A linear relationship of optical signal with the initial template of seb was obtained which could be monitored by visually or spectrophotrometrically for qualitative and quantitative detection. The limit of detection for the assay was approximately 10 2 CFU/mL of seb gene harboring target. This method is convenient compared to gel based and ELISA systems. Further, spiking studies and analysis on natural samples emphasized the robustness and applicability of developed method. Altogether, the established assay could be a reliable alternative, low-cost, viable detection tool for the routine investigation of seb from food and clinical sources.

Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types

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Pricilla DM de Matos

2010-11-01

Full Text Available A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD, agar dilution (AD, latex agglutination (LA, oxacillin agar screening (OAS and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS-98.3% (AD. The LA test showed the second lowest sensitivity (86.4%. The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

Accuracy of phenotypic methicillin susceptibility methods in the detection of Staphylococcus aureus isolates carrying different SCCmec types.

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Matos, Pricilla D M de; Schuenck, Ricardo P; Cavalcante, Fernanda S; Caboclo, Roberta M F; Santos, Kátia Regina N dos

2010-11-01

A total of 138 isolates, 118 methicillin-resistant Staphylococcus aureus (MRSA) isolates (staphylococcal cassette chromosome type II, 20 isolates, type III, 39 isolates and type IV, 59 isolates) and 20 methicillin-sensitive S. aureus isolates were evaluated by phenotypic methods: cefoxitin and oxacillin disk diffusion (DD), agar dilution (AD), latex agglutination (LA), oxacillin agar screening (OAS) and chromogenic agar detection. All methods showed 100% specificity, but only the DD tests presented 100% sensitivity. The sensitivity of the other tests ranged from 82.2% (OAS)-98.3% (AD). The LA test showed the second lowest sensitivity (86.4%). The DD test showed high accuracy in the detection of MRSA isolates, but there was low precision in the detection of type IV isolates by the other tests, indicating that the genotypic characteristics of the isolates should be considered.

Subinhibitory concentrations of thymol reduce enterotoxins A and B and alpha-hemolysin production in Staphylococcus aureus isolates.

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Jiazhang Qiu

Full Text Available BACKGROUND: Targeting bacterial virulence factors is now gaining interest as an alternative strategy to develop new types of anti-infective agents. It has been shown that thymol, when used at low concentrations, can inhibit the TSST-1 secretion in Staphylococcus aureus. However, there are no data on the effect of thymol on the production of other exotoxins (e.g., alpha-hemolysin and enterotoxins by S. aureus. METHODOLOGY/PRINCIPAL FINDINGS: Secretion of alpha-hemolysin, SEA and SEB in both methicillin-sensitive and methicillin-resistant S. aureus isolates cultured with graded subinhibitory concentrations of thymol was detected by immunoblot analysis. Hemolysin and tumor necrosis factor (TNF release assays were performed to elucidate the biological relevance of changes in alpha-hemolysin, SEA and SEB secretion induced by thymol. In addition, the influence of thymol on the transcription of hla, sea, and seb (the genes encoding alpha-hemolysin, SEA and SEB, respectively was analyzed by quantitative RT-PCR. Thymol inhibited transcription of hla, sea and seb in S. aureus, resulting in a reduction of alpha-hemolysin, SEA and SEB secretion and, thus, a reduction in hemolytic and TNF-inducing activities. CONCLUSIONS/SIGNIFICANCE: Subinhibitory concentrations of thymol decreased the production of alpha-hemolysin, SEA and SEB in both MSSA and MRSA in a dose-dependent manner. These data suggest that thymol may be useful for the treatment of S. aureus infections when used in combination with beta-lactams and glycopeptide antibiotics, which induce expression of alpha-hemolysin and enterotoxins at subinhibitory concentrations. Furthermore, the structure of thymol may potentially be used as a basic structure for development of drugs aimed against these bacterial virulence factors.

Impact of sub-inhibitory antibiotics on fibronectin-mediated host cell adhesion and invasion by Staphylococcus aureus

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Rasigade Jean

2011-12-01

Full Text Available Abstract Background Staphylococcus aureus is a well-armed pathogen prevalent in severe infections such as endocarditis and osteomyelitis. Fibronectin-binding proteins A and B, encoded by fnbA/B, are major pathogenesis determinants in these infections through their involvement in S. aureus adhesion to and invasion of host cells. Sub-minimum inhibitory concentrations (sub-MICs of antibiotics, frequently occurring in vivo because of impaired drug diffusion at the infection site, can alter S. aureus phenotype. We therefore investigated their impact on S. aureus fibronectin-mediated adhesiveness and invasiveness. Methods After in vitro challenge of S. aureus 8325-4 and clinical isolates with sub-MICs of major anti-staphylococcal agents, we explored fnbA/B transcription levels, bacterial adhesiveness to immobilised human fibronectin and human osteoblasts in culture, and bacterial invasion of human osteoblasts. Results Oxacillin, moxifloxacin and linezolid led to the development of a hyper-adhesive phenotype in the fibronectin adhesion assay that was consistent with an increase in fnbA/B transcription. Conversely, rifampin treatment decreased fibronectin binding in all strains tested without affecting fnbA/B transcription. Gentamicin and vancomycin had no impact on fibronectin binding or fnbA/B transcription levels. Only oxacillin-treated S. aureus displayed a significantly increased adhesion to cultured osteoblasts, but its invasiveness did not differ from that of untreated controls. Conclusion Our findings demonstrate that several antibiotics at sub-MICs modulate fibronectin binding in S. aureus in a drug-specific fashion. However, hyper- and hypo- adhesive phenotypes observed in controlled in vitro conditions were not fully confirmed in whole cell infection assays. The relevance of adhesion modulation during in vivo infections is thus still uncertain and requires further investigations.

Magnetically Assisted Surface-Enhanced Raman Spectroscopy for the Detection of Staphylococcus aureus Based on Aptamer Recognition.

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Wang, Junfeng; Wu, Xuezhong; Wang, Chongwen; Shao, Ningsheng; Dong, Peitao; Xiao, Rui; Wang, Shengqi

2015-09-23

A magnetically assisted surface-enhanced Raman scattering (SERS) biosensor for single-cell detection of S. aureus on the basis of aptamer recognition is reported for the first time. The biosensor consists of two basic elements including a SERS substrate (Ag-coated magnetic nanoparticles, AgMNPs) and a novel SERS tag (AuNR-DTNB@Ag-DTNB core-shell plasmonic NPs or DTNB-labeled inside-and-outside plasmonic NPs, DioPNPs). Uniform, monodisperse, and superparamagnetic AgMNPs with favorable SERS activity and magnetic responsiveness are synthesized by using polymer polyethylenimine. AgMNPs use magnetic enrichment instead of repeated centrifugation to prevent sample sedimentation. DioPNPs are designed and synthesized as a novel SERS tag. The Raman signal of DioPNPs is 10 times stronger than that of the commonly used SERS tag AuNR-DTNB because of the double-layer DTNB and the LSPR position adjustment to match the given laser excitation wavelength. Consequently, a strong SERS enhancement is achieved. Under the optimized aptamer density and linker length, capture by aptamer-modified AgMNPs can achieve favorable bacteria arrest (up to 75%). With the conventional Raman spectroscopy, the limit of detection (LOD) is 10 cells/mL for S. aureus detection, and a good linear relationship is also observed between the SERS intensity at Raman peak 1331 cm(-1) and the logarithm of bacteria concentrations ranging from 10(1) to 10(5) cells/mL. With the help of the newly developed SERS mapping technique, single-cell detection of S. aureus is easily achieved.

Staphylococcus aureus detection in the mouth of housekeepers Detección de Staphylococcus aureus en la boca de trabajadores de la limpieza hospitalaria Detecção de Staphylococcus aureus na boca de trabalhadores da limpeza hospitalar

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Elaine Drehmer de Almeida Cruz

2011-02-01

Full Text Available This study assessed the prevalence of colonization by Staphylococcus aureus in hospital housekeepers, and their knowledge and beliefs regarding this problem. Three saliva samples were collected and a questionnaire regarding knowledge and beliefs was applied. Of the 92 workers, 63 (68.5% participated in the study; 20 were not and 43 were colonized; 13 by methicillin resistant Staphylococcus aureus and 30 by methicillin sensitive Staphylococcus aureus. Persistent carrier status of methicillin resistant Staphylococcus aureus was detected in 15.4% of cases. Low knowledge and perception of occupational risk were observed. The mouth was identified as an important reservoir of methicillin resistant Staphylococcus aureus. Analyzing knowledge and beliefs, as well as the state of carrier, is an important strategy to be added to educational actions for the prevention of workers' colonization.Este estudio evaluó la prevalencia de la colonización por Staphylococcus aureus en trabajadores de limpieza hospitalaria, y su conocimiento y creencias acerca de la problemática. Fueron recolectadas tres muestras de saliva y aplicado un cuestionario referente al conocimiento y creencias. De 92 trabajadores, 63 (68,5% participaron del estudio; 20 se presentaron no colonizados y 43 colonizados; 13 para Staphylococcus aureus resistente a la meticilina y 30 para Staphylococcus aureus sensibles a la meticilina. El estado de portador persistente por Staphylococcus aureus resistente a la meticilina fue detectado en 15,4% de los casos. Bajo conocimiento y percepción del riesgo ocupacional fueron observados. La boca fue identificada como importante reservatorio de Staphylococcus aureus resistente a la meticilina. Analizar el conocimiento y creencias juntamente con la investigación del estado de portador es una importante estrategia a ser agregada a las acciones educativas para la prevención de la colonización de trabajadores.Este estudo avaliou a prevalência da coloniza

Global transcriptional response of methicillin-resistant Staphylococcus aureus to thioridazine

DEFF Research Database (Denmark)

Bonde, Mette; Jacobsen, Kirstine; Kolmos, Hans Jørn

Few drugs are available against methicillin-resistant S. aureus (MRSA), and decreased susceptibility among staphylococci to newly introduced agents such as linezolid, daptomycin, and tigecycline has been observed [1-3]. Consequently, new treatment strategies are urgently needed. Thioridazine...

Lysionotin attenuates Staphylococcus aureus pathogenicity by inhibiting α-toxin expression.

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Teng, Zihao; Shi, Dongxue; Liu, Huanyu; Shen, Ziying; Zha, Yonghong; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

2017-09-01

α-Toxin, one of the best known pore-forming proteins produced by Staphylococcus aureus (S. aureus), is a critical virulence factor in multiple infections. The necessity of α-toxin for S. aureus pathogenicity suggests that this toxin is an important target for the development of a potential treatment strategy. In this study, we showed that lysionotin, a natural compound, can inhibit the hemolytic activity of culture supernatants by S. aureus by reducing α-toxin expression. Using real-time PCR analysis, we showed that transcription of hla (the gene encoding α-toxin) and agr (the locus regulating hla) was significantly inhibited by lysionotin. Lactate dehydrogenase and live/dead assays indicated that lysionotin effectively protected human alveolar epithelial cells against S. aureus, and in vivo studies also demonstrated that lysionotin can protect mice from pneumonia caused by S. aureus. These findings suggest that lysionotin is an efficient inhibitor of α-toxin expression and shows significant protection against S. aureus in vitro and in vivo. This study supports a potential strategy for the treatment of S. aureus infection by inhibiting the expression of virulence factors and indicates that lysionotin may be a potential treatment for S. aureus pneumonia.

Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis Detecção de resistência a meticilina e produção do fator slime por Staphylococcus aureus em mastite bovina

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Alper Ciftci

2009-06-01

Full Text Available This study aimed to detect methicillin resistant and slime producing Staphylococcus aureus in cases of bovine mastitis. A triplex PCR was optimized targetting 16S rRNA, nuc and mecA genes for detection of Staphylococcus species, S. aureus and methicillin resistance, respectively. Furthermore, for detection of slime producing strains, a PCR assay targetting icaA and icaD genes was performed. In this study, 59 strains were detected as S. aureus by both conventional tests and PCR, and 13 of them were found to be methicillin resistant and 4 (30.7% were positive for mecA gene. Although 22 of 59 (37.2% S. aureus isolates were slime-producing in Congo Red Agar, in PCR analysis only 15 were positive for both icaA and icaD genes. Sixteen and 38 out of 59 strains were positive for icaA and icaD gene, respectively. Only 2 of 59 strains were positive for both methicillin resistance and slime producing, phenotypically, suggesting lack of correlation between methicillin resistance and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. Furthermore, only PCR targetting icaA and icaD may not sufficient to detect slime production and further studies targetting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains.Este estudo objetivou a detecção de Staphylococcus aureus resistente a meticilina e produtor do fator slime em casos de mastite bovina. Um PCR triplex foi otimizado, com alvo no genes 16SrRNA, nuc e mecA para detecção de Staphylococcus spp, S. aureus e resistencia a meticilina, respectivamente. Para detecção das cepas produtoras do fator slime, empregou-se um PCR com alvo nos genes icaA e icaD. No estudo, 59 cepas foram identificadas como S. aureus por testes convencionais e PCR, sendo 13 resistentes a meticilina e quatro positivas para o gene mecA. Embora 22 das 59 cepas

Rapid single cell detection of Staphylococcus aureus by aptamer-conjugated gold nanoparticles

OpenAIRE

Chang, Yi-Chung; Yang, Chia-Ying; Sun, Ruei-Lin; Cheng, Yi-Feng; Kao, Wei-Chen; Yang, Pan-Chyr

2013-01-01

Staphylococcus aureus is one of the most important human pathogens, causing more than 500,000 infections in the United States each year. Traditional methods for bacterial culture and identification take several days, wasting precious time for patients who are suffering severe bacterial infections. Numerous nucleic acid-based detection methods have been introduced to address this deficiency; however, the costs and requirement for expensive equipment may limit the widespread use of such technol...

Antibiotic resistance and molecular analysis of Staphylococcus aureus isolated from cow's milk and dairy products in northeast Brazil.

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Silveira-Filho, Vladimir M; Luz, Isabelle S; Campos, Ana Paula F; Silva, Wellington M; Barros, Maria Paloma S; Medeiros, Elizabeth S; Freitas, Manuela F L; Mota, Rinaldo A; Sena, Maria J; Leal-Balbino, Tereza C

2014-04-01

This work aimed to assess the clonal distribution among 94 strains of Staphylococcus aureus isolated from cow's milk, raw cheese, and a milking machine in 12 dairy farms in northeast Brazil, by analyzing different typing methods and detecting resistance and toxigenic profiles. For the first time, isolates of this region were assessed simultaneously by the polymorphism of the 3'-end coa gene and 16S-23S rDNA, pulsed-field gel electrophoresis, antibiotic resistance phenotyping, and toxigenic arsenal. Although pulsed-field gel electrophoresis patterns showed a wider variation (discriminatory index 0.83) than the PCR-based methods, the internal transcribed spacer-PCR proved to be a useful and inexpensive procedure for conducting epidemiological surveys of S. aureus on a regional scale. Each dairy farm had its own resistance profile, and in two herds, 63% of the strains were multiresistant, probably due to the indiscriminate use of antibiotics in bovine mastitis treatment. No methicillin-resistant S. aureus strains were detected in this study; however, 93.6% of S. aureus strains harbored variable profiles of staphylococcal enterotoxin genes seg, seh, sei, and sej. Transcriptional analysis revealed that 53.3% of staphylococcal enterotoxin genes actually transcribed, pointing out the food poisoning risk of these dairy products to consumers in the region. Based on the detection of the most prevalent clones in a herd or region, appropriate antibiotic therapy and specific immunization can be used for the treatment and control of staphylococcal mastitis.

Detection of the enterotoxigenic genes (sei,sej) in Staphylococcus aureus isolates from bovine mastitis milk in the West Azerbaijan of Iran.

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Ahmady, Malahat; Kazemi, Sahar

2013-07-01

Staphylococcus aureus is a major causative pathogen of clinical and subclinical mastitis of dairy domestic ruminants. This organism produces a variety of extracellular toxins and virulence factors such as enterotoxin SEI and SEJ that contribute to its pathogenic potential. In this study 25 S. aureus isolates obtained from four dairy herds of Urmia region which is located in West Azerbaijan province in Iran. The tested isolates were identified on the basis of the cultural and biochemical properties, as well as amplification of the aroA gene which is specific for S. aureus . All isolates were also analyzed for the presence of the SEI ( sei ) and SEJ ( sej ) encoding genes using polymerase chain reaction (PCR). Seven positive isolates were detected for sei , but sej gene was not detected in any of the total number of 25 isolates. The present study revealed that the PCR amplification of the aroA gene could be used as a powerful tool for identification of S. aureus from the cases of bovine mastitis. Results of the present study also showed that the strains of S. aureus which cause mastitis can potentially produce enterotoxin SEI. Overall, our results suggest that it is of special importance to follow the presence of enterotoxin-producing S. aureus in other dairy products, especially for protecting the consumers from staphylococcal food poisoning.

Multiple displacement amplification as an adjunct to PCR-based detection of Staphylococcus aureus in synovial fluid

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Johnson Sandra

2010-10-01

Full Text Available Abstract Background Detection of bacterial nucleic acids in synovial fluid following total joint arthroplasty with suspected infection can be difficult; among other technical challenges, inhibitors in the specimens require extensive sample preparation and can diminish assay sensitivity even using polymerase chain reaction (PCR-based methods. To address this problem a simple protocol for prior use of multiple displacement amplification (MDA as an adjunct to PCR was established and tested on both purified S. aureus DNA as well as on clinical samples known to contain S. aureus nucleic acids. Findings A single round of MDA on purified nucleic acids resulted in a > 300 thousand-fold increase in template DNA on subsequent quantitative PCR (qPCR analysis. MDA use on clinical samples resulted in at least a 100-fold increase in sensitivity on subsequent qPCR and required no sample preparation other than a simple alkali/heat lysis step. Mixed samples of S. aureus DNA with a 103 - 104-fold excess of human genomic DNA still allowed for MDA amplification of the minor bacterial component to the threshold of detectability. Conclusion MDA is a promising technique that may serve to significantly enhance the sensitivity of molecular assays in cases of suspected joint infection while simultaneously reducing the specimen handling required.

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment

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Tian Ding

2017-05-01

Full Text Available This study firstly developed a multiplex real-time PCR (RT-PCR technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus, Listeria monocytogenes (L. monocytogenes and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water in one reaction. Brain heart infusion (BHI broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

Comparison of real-time PCR with disk diffusion, agar screen and E-test methods for detection of methicillin-resistant Staphylococcus aureus.

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Shariati, Laleh; Validi, Majid; Tabatabaiefar, Mohammad Amin; Karimi, Ali; Nafisi, Mohammad Reza

2010-12-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the "gold standard" comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.

An Electrochemical Immunosensor for Detection of Staphylococcus aureus Bacteria Based on Immobilization of Antibodies on Self-Assembled Monolayers-Functionalized Gold Electrode

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Abderrazak Maaref

2012-10-01

Full Text Available The detection of pathogenic bacteria remains a challenge for the struggle against biological weapons, nosocomial diseases, and for food safety. In this research, our aim was to develop an easy-to-use electrochemical immunosensor for the detection of pathogenic Staphylococcus aureus ATCC25923. The biosensor was elaborated by the immobilization of anti-S. aureus antibodies using a self-assembled monolayer (SAMs of 3-Mercaptopropionic acid (MPA. These molecular assemblies were spontaneously formed by the immersion of the substrate in an organic solvent containing the SAMs that can covalently bond to the gold surface. The functionalization of the immunosensor was characterized using two electrochemical techniques: cyclic voltammetry (CV and electrochemical impedance spectroscopy (EIS. Here, the analysis was performed in phosphate buffer with ferro/ferricyanide as the redox probe. The EIS technique was used for affinity assays: antibody-cell binding. A linear relationship between the increment in the electron transfer resistance (RCT and the logarithmic value of S. aureus concentration was observed between 10 and 106 CFU/mL. The limit of detection (LOD was observed at 10 CFU/mL, and the reproducibility was calculated to 8%. Finally, a good selectivity versus E. coli and S. epidermidis was obtained for our developed immunosensor demonstrating its specificity towards only S. aureus.

Detection of oxacillin-susceptible mecA-positive Staphylococcus aureus isolates by use of chromogenic medium MRSA ID.

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Kumar, V Anil; Steffy, Katherin; Chatterjee, Maitrayee; Sugumar, Madhan; Dinesh, Kavitha R; Manoharan, Anand; Karim, Shamsul; Biswas, Raja

2013-01-01

Reports of oxacillin-susceptible mecA-positive Staphylococcus aureus strains are on the rise. Because of their susceptibility to oxacillin and cefoxitin, it is very difficult to detect them by using routine phenotypic methods. We describe two such isolates that were detected by chromogenic medium and confirmed by characterization of the mecA gene element.

DETECTION OF BACTERIAL SMALL TRANSCRIPTS FROM RNA-SEQ DATA: A COMPARATIVE ASSESSMENT.

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Peña-Castillo, Lourdes; Grüell, Marc; Mulligan, Martin E; Lang, Andrew S

2016-01-01

Small non-coding RNAs (sRNAs) are regulatory RNA molecules that have been identified in a multitude of bacterial species and shown to control numerous cellular processes through various regulatory mechanisms. In the last decade, next generation RNA sequencing (RNA-seq) has been used for the genome-wide detection of bacterial sRNAs. Here we describe sRNA-Detect, a novel approach to identify expressed small transcripts from prokaryotic RNA-seq data. Using RNA-seq data from three bacterial species and two sequencing platforms, we performed a comparative assessment of five computational approaches for the detection of small transcripts. We demonstrate that sRNA-Detect improves upon current standalone computational approaches for identifying novel small transcripts in bacteria.

Staphylococcus aureus and staphylococcal enterotoxin A in breaded chicken products: detection and behavior during the cooking process.

Science.gov (United States)

Pepe, Olimpia; Blaiotta, Giuseppe; Bucci, Francesca; Anastasio, Marilena; Aponte, Maria; Villani, Francesco

2006-11-01

In this study we examined the presence of Staphylococcus aureus and staphylococcal enterotoxin A (SEA) in 20 industrial breaded chicken products obtained from different retail butchers and supermarket stores in Italy. The levels of contamination in the products analyzed were quite low, although the pH values and water activities (a(w)) in the samples considered were in ranges favorable for S. aureus growth. As demonstrated by phenotypic and molecular characterization, in spite of the high percentage of coagulase-positive Staphylococcus strains, only three strains could be referred to the species S. aureus. Moreover, all the strains were negative in PCR assays targeting staphylococcal enterotoxin genes (seA to seE, seG to seJ, and seM to seO), as well as the toxic shock syndrome toxin 1 gene, and no SEA was detected in the retail breaded chicken samples analyzed by a reversed passive latex agglutination assay or by Western blotting. Hence, we evaluated the thermal resistance of two strains of SEA-producing S. aureus in a laboratory-scale preparation of precooked breaded chicken cutlets. The heat treatment employed in the manufacture determined the inactivation of S. aureus cells, but the preformed SEA remained active during product storage at 4 degrees C. The presence of the staphylococci and, in particular, of S. aureus in the retail breaded chicken products analyzed is a potential health risk for consumers since the pH and a(w) values of these kinds of products are favorable for S. aureus growth. The thermal process used during their manufacture can limit staphylococcal contamination but cannot eliminate preformed toxins.

Comparative Effects of Food Preservatives on the Production of Staphylococcal Enterotoxin I from Staphylococcus aureus Isolate

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Yanying Zhao

2017-01-01

Full Text Available Staphylococcal enterotoxin I (SEI is associated with staphylococcal food poisoning, but little is known about different food preservatives on the production of SEI. In this study, the effect of different food preservatives (sodium nitrite, polylysine, chitosan, and tea catechin on the bacteria growth, sei gene expression, and extracellular SEI production of Staphylococcus aureus isolate H4 was detected in tryptone soya broth (TSB culture. Our results showed that all of these preservatives depressed S. aureus H4 growth and the order of inhibitory effect was 0.8 g/L tea catechin > 6 g/L chitosan > 0.25 g/L polylysine > 0.4 g/L tea catechin > 0.15 g/L sodium nitrite. Furthermore, 0.25 g/L polylysine or 0.15 g/L sodium nitrite did not significantly alter sei gene transcription, while 6 g/L chitosan obviously increased the relative mRNA level of sei gene expression. 0.4 g/L tea catechin remarkably inhibited sei gene transcription. In addition, 0.15 g/L sodium nitrite and 6 g/L chitosan significantly enhanced SEI secretion. 0.25 g/L polylysine, especially 0.4 g/L tea catechin, sharply inhibited the level of SEI secretion. The results indicated that tea catechin not only suppressed Staphylococcus aureus growth, but also inhibited SEI production and secretion, suggesting that tea catechin may be better than sodium nitrite, polylysine, or chitosan for keeping the food from the contamination of SEI. These investigations would be useful for food industry to provide safer food products due to S. aureus enterotoxins-related control strategy.

Microbiological evaluation of a new growth-based approach for rapid detection of methicillin-resistant Staphylococcus aureus

NARCIS (Netherlands)

von Eiff, Christof; Maas, Dominik; Sander, Gunnar; Friedrich, Alexander W; Peters, Georg; Becker, Karsten

OBJECTIVES: Recently, a rapid screening tool for methicillin-resistant Staphylococcus aureus (MRSA) has been introduced that applies a novel detection technology allowing the rapid presence or absence of MRSA to be determined from an enrichment broth after only a few hours of incubation. To evaluate

Identification of single nucleotide polymorphisms associated with hyperproduction of alpha-toxin in Staphylococcus aureus.

Directory of Open Access Journals (Sweden)

Xudong Liang

2011-04-01

Full Text Available The virulence factor α-toxin (hla is needed by Staphylococcus aureus in order to cause infections in both animals and humans. Although the complicated regulation of hla expression has been well studied in human S. aureus isolates, the mechanisms of of hla regulation in bovine S. aureus isolates remain undefined. In this study, we found that many bovine S. aureus isolates, including the RF122 strain, generate dramatic amounts of α-toxin in vitro compared with human clinical S. aureus isolates, including MRSA WCUH29 and MRSA USA300. To elucidate potential regulatory mechanisms, we analyzed the hla promoter regions and identified predominant single nucleotide polymorphisms (SNPs at positions -376, -483, and -484 from the start codon in α-toxin hyper-producing isolates. Using site-directed mutagenesis and hla promoter-gfp-luxABCDE dual reporter approaches, we demonstrated that the SNPs contribute to the differential control of hla expression among bovine and human S. aureus isolates. Using a DNA affinity assay, gel-shift assays and a null mutant, we identified and revealed that an hla positive regulator, SarZ, contributes to the involvement of the SNPs in mediating hla expression. In addition, we found that the bovine S. aureus isolate RF122 exhibits higher transcription levels of hla positive regulators, including agrA, saeR, arlR and sarZ, but a lower expression level of hla repressor rot compared to the human S. aureus isolate WCUH29. Our results indicate α-toxin hyperproduction in bovine S. aureus is a multifactorial process, influenced at both the genomic and transcriptional levels. Moreover, the identification of predominant SNPs in the hla promoter region may provide a novel method for genotyping the S. aureus isolates.

Comparative Efficacy of Ceftaroline with Linezolid against Staphylococcus Aureus and Methicillin Resistant Staphylococcus Aureus

International Nuclear Information System (INIS)

Hafeez, A.; Munir, T.; Rehman, S.; Najeeb, S.; Gilani, M.; Latif, M.; Ansari, M.; Saad, N.

2015-01-01

Objective:To compare the in vitro antimicrobial efficacy of ceftaroline with linezolid against Staphylococcus aureus and methicillin resistant Staphylococcus aureus. Study Design: Quasi-experimental study. Place and Duration of Study: Microbiology Department, Army Medical College, Rawalpindi, from January to December 2013. Methodology: Clinical samples from respiratory tract, blood, pus and various catheter tips routinely received in the Department of Microbiology, Army Medical College, Rawalpindi were innoculated on blood and MacConkey agar. Staphylococcus aureus was identified by colony morphology, Gram reaction, catalase test and coagulase test. Methicillin resistant Staphylococcus aureus detection was done by modified Kirby Bauer disc diffusion method using cefoxitin disc (30g) and the isolates were considered methicillin resistant if the zone of inhibition around cefoxitin disc was /sup 2/ 21 mm. Bacterial suspensions of 56 Staphylococcus aureus isolates and 50 MRSA isolates were prepared, which were standardized equal to 0.5 McFarland's turbidity standard and inoculated on Mueller-Hinton agar plates followed by application of ceftaroline and linezolid disc (Oxoid, UK), according to manufacturer's instructions. The plates were then incubated at 37 Degree C aerobically for 18 - 24 hours. Diameters of inhibition zone were measured and interpretated as per Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Out of 106 isolates all of the 56 Staphylococcus aureus (100%) were sensitive to ceftaroline and linezolid. However, out of 50 methicillin resistant Staphylococcus aureus, 48 (96%) were sensitive to ceftaroline whereas, 49 (98%) were sensitive to linezolid. Conclusion: Ceftaroline is equally effective as linezolid against Staphylococcus aureus and methicillin resistant Staphylococcus aureus. (author)

Comparison of different phenotypic methods of detection of methicillin resistance in staphylococcus aureus with the molecular detection of mec-a gene

International Nuclear Information System (INIS)

Zeeshan, M.; Jabeen, K.; Irfan, S.; Parween, Z.; Zafar, A.

2007-01-01

To evaluate accuracy, cost-effectiveness and ease to perform different phenotypic methods i.e. Cefoxitin 30 micro g disc, Oxacillin 1micro g disc and Oxacillin agar screening plate (6 micro g/ml) for early and accurate identification of MRSA by comparing with the detection of mec-A gene in our clinical isolates. Out of 200 clinical samples, conventional Polymerase Chain Reaction (PCR) was done on 62 pure biochemically identified S. aureus isolates for mec-A gene detection. Phenotypic methods for detecting methicillin sensitivity (Cefoxitin 30 microg disc, Oxacillin 1 micro g disc and Oxacillin agar screening plate) were also used according to the recommended incubation time, duration and temperature on the same isolates. Out of 62 isolates of S. aureus, mec-A gene were detected (MRSA) in 32, whereas 30 were mec-A gene negative (MSSA). Cefoxitin disc and agar screening plate correctly identify all MRSA isolates with the sensitivity and specificity of 100%. Single isolate was false, positively detected as sensitive with Oxacillin 1g disc, due to which, the sensitivity and negative predictive value of this method were reduced to 96.9% and 96.8% respectively, while positive predictive value and specificity remained 100%. Comparing different phenotypic methods for MRSA screening in routine microbiology laboratory, Cefoxitin disc and Oxacillin agar screening has better sensitivity and specificity comparative to Oxacillin disc. However, Cefoxitin disc can be preferred especially for small laboratories because it is easy to perform, do not require special technique and media preparation is consequently more cost-effective. (author)

Predictive value and cost-effectiveness analysis of a rapid polymerase chain reaction for preoperative detection of nasal carriage of Staphylococcus aureus.

Science.gov (United States)

Shrestha, Nabin K; Shermock, Kenneth M; Gordon, Steven M; Tuohy, Marion J; Wilson, Deborah A; Cwynar, Roberta E; Banbury, Michael K; Longworth, David L; Isada, Carlos M; Mawhorter, Steven D; Procop, Gary W

2003-05-01

To determine the accuracy and cost-effectiveness of a polymerase chain reaction (PCR) for detecting nasal carriage of Staphylococcus aureus directly from clinical specimens. CROSS-SECTIONAL STUDY: This occurred in a tertiary-care hospital in Cleveland, Ohio, and included 239 consecutive patients who were scheduled for a cardiothoracic surgical procedure. Conventional cultures and a PCR for S. aureus from nasal swabs were used as measurements. COST-EFFECTIVENESS ANALYSIS: Data sources were market prices and Bureau of Labor Statistics. The time horizon was the maximum period for availability of culture results (3 days). Interventions included universal mupirocin therapy without testing; initial therapy, with termination if PCR negative (treat-PCR); initial therapy, with termination if culture negative (treat-culture); treat PCR-positive carriers (PCR-guided treatment); and treat culture-positive carriers (culture-guided treatment). The perspective was institutional and costs and the length of time to treatment were outcome measures. Sixty-seven (28%) of the 239 swabs grew S. aureus. Rapid PCR was 97.0% sensitive and 97.1% specific for the detection of S. aureus. For populations with prevalences of nasal S. aureus carriage of up to 50%, the PCR assay had negative predictive values of greater than 97%. PCR-guided treatment had the lowest incremental cost-effectiveness ratio (1.93 dollars per additional day compared with the culture strategy). Among immediate treatment strategies, treat-PCR was most cost-effective. The universal therapy strategy cost 38.19 dollars more per additional day gained with carrier identification compared with the PCR strategy. Rapid real-time PCR is an accurate, rapid, and cost-effective method for identifying S. aureus carriers for preoperative intervention.

Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus.

Science.gov (United States)

Irazoqui, Javier E; Troemel, Emily R; Feinbaum, Rhonda L; Luhachack, Lyly G; Cezairliyan, Brent O; Ausubel, Frederick M

2010-07-01

The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with

Distinct pathogenesis and host responses during infection of C. elegans by P. aeruginosa and S. aureus.

Directory of Open Access Journals (Sweden)

Javier E Irazoqui

2010-07-01

Full Text Available The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs. Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C

Dual-color upconversion fluorescence and aptamer-functionalized magnetic nanoparticles-based bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus.

Science.gov (United States)

Duan, Nuo; Wu, Shijia; Zhu, Changqing; Ma, Xiaoyuan; Wang, Zhouping; Yu, Ye; Jiang, Yuan

2012-04-20

A sensitive luminescent bioassay for the simultaneous detection of Salmonella Typhimurium and Staphylococcus aureus was developed using aptamer-conjugated magnetic nanoparticles (MNPs) for both recognition and concentration elements and using upconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The bioassay system was fabricated by immobilizing aptamer 1 and aptamer 2 onto the surface of MNPs, which were employed to capture and concentrate S. Typhimurium and S. aureus. NaY(0.78)F(4):Yb(0.2),Tm(0.02) UCNPs modified aptamer 1 and NaY(0.28)F(4):Yb(0.70),Er(0.02) UCNPs modified aptamer 2 further were bond onto the captured bacteria surface to form sandwich-type complexes. Under optimal conditions, the correlation between the concentration of S. Typhimurium and the luminescent signal was found to be linear within the range of 10(1)-10(5) cfu mL(-1) (R(2)=0.9964), and the signal was in the range of 10(1)-10(5) cfu mL(-1) (R(2)=0.9936) for S. aureus. The limits of detection of the developed method were found to be 5 and 8 cfu mL(-1) for S. Typhimurium and S. aureus, respectively. The ability of the bioassay to detect S. Typhimurium and S. aureus in real water samples was also investigated, and the results were compared to the experimental results from the plate-counting methods. Improved by the magnetic separation and concentration effect of MNPs, the high sensitivity of UCNPs, and the different emission lines of Yb/Er- and Yb/Tm-doped NaYF(4) UCNPs excited by a 980 nm laser, the present method performs with both high sensitivity and selectivity for the two different types of bacteria. Copyright © 2012 Elsevier B.V. All rights reserved.

Diverse modulation of spa transcription by cell wall active antibiotics in Staphylococcus aureus

DEFF Research Database (Denmark)

Nielsen, Lene Nørby; Roggenbuck, Michael; Haaber, Jakob Krause

2012-01-01

ABSTRACT: BACKGROUND: The aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus. FINDINGS: LacZ promoter fusions of genes related to staphylococ......ABSTRACT: BACKGROUND: The aim of this study was to investigate the effect of various classes of clinically relevant antibiotics at sub-lethal concentrations on virulence gene expression and biofilm formation in Staphylococcus aureus. FINDINGS: LacZ promoter fusions of genes related...... to staphylococcal virulence were used to monitor the effects of antibiotics on gene expression in a disc diffusion assay. The selected genes were hla and spa encoding alpha-hemolysin and Protein A, respectively and RNAIII, the effector molecule of the agr quorum sensing system. The results were confirmed...... by quantitative real-time PCR. Additionally, we monitored the effect of subinhibitory concentrations of antibiotics on the ability of S. aureus to form biofilm in a microtiter plate assay. The results show that sub-lethal antibiotic concentrations diversely modulate expression of RNAIII, hla and spa. Consistently...

New method for early detection of two random amplified polymorphic DNA (RAPD groups of Staphylococcus aureus causing bovine mastitis infection in Paraná State, Brazil

Directory of Open Access Journals (Sweden)

Dicezar Gonçalves

2010-04-01

Full Text Available The aim of this work was to develop a fast and accurate molecular approach to allow early detection of two RAPD groups of S. aureus causing bovine mastitis. Seventy five S. aureus isolates from infected animals were characterized by RAPD. Genomic fragments isolated from the unique bands present in either group were cloned and sequenced. Based on the DNA sequences, specific primers were designed to allow for the simultaneous detection of either group by multiplex PCR of S. aureus DNA isolated from clinical and subclinical bovine mastitis. Results showed that these proposed primers set could be used to detect various clinical and subclinical S. aureus isolates as well as the detection of the microorganism in bulk milk. Their use as a specific method for effective and early diagnostic tool for S. aureus infection in dairy herds is suggested.Esta pesquisa objetivou o desenvolvimento de técnica rápida e eficiente para diagnosticar precocemente diferentes linhagens de S. aureus causadoras de mastite bovina. Como resultados da metodologia empregada, foram isoladas duas linhagens destas bactérias que causam diferentes tipos de mastite bovina. Os fragmentos de DNA genômico caracterizando ambas as linhagens, por meio de RAPD foram inseridos em vetor plasmidial pGEM e clonados por meio de clones T10 F1 de Escherichia coli. As seqüências obtidas permitiram desenhar iniciadores específicos para o reconhecimento de ambas as linhagens, os quais foram testados com amostras de S. aureus e com outras linhagens próximas. O diagnóstico por meios moleculares, pode ser realizado diretamente de amostras coletadas de rebanhos leiteiros assim como dos equipamentos de ordenha. A significância deste estudo consiste em um rápido e acurado método para localizar animais infectados, representando importante ferramenta no manejo do rebanho, na redução de custos com tratamentos e, rápida recuperação de rebanhos infectados.

Characterization of staphylococci in urban wastewater treatment plants in Spain, with detection of methicillin resistant Staphylococcus aureus ST398

International Nuclear Information System (INIS)

Gómez, Paula; Lozano, Carmen; Benito, Daniel; Estepa, Vanesa; Tenorio, Carmen; Zarazaga, Myriam; Torres, Carmen

2016-01-01

The objective of this study was to determine the prevalence of Staphylococcus in urban wastewater treatment plants (UWTP) of La Rioja (Spain), and to characterize de obtained isolates. 16 wastewater samples (8 influent, 8 effluent) of six UWTPs were seeded on mannitol-salt-agar and oxacillin-resistance-screening-agar-base for staphylococci and methicillin-resistant Staphylococcus aureus recovery. Antimicrobial susceptibility profile was determined for 16 antibiotics and the presence of 35 antimicrobial resistance genes and 14 virulence genes by PCR. S. aureus was typed by spa, agr, and multilocus-sequence-typing, and the presence of immune-evasion-genes cluster was analyzed. Staphylococcus spp. were detected in 13 of 16 tested wastewater samples (81%), although the number of CFU/mL decreased after treatment. 40 staphylococci were recovered (1–5/sample), and 8 of them were identified as S. aureus being typed as (number of strains): spa-t011/agr-II/ST398 (1), spa-t002/agr-II/ST5 (2), spa-t3262/agr-II/ST5 (1), spa-t605/agr-II/ST126 (3), and spa-t878/agr-III/ST2849 (1). S. aureus ST398 strain was methicillin-resistant and showed a multidrug resistance phenotype. Virulence genes tst, etd, sea, sec, seg, sei, sem, sen, seo, and seu, were detected among S. aureus and only ST5 strains showed genes of immune evasion cluster. Thirty-two coagulase-negative Staphylococcus of 12 different species were recovered (number of strains): Staphylococcus equorum (7), Staphylococcus vitulinus (4), Staphylococcus lentus (4), Staphylococcus sciuri (4), Staphylococcus fleurettii (2), Staphylococcus haemolyticus (2), Staphylococcus hominis (2), Staphylococcus saprophyticus (2), Staphylococcus succinus (2), Staphylococcus capitis (1), Staphylococcus cohnii (1), and Staphylococcus epidermidis (1). Five presented a multidrug resistance phenotype. The following resistance and virulence genes were found: mecA, lnu(A), vga(A), tet(K), erm(C), msr(A)/(B), mph(C), tst, and sem. We found that

Methicillin-resistant Staphylococcus aureus: laboratory detection methods in use in the Republic of Ireland and Northern Ireland.

LENUS (Irish Health Repository)

Humphreys, H

2002-01-01

There is no universally agreed laboratory protocol for the detection of methicillin-resistant Staphylococcus aureus (MRSA) and hence a variety of approaches are used. As part of an all-island survey of MRSA in the Republic of Ireland (the South) and Northern Ireland (the North), a questionnaire was circulated to 14 participating laboratories in the North and 49 in the South, to determine the methods used to isolate MRSA from clinical specimens, identify S. aureus and test for susceptibility to methicillin. Almost two-thirds (64%) of laboratories in the North but only 16% of laboratories in the South use enrichment culture. There is heavy reliance on commercial kits to confirm the identification of S. aureus in the South but all laboratories in the North use the staphylocoagulase test. More than 90% of all laboratories use a disc method for susceptibility testing and 71% of laboratories in the North supplement this with the E-test; however, a range of methicillin disk concentrations are in use. There is a need to review current laboratory methods used to detect MRSA, with follow-up audit on their implementation. Additional resources may be needed in some laboratories to comply with revised guidelines, and reference facilities are required to assess new commercially available techniques and to confirm the identification of unusual or difficult strains.

Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) at ambient freshwater beaches

Science.gov (United States)

Fogarty, Lisa R.; Haack, Sheridan K.; Johnson, Heather E.; Brennan, Angela K.; Isaacs, Natasha M.; Spencer, Chelsea

2015-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) are a threat to human health worldwide, and although detected at marine beaches, they have been largely unstudied at freshwater beaches. Genes indicating S. aureus (SA; femA) and methicillin resistance (mecA) were detected at 11 and 12 of 13 US Great Lakes beaches and in 18% or 27% of 287 recreational water samples, respectively. Eight beaches had mecA + femA (potential MRSA) detections. During an intensive study, higher bather numbers, staphylococci concentrations, and femA detections were found in samples collected after noon than before noon. Local population density, beach cloud cover, and beach wave height were significantly correlated with SA or MRSA detection frequency. The Panton-Valentine leukocidin gene, associated with community-acquired MRSA, was detected in 12 out of 27 potential MRSA samples. The femA gene was detected less frequently at beaches that met US enterococci criteria or EU enterococci ‘excellent’ recreational water quality, but was not related to Escherichia coli-defined criteria. Escherichia coli is often the only indicator used to determine water quality at US beaches, given the economic and healthcare burden that can be associated with infections caused by SA and MRSA, monitoring of recreational waters for non-fecal bacteria such as staphylococci and/or SA may be warranted.

[Evaluation of four methods for detecting methicillin-resistant Staphylococcus aureus isolates from clinical specimens at a regional hospital in Mexico].

Science.gov (United States)

Acosta-Pérez, Gabriel; Rodríguez-Ábrego, Gabriela; Longoria-Revilla, Ernesto; Castro-Mussot, María Eugenia

2012-01-01

To estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical isolates and to compare different methods for detection of MRSA in a lab with limited available personnel and resources. 140 Staphylococcus aureus strains isolated from patients in several departments were assayed for β-lactamase production, MIC-Vitek 2 oxacillin, ChromID MRSA, disk diffusion in agar for cefoxitin 30 μg and PBP2a detection. The results of conventional tests were compared with the "gold standard" PCR test for mecA gene. Cohen´s kappa index was also calculated in order to evaluate the intra assay agreement between the used methods. The found prevalence was 90.7%. Sensitivity and specificity were: disk diffusion for cefoxitin 97 and 92% respectively, MIC Vitek 2-XL 97 and 69%, ChromoID MRSA 97 and 85%, and PBP2a detection 98 and 100%. All methods are very good for detecting MRSA, choosing a method to use will depend on each laboratory infrastructure.

Detection of Methicillin Resistant Staphylococcus aureus and Determination of Minimum Inhibitory Concentration of Vancomycin for Staphylococcus aureus Isolated from Pus/Wound Swab Samples of the Patients Attending a Tertiary Care Hospital in Kathmandu, Nepal

Directory of Open Access Journals (Sweden)

Raghabendra Adhikari

2017-01-01

Full Text Available The present study was conducted to evaluate the performance of cefoxitin disc diffusion method and oxacillin broth microdilution method for detection of methicillin resistant S. aureus (MRSA, taking presence of mecA gene as reference. In addition, inducible clindamycin resistance and beta-lactamase production were studied and minimum inhibitory concentration (MIC of vancomycin for S. aureus isolates was determined. A total of 711 nonrepeated pus/wound swab samples from different anatomic locations were included in the study. The Staphylococcus aureus was identified on the basis of colony morphology, Gram’s stain, and biochemical tests. A total of 110 (15.47% S. aureus isolates were recovered, of which 39 (35.50% isolates were identified as MRSA by cefoxitin disc diffusion method. By oxacillin broth microdilution method, 31.82% of the Staphylococcus aureus isolates were found to be MRSA. However, mecA gene was present in only 29.1% of the isolates. Further, beta-lactamase production was observed in 71.82% of the isolates, while inducible clindamycin resistance was found in 10% of S. aureus isolates. The MIC value of vancomycin for S. aureus ranged from 0.016 μg/mL to 1 μg/mL. On the basis of the absolute sensitivity (100%, both phenotypic methods could be employed for routine diagnosis of MRSA in clinical microbiology laboratory; however cefoxitin disc diffusion could be preferred over MIC method considering time and labour factor.

The Isolation and Detection of Staphylococcus aureus Enterotoxins A-E and TSST-1 Genes from Different Sources by PCR Method

Directory of Open Access Journals (Sweden)

Norouzi J

2012-09-01

Full Text Available Background and Objectives: Enterotoxins and toxic shock syndrome toxin–1 (TSST-1 are an important virulence factor of S. aureus. The purpose of this study was to analyze the presence of S. aureus enterotoxin (sea-see and tst genes in the samples that collected from different sources with PCR method. Methods: During 5 months from 150 collected samples, 80 strains were identified as S. aureus. PCR reaction was used for investigation on the presence of genes for staphylococcal enterotoxins (A-E and toxic shock syndrome toxin-1 (TSST-1. Results: Fifty three samples (66.25% out of 80 samples were positive for one or more ETs and TSST-1 genes. of these positive strains, 17 (32.07% were positive for sea 39 (73.58% for seb, 30 (56.6% for sec, 2 (3.7% for sed, 21 (39.62% for see, and 14 (26.41% for tst. Enterotoxins and tst in 40 samples (75.47% observed.Conclusion: In this study, high prevalence of S. aureus, its enterotoxin, and tst genes were observed in clinical samples, food samples, and healthy people. This fact emphasizes on the role of human as original source and carrier of S. aureus. Also, use of PCR reaction for detection of these genes in S. aureus that isolated from various sources is recommended.

Quantitative detection of Staphylococcus aureus and Enterococcus faecalis DNA in blood to diagnose bacteremia in patients in the intensive care unit

NARCIS (Netherlands)

Peters, Remco P. H.; van Agtmael, Michiel A.; Gierveld, Sonja; Danner, Sven A.; Groeneveld, A. B. Johan; Vandenbroucke-Grauls, Christina M. J. E.; Savelkoul, Paul H. M.

2007-01-01

Direct detection of bacterial DNA in blood offers a fast alternative to blood culture and is presumably unaffected by the prior use of antibiotics. We evaluated the performance of two real-time PCR assays for the quantitative detection of Staphylococcus aureus bacteremia and for Enterococcus

Staphylococcus aureus Transcriptome Architecture : From Laboratory to Infection-Mimicking Conditions

NARCIS (Netherlands)

Maeder, Ulrike; Nicolas, Pierre; Depke, Maren; Pane-Farre, Jan; Debarbouille, Michel; van der Kooi-Pol, Magdalena M.; Guerin, Cyprien; Derozier, Sandra; Hiron, Aurelia; Jarmer, Hanne; Leduc, Aurelie; Michalik, Stephan; Reilman, Ewoud; Schaffer, Marc; Schmidt, Frank; Bessieres, Philippe; Noirot, Philippe; Hecker, Michael; Msadek, Tarek; Voelker, Uwe; van Dijl, Jan Maarten

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S.

Characterization of staphylococci in urban wastewater treatment plants in Spain, with detection of methicillin resistant Staphylococcus aureus ST398.

Science.gov (United States)

Gómez, Paula; Lozano, Carmen; Benito, Daniel; Estepa, Vanesa; Tenorio, Carmen; Zarazaga, Myriam; Torres, Carmen

2016-05-01

The objective of this study was to determine the prevalence of Staphylococcus in urban wastewater treatment plants (UWTP) of La Rioja (Spain), and to characterize de obtained isolates. 16 wastewater samples (8 influent, 8 effluent) of six UWTPs were seeded on mannitol-salt-agar and oxacillin-resistance-screening-agar-base for staphylococci and methicillin-resistant Staphylococcus aureus recovery. Antimicrobial susceptibility profile was determined for 16 antibiotics and the presence of 35 antimicrobial resistance genes and 14 virulence genes by PCR. S. aureus was typed by spa, agr, and multilocus-sequence-typing, and the presence of immune-evasion-genes cluster was analyzed. Staphylococcus spp. were detected in 13 of 16 tested wastewater samples (81%), although the number of CFU/mL decreased after treatment. 40 staphylococci were recovered (1-5/sample), and 8 of them were identified as S. aureus being typed as (number of strains): spa-t011/agr-II/ST398 (1), spa-t002/agr-II/ST5 (2), spa-t3262/agr-II/ST5 (1), spa-t605/agr-II/ST126 (3), and spa-t878/agr-III/ST2849 (1). S. aureus ST398 strain was methicillin-resistant and showed a multidrug resistance phenotype. Virulence genes tst, etd, sea, sec, seg, sei, sem, sen, seo, and seu, were detected among S. aureus and only ST5 strains showed genes of immune evasion cluster. Thirty-two coagulase-negative Staphylococcus of 12 different species were recovered (number of strains): Staphylococcus equorum (7), Staphylococcus vitulinus (4), Staphylococcus lentus (4), Staphylococcus sciuri (4), Staphylococcus fleurettii (2), Staphylococcus haemolyticus (2), Staphylococcus hominis (2), Staphylococcus saprophyticus (2), Staphylococcus succinus (2), Staphylococcus capitis (1), Staphylococcus cohnii (1), and Staphylococcus epidermidis (1). Five presented a multidrug resistance phenotype. The following resistance and virulence genes were found: mecA, lnu(A), vga(A), tet(K), erm(C), msr(A)/(B), mph(C), tst, and sem. We found that

Label-free, electrochemical detection of methicillin-resistant staphylococcus aureus DNA with reduced graphene oxide-modified electrodes

KAUST Repository

Wang, Zhijuan

2011-05-01

Reduced graphene oxide (rGO)-modified glassy carbon electrode is used to detect the methicillin-resistant Staphylococcus aureus (MRSA) DNA by using electrochemical impedance spectroscopy. Our experiments confirm that ssDNA, before and after hybridization with target DNA, are successfully anchored on the rGO surface. After the probe DNA, pre-adsorbed on rGO electrode, hybridizes with target DNA, the measured impedance increases dramatically. It provides a new method to detect DNA with high sensitivity (10-13M, i.e., 100 fM) and selectivity. © 2011 Elsevier B.V.

An empirical strategy to detect bacterial transcript structure from directional RNA-seq transcriptome data.

Science.gov (United States)

Wang, Yejun; MacKenzie, Keith D; White, Aaron P

2015-05-07

As sequencing costs are being lowered continuously, RNA-seq has gradually been adopted as the first choice for comparative transcriptome studies with bacteria. Unlike microarrays, RNA-seq can directly detect cDNA derived from mRNA transcripts at a single nucleotide resolution. Not only does this allow researchers to determine the absolute expression level of genes, but it also conveys information about transcript structure. Few automatic software tools have yet been established to investigate large-scale RNA-seq data for bacterial transcript structure analysis. In this study, 54 directional RNA-seq libraries from Salmonella serovar Typhimurium (S. Typhimurium) 14028s were examined for potential relationships between read mapping patterns and transcript structure. We developed an empirical method, combined with statistical tests, to automatically detect key transcript features, including transcriptional start sites (TSSs), transcriptional termination sites (TTSs) and operon organization. Using our method, we obtained 2,764 TSSs and 1,467 TTSs for 1331 and 844 different genes, respectively. Identification of TSSs facilitated further discrimination of 215 putative sigma 38 regulons and 863 potential sigma 70 regulons. Combining the TSSs and TTSs with intergenic distance and co-expression information, we comprehensively annotated the operon organization in S. Typhimurium 14028s. Our results show that directional RNA-seq can be used to detect transcriptional borders at an acceptable resolution of ±10-20 nucleotides. Technical limitations of the RNA-seq procedure may prevent single nucleotide resolution. The automatic transcript border detection methods, statistical models and operon organization pipeline that we have described could be widely applied to RNA-seq studies in other bacteria. Furthermore, the TSSs, TTSs, operons, promoters and unstranslated regions that we have defined for S. Typhimurium 14028s may constitute valuable resources that can be used for

Sensitive detection of viral transcripts in human tumor transcriptomes.

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Sven-Eric Schelhorn

Full Text Available In excess of 12% of human cancer incidents have a viral cofactor. Epidemiological studies of idiopathic human cancers indicate that additional tumor viruses remain to be discovered. Recent advances in sequencing technology have enabled systematic screenings of human tumor transcriptomes for viral transcripts. However, technical problems such as low abundances of viral transcripts in large volumes of sequencing data, viral sequence divergence, and homology between viral and human factors significantly confound identification of tumor viruses. We have developed a novel computational approach for detecting viral transcripts in human cancers that takes the aforementioned confounding factors into account and is applicable to a wide variety of viruses and tumors. We apply the approach to conducting the first systematic search for viruses in neuroblastoma, the most common cancer in infancy. The diverse clinical progression of this disease as well as related epidemiological and virological findings are highly suggestive of a pathogenic cofactor. However, a viral etiology of neuroblastoma is currently contested. We mapped 14 transcriptomes of neuroblastoma as well as positive and negative controls to the human and all known viral genomes in order to detect both known and unknown viruses. Analysis of controls, comparisons with related methods, and statistical estimates demonstrate the high sensitivity of our approach. Detailed investigation of putative viral transcripts within neuroblastoma samples did not provide evidence for the existence of any known human viruses. Likewise, de-novo assembly and analysis of chimeric transcripts did not result in expression signatures associated with novel human pathogens. While confounding factors such as sample dilution or viral clearance in progressed tumors may mask viral cofactors in the data, in principle, this is rendered less likely by the high sensitivity of our approach and the number of biological replicates

Real-Time Detection of Staphylococcus Aureus Using Whispering Gallery Mode Optical Microdisks

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Hala Ghali

2016-05-01

Full Text Available Whispering Gallery Mode (WGM microresonators have recently been studied as a means to achieve real-time label-free detection of biological targets such as virus particles, specific DNA sequences, or proteins. Due to their high quality (Q factors, WGM resonators can be highly sensitive. A biosensor also needs to be selective, requiring proper functionalization of its surface with the appropriate ligand that will attach the biomolecule of interest. In this paper, WGM microdisks are used as biosensors for detection of Staphylococcus aureus. The microdisks are functionalized with LysK, a phage protein specific for staphylococci at the genus level. A binding event on the surface shifts the resonance peak of the microdisk resonator towards longer wavelengths. This reactive shift can be used to estimate the surface density of bacteria that bind to the surface of the resonator. The limit of detection of a microdisk with a Q-factor around 104 is on the order of 5 pg/mL, corresponding to 20 cells. No binding of Escherichia coli to the resonators is seen, supporting the specificity of the functionalization scheme.

Molecular Typing of Staphylococcus aureus Isolated from Patients with Autosomal Dominant Hyper IgE Syndrome

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Inka Sastalla

2017-06-01

Full Text Available Autosomal dominant hyper IgE syndrome (AD-HIES is a primary immunodeficiency caused by a loss-of-function mutation in the Signal Transducer and Activator of Transcription 3 (STAT3. This immune disorder is clinically characterized by increased susceptibility to cutaneous and sinopulmonary infections, in particular with Candida and Staphylococcus aureus. It has recently been recognized that the skin microbiome of patients with AD-HIES is altered with an overrepresentation of certain Gram-negative bacteria and Gram-positive staphylococci. However, these alterations have not been characterized at the species- and strain-level. Since S. aureus infections are influenced by strain-specific expression of virulence factors, information on colonizing strain characteristics may provide insights into host-pathogen interactions and help guide management strategies for treatment and prophylaxis. The aim of this study was to determine whether the immunodeficiency of AD-HIES selects for unique strains of colonizing S. aureus. Using multi-locus sequence typing (MLST, protein A (spa typing, and PCR-based detection of toxin genes, we performed a detailed analysis of the S. aureus isolates (n = 13 found on the skin of twenty-one patients with AD-HIES. We found a low diversity of sequence types, and an abundance of strains that expressed methicillin resistance, Panton-Valentine leukocidin (PVL, and staphylococcal enterotoxins K and Q (SEK, SEQ. Our results indicate that patients with AD-HIES may often carry antibiotic-resistant strains that harbor key virulence factors.

Contribution of SecDF to Staphylococcus aureus resistance and expression of virulence factors

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Berger-Bächi Brigitte

2011-04-01

Full Text Available Abstract Background SecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined. Results Markerless deletion mutants were constructed to analyze the impact of the so far uncharacterized RND proteins in S. aureus. While the lack of Sa2056 and Sa2339 caused no phenotype regarding growth and resistance, the secDF mutant resulted in a pleiotropic phenotype. The secDF mutant was cold sensitive, but grew normally in rich medium at 37°C. Resistance to beta-lactams, glycopeptides and the RND substrates acriflavine, ethidium bromide and sodium dodecyl sulfate was reduced. The secDF mutant showed an aberrant cell separation and increased spontaneous and Triton X-100 induced autolysis, although the amounts of penicillin-binding proteins in the membrane were unchanged. The impact of secDF deletion on transcription and expression of specific virulence determinants varied: While coagulase transcription and activity were reduced, the opposite was observed for the autolysin Atl. A reduction of the transcription of the cell wall anchored protein A (spa was also found. The accumulation of SpA in the membrane and lowered amounts in the cell wall pointed to an impaired translocation. Conclusions The combination of different effects of secDF deletion on transcription, regulation and translocation lead to impaired cell division, reduced resistance and altered expression of virulence determinants suggesting SecDF to be of major relevance in S. aureus. Thus SecDF could be a potential target for the control and eradication of S. aureus in the future.

Detection of ST772 Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (Bengal Bay clone and ST22 S. aureus isolates with a genetic variant of elastin binding protein in Nepal

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R.H. Pokhrel

2016-05-01

Full Text Available Genetic characteristics were analysed for recent clinical isolates of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA respectively in Kathmandu, Nepal. MRSA isolates harbouring Panton-Valentine leukocidin (PVL genes were classified into ST1, ST22 and ST88 with SCCmec-IV and ST772 with SCCmec-V (Bengal Bay clone, while PVL-positive MSSA into ST22, ST30 and ST772. ST22 isolates (PVL-positive MRSA and MSSA, PVL-negative MRSA possessed a variant of elastin binding protein gene (ebpS with an internal deletion of 180 bp, which was similar to that reported for ST121 S. aureus previously outside Nepal. Phylogenetic analysis indicated that the ebpS variant in ST22 might have occurred independently of ST121 strains. This is the first report of ST772 PVL-positive MRSA in Nepal and detection of the deletion variant of ebpS in ST22 S. aureus.

Prevalence and Characterization of Staphylococcus aureus Strains in the Pork Chain Supply in Chile.

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Velasco, Valeria; Vergara, José L; Bonilla, Ana M; Muñoz, Javier; Mallea, Alejandra; Vallejos, Diego; Quezada-Aguiluz, Mario; Campos, Jorge; Rojas-García, Pedro

2018-05-01

The detection of methicillin-resistant Staphylococcus aureus (MRSA) and other emerging strains in meat-producing animals and retail meat has increased the risk of contamination of food. The aim of this study was to determine the prevalence and characterize S. aureus strains isolated from the pork chain supply in Chile. A total of 487 samples were collected: 332 samples from pigs at farms and slaughterhouses (nasal, n = 155; skin, n = 177); 85 samples from carcasses at slaughterhouses; and 70 meat samples at supermarkets and retail stores. The isolation of S. aureus was carried out by selective enrichment and culture media. Biochemical testing (API ® Staph) and PCR (detection of the nuc and mecA genes) were used to confirm S. aureus and MRSA strains. The agglutination test was used to determine the protein PBP2'. Enterotoxins (SEA, SEB, SEC, SED) were determined by agglutination test and the se genes by PCR method. Oxacillin and cefoxitin susceptibility testing were carried out using the diffusion method. The overall prevalence of S. aureus in the pork meat supply was 33.9%. A higher prevalence was detected on carcasses (56.5%), in pigs sampled at farms (40.6%) than in pigs sampled at slaughterhouses (23.3%) and in nonpackaged retail meat (43.1%) than packaged retail meat (5.3%) (p ≤ 0.05). No significant differences (p > 0.05) were found between the prevalence in pigs (28.3%) and pork meat (32.9%) and between natural pig farming (33.3%) and conventional production (52.8%). The mecA gene and the protein PBP2' were not detected in S. aureus strains. Two S. aureus strains exhibited oxacillin and cefoxitin resistance, and one S. aureus strain was resistant to cefoxitin. One S. aureus strain isolated from a meat sample was positive for enterotoxin SEB. Although the mecA gene was not detected, oxacillin-resistant and seb-producing S. aureus strains were detected, which represent a risk in the pork chain supply.

Simultaneous detection of Staphylococcus aureus and Salmonella typhimurium using multicolor time-resolved fluorescence nanoparticles as labels.

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Wang, Xiaole; Huang, Yukun; Wu, Shijia; Duan, Nuo; Xu, Baocai; Wang, Zhouping

2016-11-21

Foodborne illnesses caused by Staphylococcus aureus and Salmonella typhimurium are common public health issues worldwide, affecting both developing and developed countries. In this study, aptamers labeled with multicolor lanthanide-doped time-resolved fluorescence (TRFL) nanoparticles were used as signal probes, and immobilized by Fe 3 O 4 magnetic nanoparticles were used as the capture probes. The signal probes were bonded onto the captured bacteria by the recognition of aptamer to form the sandwich-type complex. Under the optimal conditions, TRFL intensity at 544nm was used to quantify S. typhimurium (y=10,213×-12,208.92, R 2 =0.9922) and TRFL intensity at 615nm for S. aureus (y=4803.20×-1933.87, R 2 =0.9982) in the range of 10 2 -10 5 CFU/ml. Due to the magnetic separation and concentration of Fe 3 O 4 nanoparticles, detection limits of the developed method were found to be 15, 20CFU/ml for S. typhimurium and S. aureus, respectively. The application of this bioassay in milk was also investigated, and results were consistent with those of plate-counting method. Therefore, this simple and rapid method owns a great potential in the application for the multiplex analysis in food safety. Copyright © 2016 Elsevier B.V. All rights reserved.

Staphylococcus aureus growth and enterotoxin production in different types of milk

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Bohdana Janštová, Jr.

2012-01-01

Full Text Available The aim of our study was to assess Staphylococcus aureus growth and the time of first detection of staphylococcal enterotoxins type A, B and C (SEA, SEB, SEC in different type of milk, depending on the strain and storage conditions. Raw, pasteurized, and UHT milk were inoculated with three strains of S. aureus, and growth patterns were determined by the plate method in accordance with EN ISO 6888-1. Baird-Parker agar medium was used for the detection of S. aureus and the Enzyme Linked Fluorescent Assay (ELFA used with a miniVIDAS analyzer tested the production of staphylococcal enterotoxins. The results of model experiments showed the dependence of the growth rate and subsequent production of staphylococcal enterotoxins on incubation (storage temperature, S. aureus strain, and type of milk. A significant finding was that the growth of S. aureus and production of enterotoxins in raw milk was inhibited by natural microflora, and production of enterotoxins was therefore not detected in raw milk within 102 hours of storage either at 15 °C or 22 °C. The highest risk of SEs production is associated with secondary contamination of pasteurized and UHT milk when stored at room temperature, where production was first detected after 12 hours of incubation.

Design and fabrication of an electrochemical aptasensor using Au nanoparticles/carbon nanoparticles/cellulose nanofibers nanocomposite for rapid and sensitive detection of Staphylococcus aureus.

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Ranjbar, Saba; Shahrokhian, Saeed

2018-04-27

Since that pathogenic bacteria are major threats to human health, this paper describes the fabrication of an effective and durable sensing platform based on gold nanoparticles/carbon nanoparticles/cellulose nanofibers nanocomposite (AuNPs/CNPs/CNFs) at the surface of glassy carbon electrode for sensitive and selective detection of Staphylococcus aureus (S. aureus). The AuNPs/CNPs/CNFs nanocomposite with the high surface area, excellent conductivity, and good biocompatibility was used for self-assembled of the thiolated specific S. aureus aptamer as a sensing element. The surface morphology of AuNPs/CNPs/CNFs nanocomposite was characterized with field emission scanning electron microscopy (FESEM), energy dispersive spectroscopy (EDS), dynamic light scattering (DLS) and ultraviolet-visible (UV-Vis) spectrophotometric methods. Each aptasensor modification step was monitored with cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. The fabricated aptasensor exhibited a wide linear dynamic range (1.2 × 10 1 to 1.2 × 10 8 ) CFU mL -1 with a LOD of 1 CFU mL -1 and was be capable to accurate detection and determination of Staphylococcus aureus in human blood serum as a clinical sample with a complex matrix. Copyright © 2018 Elsevier B.V. All rights reserved.

Environmental contamination with Staphylococcus aureus at a large, Midwestern university campus.

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Thapaliya, Dipendra; Taha, Mohammed; Dalman, Mark R; Kadariya, Jhalka; Smith, Tara C

2017-12-01

Staphylococcus aureus can cause minor to severe life-threatening infections. The changing epidemiology of S. aureus is of public health concern due to the emergence of multi-drug resistant (MDR) strains. Environmental surfaces play a crucial role in the transmission of S. aureus. The objective of this study was to examine environmental contamination and molecular characteristics of S. aureus in health professional-associated (HPA) and non-health professional-associated (NHPA) buildings at a large university. A total of 152 environmental surface samples were collected from two HPA and two NHPA campus buildings. Bacterial culture and diagnostics were done using standard microbiology methods. Polymerase chain reaction was conducted to detect mecA and PVL genes. All isolates were spa typed. A subset of isolates was characterized via multi-locus sequence typing (MLST). All S. aureus isolates were tested for antibiotic susceptibility. The overall contamination of S. aureus and methicillin-resistant S. aureus (MRSA) was 22.4% (34/152) and 5.9% (9/152) respectively. Similar prevalence of contamination was found in HPA and NHPA buildings. A total of 17 different spa types were detected among 34 S. aureus isolates. The majority of the MRSA isolates belonged to clonal complex (CC) 8. One isolate was positive for PVL. Eleven different sequence types (STs) were detected from 17 tested isolates. ST8 was the most common. Twelve isolates (35.3%) were MDR. Almost 27% (9/34) of the isolates were MRSA. The highest contamination of S. aureus was found in high hand-touch areas such as door knobs, suggesting that human interaction in crowded environments such as academic institutions plays a crucial role in S. aureus/MRSA transmission via inanimate objects. Additionally, more than one-third of the isolates were MDR. These data reinforce the need to implement effective prevention strategies outside the healthcare setting to decrease the incidence of drug-resistant S. aureus infections

Co-detection of Panton-Valentine Leukocidin and cotrimoxazole resistance in Staphylococcus aureus: Implications for HIV-patients' care

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Christian eKraef

2015-02-01

Full Text Available Patients infected with the human immunodeficiency virus (HIV are frequently exposed to antimicrobial agents. This might have an impact on the resistance profile, genetic background and virulence factors of colonizing Staphylococcus aureus. Sub-Saharan Africa is considered to be endemic for Panton-Valentine leukocidin (PVL positive S. aureus which can be associated with skin and soft tissue infections. We compared S. aureus from nasal and pharyngeal swabs from HIV patients (n=141 and healthy controls (n=206 in Gabon in 2013, and analyzed determinants of colonization with PVL positive isolates in a cross-sectional study. S. aureus isolates were screened for the presence of selected virulence factors (incl. PVL and were subjected to antimicrobial susceptibility testing and genotyping. In HIV patients, S. aureus was more frequently detected (36.9 vs. 31.6% and the isolates were more frequently PVL positive than in healthy controls (42.1 vs. 23.2%. The presence of PVL was associated with cotrimoxazole resistance (OR=25.1, p<0.001 and the use of cotrimoxazole was a risk factor for colonization with PVL positive isolates (OR=2.5, p=0.06. PVL positive isolates were associated with the multilocus sequence types ST15 (OR=5.6, p<0.001 and ST152 (OR=62.1, p<0.001.Participants colonized with PVL positive isolates reported more frequently skin and soft tissue infection (SSTI in the past compared to carriers of PVL negative isolates (OR=2.7, p=0.01. In conclusion, the novelty of our study is that cotrimoxazole might increase the risk of SSTI in regions where cotrimoxazole resistance is high and associated with PVL. This finding needs to be confirmed in prospective studies.

Chemical composition of fennel essential oil and its impact on Staphylococcus aureus exotoxin production.

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Qiu, Jiazhang; Li, Hongen; Su, Hongwei; Dong, Jing; Luo, Mingjing; Wang, Jianfeng; Leng, Bingfeng; Deng, Yanhong; Liu, Juxiong; Deng, Xuming

2012-04-01

In this study, fennel oil was isolated by hydrodistillation, and the chemical composition was determined by gas chromatography/mass spectral analysis. The antimicrobial activity of fennel oil against Staphylococcus aureus was evaluated by broth microdilution. A haemolysis assay, tumour necrosis factor (TNF) release assay, western blot, and real-time reverse transcription (RT)-PCR were applied to investigate the influence of fennel oil on the production of S. aureus virulence-related exoproteins. The data show that fennel oil, which contains a high level of trans-anethole, was active against S. aureus, with MICs ranging from 64 to 256 μg/ml. Furthermore, fennel oil, when used at subinhibitory concentrations, could dose-dependently decrease the expression of S. aureus exotoxins, including α-toxin, Staphylococcal enterotoxins (SEs) and toxic shock syndrome toxin 1 (TSST-1).

In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

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Ka L. Hong

2015-01-01

Full Text Available Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX variant previously developed by our laboratory was utilized to select a single-stranded DNA molecular recognition element (MRE targeting alpha toxin with high affinity and specificity. At the end of the 12-round selection, the selected MRE had an equilibrium dissociation constant (Kd of 93.7 ± 7.0 nM. Additionally, a modified sandwich enzyme-linked immunosorbent assay (ELISA was developed by using the selected ssDNA MRE as the toxin-capturing element and a sensitive detection of 200 nM alpha toxin in undiluted human serum samples was achieved.

Pseudomonas aeruginosa induces pigment production and enhances virulence in a white phenotypic variant of Staphylococcus aureus.

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Antonic, Vlado; Stojadinovic, Alexander; Zhang, Binxue; Izadjoo, Mina J; Alavi, Mohammad

2013-01-01

Staphyloxanthin is a virulence factor which protects Staphylococcus aureus in stress conditions. We isolated two pigment variants of S. aureus and one strain of Pseudomonas aeruginosa from a single wound infection. S. aureus variants displayed white and yellow colony phenotypes. The sequence of the operons for staphyloxanthin synthesis indicated that coding and promoter regions were identical between the two pigment variants. Quorum sensing controls pigment synthesis in some bacteria. It is also shown that P. aeruginosa quorum-sensing molecules affect S. aureus transcription. We explored whether the co-infecting P. aeruginosa can affect pigment production in the white S. aureus variant. In co-culture experiments between the white variants and a selected number of Gram-positive and Gram-negative bacteria, only P. aeruginosa induced pigment production in the white variant. Gene expression analysis of the white variant did not indicate upregulation of the crtM and other genes known to be involved in pigment production (sigB, sarA, farnesyl pyrophosphate synthase gene [FPP-synthase], hfq). In contrast, transcription of the catalase gene was significantly upregulated after co-culture. P. aeruginosa-induced pigment synthesis and catalase upregulation correlated with increased resistance to polymyxin B, hydrogen peroxide, and the intracellular environment of macrophages. Our data indicate the presence of silent but functional staphyloxanthin synthesis machinery in a white phenotypic variant of S. aureus which is activated by a co-infecting P. aeruginosa via inter-species communication. Another S. aureus virulence factor, catalase is also induced by this co-infecting bacterium. The resulting phenotypic changes are directly correlated with resistance of the white variant to stressful conditions.

Phenotypic and genotypic detection of methicillin-resistant Staphylococcus aureus in hunting dogs in Maiduguri metropolitan, Borno State, Nigeria

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Muhammad Mustapha

2016-05-01

Full Text Available Aim: To determine the presence of MRSA in hunting dogs in Maiduguri metropolitan. Materials and Methods: Phenotypic methods used includes microscopic technique, colony morphology study, catalase-coagulase tests, and the use of mannitol salt agar test, oxacillin resistance screening agar base, and antibiotic susceptibility testing methods. Genotypic approach was used for deoxyribonucleic acid extraction, and the presence of nuc and mecA gene was detected using polymerase chain reaction (PCR techniques. Results: Examination of 416 swab samples from nasal and perineal region of dogs revealed a total of 79.5% of S. aureus, where 62.5% of the isolates were MRSA. Molecular analysis revealed that 7nuc genes specific for S. aureus from 20 presumptive MRSA assay were all mecA PCR negative. The isolates were sensitive to gentamicin and ciprofloxacin but proved resistant to cefoxitin and oxacillin. Conclusion: High isolation rate of MRSA was found in hunting dogs. Significant level (p<0.05 of MRSA was isolated in the nasal cavity of hunting dogs than its perineum. Only nuc genes were detected from the MRSA isolates.

Phenotypic and genotypic detection of methicillin-resistant Staphylococcus aureus in hunting dogs in Maiduguri metropolitan, Borno State, Nigeria.

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Mustapha, Muhammad; Bukar-Kolo, Yachilla Maryam; Geidam, Yaqub Ahmed; Gulani, Isa Adamu

2016-05-01

To determine the presence of MRSA in hunting dogs in Maiduguri metropolitan. Phenotypic methods used includes microscopic technique, colony morphology study, catalase-coagulase tests, and the use of mannitol salt agar test, oxacillin resistance screening agar base, and antibiotic susceptibility testing methods. Genotypic approach was used for deoxyribonucleic acid extraction, and the presence of nuc and mecA gene was detected using polymerase chain reaction (PCR) techniques. Examination of 416 swab samples from nasal and perineal region of dogs revealed a total of 79.5% of S. aureus, where 62.5% of the isolates were MRSA. Molecular analysis revealed that 7nuc genes specific for S. aureus from 20 presumptive MRSA assay were all mecA PCR negative. The isolates were sensitive to gentamicin and ciprofloxacin but proved resistant to cefoxitin and oxacillin. High isolation rate of MRSA was found in hunting dogs. Significant level (p<0.05) of MRSA was isolated in the nasal cavity of hunting dogs than its perineum. Only nuc genes were detected from the MRSA isolates.

An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification.

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Xu, Jingguo; Guo, Jia; Maina, Sarah Wanjiku; Yang, Yumeng; Hu, Yimin; Li, Xuanxuan; Qiu, Jiarong; Xin, Zhihong

2018-05-15

An ultra-sensitive aptamer-based biosensor for the detection of staphylococcus aureus was established by adopting the nicking enzyme amplification reaction (NEAR) and the rolling circle amplification (RCA) technologies. Aptamer-probe (AP), containing an aptamer and a probe sequence, was developed to act as the recognition unit of the biosensor, which was specifically bound to S. aureus. The probe was released from AP and initiated into the subsequent DNA amplification reactions where S. aureus was present, converting the detection of S. aureus to the investigation of probe oligonucleotide. The RCA amplification products contained a G-quadruplex motif and formed a three dimensional structure in presence of hemin. The G4/hemin complex showed horseradish peroxidase (HRP)-mimic activity and catalyzed the chemiluminescence reaction of luminol mediated by H 2 O 2 . The results showed that the established biosensor could detect S. aureus specifically with a good linear correlation at 5-10 4  CFU/mL. The signal values based on NEAR-RCA two-step cycle were boosted acutely, much higher than that relied on one-cycle magnification. The limit of detection (LoD) was determined to be as low as 5 CFU/mL. The established aptasensor exhibited a good discrimination of living against dead S. aureus, and can be applied to detect S. aureus in the food industry. Copyright © 2018 Elsevier Inc. All rights reserved.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA.

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Stoltenburg, Regina; Krafčiková, Petra; Víglaský, Viktor; Strehlitz, Beate

2016-09-21

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting Protein A on the cell surface. The full-length aptamer and one of its truncated variants could be demonstrated to specifically bind to Protein A-expressing intact cells of S. aureus, and thus have the potential to expand the portfolio of aptamers that can act as an analytical agent for the specific recognition and rapid detection of the bacterial pathogen. The functionality of the aptamer was found to be based on a very complex, but also highly variable structure. Two structural key elements were identified. The aptamer sequence contains several G-clusters allowing folding into a G-quadruplex structure with the potential of dimeric and multimeric assembly. An inverted repeat able to form an imperfect stem-loop at the 5'-end also contributes essentially to the aptameric function.

Molecular detection of Staphylococcus aureus resistant to temperature in milk and its products

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Sutejo, Stephani Valentina Harda; Amarantini, Charis; Budiarso, Tri Yahya

2017-11-01

Contamination of Staphylococcus aureus on milk can cause intoxication and infection by Staphylococcal enterotoxin. It has nuc gene, coding thermonuclease enzyme (TNase) that is responsible for nature of resistance in the heating process. This study was conducted to identify nuc gene of as S. aureus isolated from milk and its products like ultra-high temperature, sterile milk, sweetened condensed milk, formula milk, café/milk street traders and fresh milk. Biochemical identification was conducted by using carbohydrate fermentation tests and confirmed by API Staph. Molecular confirmation by amplification of nuc gene using PCR. Based on the results of confirmation using API Staph, all isolates were confirmed as S. aureus with index determinant percentage of 97%. An amplicon product of 270 bp was gained in all isolates. It is concluded that isolate of S. aureus has nuc gene.

Rapid Detection of Staphylococcus aureus and Methicillin-Resistant S. aureus in Atopic Dermatitis by Using the BD Max StaphSR Assay.

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Lee, Mi Kyung; Park, Kui Young; Jin, Taewon; Kim, Ju Hee; Seo, Seong Jun

2017-07-01

Eczematous lesions of atopic dermatitis (AD) patients are known to be a source of Staphylococcus aureus (SA) transmission and might be a reservoir for community-associated methicillin-resistant SA (MRSA). The BD Max StaphSR (BD-SR) is a fully automated, multiplex real-time PCR assay for the direct detection and differentiation of SA and MRSA from nasal swab samples. We evaluated the detection rates of SA and MRSA from skin lesions of outpatients with AD using the BD-SR assay, and determined the usefulness of the BD-SR assay. A total of 244 skin swab samples (skin lesions of 213 outpatients with AD and normal skin of 31 healthy controls) were tested directly by using the BD-SR assay. Of the 213 samples from patients with AD, 69 (32.4%) were positive for SA, 6 (8.7%) of which were positive for MRSA. Only 1 (3.2%) of 31 samples from healthy controls was positive for SA. The BD-SR assay is effective for the rapid detection of SA and MRSA from skin swab samples, which can provide important information for managing patients with AD and preventing the spread of MRSA. © The Korean Society for Laboratory Medicine.

Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation

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Das, Sudip; Lindemann, Claudia; Young, Bernadette C.; Muller, Julius; Österreich, Babett; Ternette, Nicola; Winkler, Ann-Cathrin; Paprotka, Kerstin; Reinhardt, Richard; Allen, Elizabeth; Flaxman, Amy; Yamaguchi, Yuko; Rollier, Christine S.; van Diemen, Pauline; Blättner, Sebastian; Remmele, Christian W.; Selle, Martina; Dittrich, Marcus; Müller, Tobias; Vogel, Jörg; Ohlsen, Knut; Crook, Derrick W.; Massey, Ruth; Wilson, Daniel J.; Rudel, Thomas; Wyllie, David H.; Fraunholz, Martin J.

2016-01-01

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection. PMID:27185949

Introduction of a hydrolysis probe PCR assay for high-throughput screening of methicillin-resistant Staphylococcus aureus with the ability to include or exclude detection of Staphylococcus argenteus.

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Bogestam, Katja; Vondracek, Martin; Karlsson, Mattias; Fang, Hong; Giske, Christian G

2018-01-01

Many countries using sensitive screening methods for detection of carriage of methicillin-resistant Staphylococcus aureus (MRSA) have a sustained low incidence of MRSA infections. For diagnostic laboratories with high sample volumes, MRSA screening requires stability, low maintenance and high performance at a low cost. Herein we designed oligonucleotides for a new nuc targeted hydrolysis probe PCR to replace the standard in-house nuc SybrGreen PCR assay. This new, more time-efficient, PCR assay resulted in a 40% increase in daily sample capacity, with maintained high specificity and sensitivity. The assay was also able to detect Staphylococcus aureus clonal cluster 75 (CC75) lineage strains, recently re-classified as Staphylococcus argenteus, with a sensitivity considerably increased compared to our previous assay. While awaiting consensus if the CC75 lineage of S. aureus should be considered as S. argenteus, and whether methicillin-resistant S. argenteus should be included in the MRSA definition, many diagnostic laboratories need to update their MRSA assay sensitivity/specificity towards this lineage/species. The MRSA screening assay presented in this manuscript is comprised of nuc oligonucleotides separately targeting S. aureus and CC75 lineage strains/S. argenteus, thus providing high user flexibility for the detection of CC75 lineage strains/S. argenteus.

The impact of CodY on virulence determinant production in community-associated methicillin-resistant Staphylococcus aureus.

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Rivera, Frances E; Miller, Halie K; Kolar, Stacey L; Stevens, Stanley M; Shaw, Lindsey N

2012-01-01

Staphylococcus aureus is a leading human pathogen of both hospital and community-associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ-based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA-MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post-exponential and stationary growth. Specifically, we observe hyper-production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry-based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Kaempferol Inhibits the Primary Attachment Phase of Biofilm Formation in Staphylococcus aureus.

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Ming, Di; Wang, Dacheng; Cao, Fengjiao; Xiang, Hua; Mu, Dan; Cao, Junjie; Li, Bangbang; Zhong, Ling; Dong, Xiaoyun; Zhong, Xiaobo; Wang, Lin; Wang, Tiedong

2017-01-01

The ability to form biofilms on surfaces makes Staphylococcus aureus the main pathogenic factor in implanted medical device infections. The aim of this study was to discover a biofilm inhibitor distinct from the antibiotics used to prevent infections resulting from S. aureus biofilms. Here, we describe kaempferol, a small molecule with anti-biofilm activity that specifically inhibited the formation of S. aureus biofilms. Crystal violet (CV) staining and fluorescence microscopy clearly showed that 64 μg/ml kaempferol inhibited biofilm formation by 80%. Meanwhile, the minimum inhibitory concentration (MIC) and growth curve results indicated that kaempferol had no antibacterial activity against the tested bacterial strain. Kaempferol inhibited the primary attachment phase of biofilm formation, as determined by a fibrinogen-binding assay. Moreover, a fluorescence resonance energy transfer (FRET) assay and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analyses revealed that kaempferol reduced the activity of S. aureus sortaseA (SrtA) and the expression of adhesion-related genes. Based on these results, kaempferol provides a starting point for the development of novel anti-biofilm drugs, which may decrease the risk of bacterial drug resistance, to prevent S. aureus biofilm-related infections.

G-quadruplex aptamer targeting Protein A and its capability to detect Staphylococcus aureus demonstrated by ELONA

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Stoltenburg, Regina; Kraf?ikov?, Petra; V?glask?, Viktor; Strehlitz, Beate

2016-01-01

Aptamers for whole cell detection are selected mostly by the Cell-SELEX procedure. Alternatively, the use of specific cell surface epitopes as target during aptamer selections allows the development of aptamers with ability to bind whole cells. In this study, we integrated a formerly selected Protein A-binding aptamer PA#2/8 in an assay format called ELONA (Enzyme-Linked OligoNucleotide Assay) and evaluated the ability of the aptamer to recognise and bind to Staphylococcus aureus presenting P...

An Annotation Agnostic Algorithm for Detecting Nascent RNA Transcripts in GRO-Seq.

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Azofeifa, Joseph G; Allen, Mary A; Lladser, Manuel E; Dowell, Robin D

2017-01-01

We present a fast and simple algorithm to detect nascent RNA transcription in global nuclear run-on sequencing (GRO-seq). GRO-seq is a relatively new protocol that captures nascent transcripts from actively engaged polymerase, providing a direct read-out on bona fide transcription. Most traditional assays, such as RNA-seq, measure steady state RNA levels which are affected by transcription, post-transcriptional processing, and RNA stability. GRO-seq data, however, presents unique analysis challenges that are only beginning to be addressed. Here, we describe a new algorithm, Fast Read Stitcher (FStitch), that takes advantage of two popular machine-learning techniques, hidden Markov models and logistic regression, to classify which regions of the genome are transcribed. Given a small user-defined training set, our algorithm is accurate, robust to varying read depth, annotation agnostic, and fast. Analysis of GRO-seq data without a priori need for annotation uncovers surprising new insights into several aspects of the transcription process.

Repression of branched-chain amino acid synthesis in Staphylococcus aureus is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide.

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Kaiser, Julienne C; King, Alyssa N; Grigg, Jason C; Sheldon, Jessica R; Edgell, David R; Murphy, Michael E P; Brinsmade, Shaun R; Heinrichs, David E

2018-01-01

Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine) for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.

Repression of branched-chain amino acid synthesis in Staphylococcus aureus is mediated by isoleucine via CodY, and by a leucine-rich attenuator peptide.

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Julienne C Kaiser

2018-01-01

Full Text Available Staphylococcus aureus requires branched-chain amino acids (BCAAs; isoleucine, leucine, valine for protein synthesis, branched-chain fatty acid synthesis, and environmental adaptation by responding to their availability via the global transcriptional regulator CodY. The importance of BCAAs for S. aureus physiology necessitates that it either synthesize them or scavenge them from the environment. Indeed S. aureus uses specialized transporters to scavenge BCAAs, however, its ability to synthesize them has remained conflicted by reports that it is auxotrophic for leucine and valine despite carrying an intact BCAA biosynthetic operon. In revisiting these findings, we have observed that S. aureus can engage in leucine and valine synthesis, but the level of BCAA synthesis is dependent on the BCAA it is deprived of, leading us to hypothesize that each BCAA differentially regulates the biosynthetic operon. Here we show that two mechanisms of transcriptional repression regulate the level of endogenous BCAA biosynthesis in response to specific BCAA availability. We identify a trans-acting mechanism involving isoleucine-dependent repression by the global transcriptional regulator CodY and a cis-acting leucine-responsive attenuator, uncovering how S. aureus regulates endogenous biosynthesis in response to exogenous BCAA availability. Moreover, given that isoleucine can dominate CodY-dependent regulation of BCAA biosynthesis, and that CodY is a global regulator of metabolism and virulence in S. aureus, we extend the importance of isoleucine availability for CodY-dependent regulation of other metabolic and virulence genes. These data resolve the previous conflicting observations regarding BCAA biosynthesis, and reveal the environmental signals that not only induce BCAA biosynthesis, but that could also have broader consequences on S. aureus environmental adaptation and virulence via CodY.

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus.

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Cao, Xiaoxiao; Li, Shaohua; Chen, Liucun; Ding, Hongmei; Xu, Hua; Huang, Yanping; Li, Jie; Liu, Nongle; Cao, Weihong; Zhu, Yanjun; Shen, Beifen; Shao, Ningsheng

2009-08-01

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequences from different families were selected for further characterization by confocal imaging and flow cytometry analysis. Results showed that five aptamers demonstrated high specificity and affinity to S. aureus individually. The five aptamers recognize different molecular targets by competitive experiment. Combining these five aptamers had a much better effect than the individual aptamer in the recognition of different S. aureus strains. In addition, the combined aptamers can probe single S. aureus in pyogenic fluids. Our work demonstrates that a set of aptamers specific to one bacterium can be used in combination for the identification of the bacterium instead of a single aptamer.

Detection of Macrolide, Lincosamide and Streptogramin Resistance among Methicillin Resistant Staphylococcus aureus (MRSA in Mumbai

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Arunagiri Subramanian

2015-01-01

Full Text Available Background: The increase in incidence of Methicillin Resistant Staphyloccocus aureus (MRSA and its extraordinary potential to develop antimicrobial resistance has highlighted the need for better agents to treat such infections. This has led to a renewed interest in use of new drugs for treatment with clindamycin and quinuprsitin-dalfopristin being the preferred choice for treatment. Aim & Objectives: This study was undertaken to detect the prevalence of MacrolideLincosamide-Streptogramin (MLS resistance among clinical isolates of MRSA.Material and Methods:Two hundred and thirty clinical isolates of S. aureus were subjected to routine antibiotic susceptibility testing including cefoxitin, erythromycin and quinupristindalfopristin. Inducible resistance to clindamycin was tested by 'D' test as per Clinical and Laboratory Standards Institute (CLSI guidelines. Results: Out of all S. aureus isolates, 93.91% were identified as MRSA. In the disc diffusion testing, 81.5% of isolates showed erythromycin resistance. Among these, the prevalence of constitutive (cMLS , inducible (iMLS b b and MS-phenotype were 35.80%, 31.82% and 32.39% respectively by the D-test method. 77.8% of isolates were resistant to quinupristin-dalfopristin and the Minimum Inhibitory Concentration (MIC ranged from 4–32 µg/ml. 89.20% of isolates were resistant to both quinupristin-dalfopristin and erythromycin of which 35.03%, 35.67% and 29.30% belonged to iMLS , cMLS and MS phenotype respectively. Conclusion: The emergence of quinupristindalfopristin resistance and MLS phenotypes brings b about the need for the simple and reliable D-test in routine diagnosis and further susceptibility testing for proper antimicrobial therapy.

High resolution analysis of the human transcriptome: detection of extensive alternative splicing independent of transcriptional activity

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Rouet Fabien

2009-10-01

Full Text Available Abstract Background Commercially available microarrays have been used in many settings to generate expression profiles for a variety of applications, including target selection for disease detection, classification, profiling for pharmacogenomic response to therapeutics, and potential disease staging. However, many commercially available microarray platforms fail to capture transcript diversity produced by alternative splicing, a major mechanism for driving proteomic diversity through transcript heterogeneity. Results The human Genome-Wide SpliceArray™ (GWSA, a novel microarray platform, utilizes an existing probe design concept to monitor such transcript diversity on a genome scale. The human GWSA allows the detection of alternatively spliced events within the human genome through the use of exon body and exon junction probes to provide a direct measure of each transcript, through simple calculations derived from expression data. This report focuses on the performance and validation of the array when measured against standards recently published by the Microarray Quality Control (MAQC Project. The array was shown to be highly quantitative, and displayed greater than 85% correlation with the HG-U133 Plus 2.0 array at the gene level while providing more extensive coverage of each gene. Almost 60% of splice events among genes demonstrating differential expression of greater than 3 fold also contained extensive splicing alterations. Importantly, almost 10% of splice events within the gene set displaying constant overall expression values had evidence of transcript diversity. Two examples illustrate the types of events identified: LIM domain 7 showed no differential expression at the gene level, but demonstrated deregulation of an exon skip event, while erythrocyte membrane protein band 4.1 -like 3 was differentially expressed and also displayed deregulation of a skipped exon isoform. Conclusion Significant changes were detected independent of

Communications of Staphylococcus aureus and non-aureus Staphylococcus species from bovine intramammary infections and teat apex colonization.

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Mahmmod, Yasser S; Klaas, Ilka Christine; Svennesen, Line; Pedersen, Karl; Ingmer, Hanne

2018-05-16

The role of non-aureus staphylococci (NAS) in the risk of acquisition of intramammary infections with Staphylococcus aureus is vague and still under debate. The objectives of this study were to (1) investigate the distribution patterns of NAS species from milk and teat skin in dairy herds with automatic milking systems, and (2) examine if the isolated NAS influences the expression of S. aureus virulence factors controlled by the accessory gene regulator (agr) quorum sensing system. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabbing and aseptic quarter foremilk samples from right hind and left front quarters. Teat skin swabs were collected using the modified wet-dry method and milk samples were taken aseptically for bacterial culture. Colonies from quarters with suspicion of having NAS in milk or teat skin samples (or both) were subjected to MALDI-TOF assay for species identification. To investigate the interaction between S. aureus and NAS, 81 isolates NAS were subjected to a qualitative β-galactosidase reporter plate assay. In total, 373 NAS isolates were identified representing 105 from milk and 268 from teat skin of 284 quarters (= 142 cows). Sixteen different NAS species were identified, 15 species from teat skin and 10 species from milk. The most prevalent NAS species identified from milk were Staphylococcus epidermidis (50%), Staphylococcus haemolyticus (15%), and Staphylococcus chromogenes (11%), accounting for 76%. Meanwhile, the most prevalent NAS species from teat skin were Staphylococcus equorum (43%), S. haemolyticus (16%), and Staphylococcus cohnii (14%), accounting for 73%. Using reporter gene fusions monitoring transcriptional activity of key virulence factors and regulators, we found that out of 81 supernatants of NAS isolates, 77% reduced expression of hla, encoding a-hemolysin, 70% reduced expression of RNAIII, the key effector molecule of agr, and 61% reduced expression of spa encoding

An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

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Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

2016-10-01

Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

Methicillin-resistant Staphylococcus aureus containing mecC in Swedish dairy cows

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Unnerstad Helle Ericsson

2013-01-01

Full Text Available Abstract Background Hitherto, methicillin-resistant Staphylococcus aureus (MRSA has not been detected in Swedish cattle. However, due to the report of mecC, a novel homologue to the mecA gene, there was reason to re-evaluate susceptibility results from strain collections of Staphylococcus aureus and test suspected isolates for the presence of mecC. Findings Bovine isolates of S. aureus with elevated minimum inhibitory concentrations of beta-lactams were retrospectively tested for presence of mecC. In four of the isolates mecC was detected. Conclusion In Sweden, this is the first finding of MRSA in cattle and the first detection of MRSA harbouring mecC of domestic animal origin. MRSA in animal populations has implications as a potential reservoir with risk for spread to humans. Occurrence of MRSA among Swedish cattle appears still very limited.

Frequency of Reduced Vancomycin Susceptibility among Clinical Staphylococcus aureus Isolated in Ahvaz Iran

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Mojtaba Moosavian

2015-11-01

Full Text Available Introduction:   One   of   the   most   important   agents   in   hospital-acquired   infections   is Staphylococcus aureus. Treatment of methicillin-resistant S. aureus (MRSA infections with decreased susceptibility to vancomycin has recently been more difficult. The aim of this study was to evaluate the possible presence of vancomycin intermediate S. aureus (VISA and vancomycin- resistant S. aureus (VRSA and also to determine the frequency of MRSA in clinical specimens.Methods: In this study, 195 S. aureus isolates were collected from the patients were examined. All of the isolates were identified using standard biochemical tests.  Susceptibility of S. aureus isolates against 10 antibiotics was detected by disk diffusion method and was followed by E-test and vancomycin screen agar methods. Minimum inhibitory concentration (MIC of vancomycin was determined according to the CLSI guidelines.  Also, detection of mecA gene was performed by PCR and finally, the results were compared.Results: All of the isolates were susceptible to vancomycin (i.e. MIC range of vancomycin was between 0.25-2 µg/ml. Out of 195 S. aureus isolates, 99 isolates (50.8% were resistant to methicillin, and mecA gene was detected in 96 isolates. These results also showed that the highest and lowest resistance rate of isolates was to penicillin (96.9% and chloramphenicol (0%, respectively.Conclusion: Our findings showed that vancomycin can still be used as a valuable drug for treatment of S. aureus infections in our region. However, periodic evaluation of vancomycin MIC of S. aureus isolates is critical for monitoring MRSA and preventing the spread of VISA or VRSA among patients.

Accurate Detection of Methicillin-Resistant Staphylococcus aureus in Mixtures by Use of Single-Bacterium Duplex Droplet Digital PCR.

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Luo, Jun; Li, Junhua; Yang, Hang; Yu, Junping; Wei, Hongping

2017-10-01

Accurate and rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is needed to screen MRSA carriers and improve treatment. The current widely used duplex PCR methods are not able to differentiate MRSA from coexisting methicillin-susceptible S. aureus (MSSA) or other methicillin-resistant staphylococci. In this study, we aimed to develop a direct method for accurate and rapid detection of MRSA in clinical samples from open environments, such as nasal swabs. The new molecular assay is based on detecting the cooccurrence of nuc and mecA markers in a single bacterial cell by utilizing droplet digital PCR (ddPCR) with the chimeric lysin ClyH for cell lysis. The method consists of (i) dispersion of an intact single bacterium into nanoliter droplets, (ii) temperature-controlled release of genomic DNA (gDNA) by ClyH at 37°C, and (iii) amplification and detection of the markers ( nuc and mecA ) using standard TaqMan chemistries with ddPCR. Results were analyzed based on MRSA index ratios used for indicating the presence of the duplex-positive markers in droplets. The method was able to achieve an absolute limit of detection (LOD) of 2,900 CFU/ml for MRSA in nasal swabs spiked with excess amounts of Escherichia coli , MSSA, and other mecA -positive bacteria within 4 h. Initial testing of 104 nasal swabs showed that the method had 100% agreement with the standard culture method, while the normal duplex qPCR method had only about 87.5% agreement. The single-bacterium duplex ddPCR assay is rapid and powerful for more accurate detection of MRSA directly from clinical specimens. Copyright © 2017 American Society for Microbiology.

Guanine limitation results in CodY-dependent and -independent alteration of Staphylococcus aureus physiology and gene expression.

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King, Alyssa N; Borkar, Samiksha; Samuels, David J; Batz, Zachary; Bulock, Logan; Sadykov, Marat R; Bayles, Kenneth W; Brinsmade, Shaun R

2018-04-30

In Staphylococcus aureus , the global transcriptional regulator CodY modulates the expression of hundreds of genes in response to the availability of GTP and the branched-chain amino acids isoleucine, leucine, and valine (ILV). CodY DNA-binding activity is high when GTP and ILV are abundant. When GTP and ILV are limited, CodY's affinity for DNA drops, altering expression of CodY regulated targets. In this work, we investigated the impact of guanine nucleotides on S. aureus physiology and CodY activity by constructing a guaA null mutant (Δ guaA ). De novo biosynthesis of guanine monophosphate is abolished due to the guaA mutation; thus, the mutant cells require exogenous guanosine for growth. We also found that CodY activity was reduced when we knocked out guaA , activating the Agr two-component system and increasing secreted protease activity. Notably, in a rich, complex medium, we detected an increase in alternative sigma factor B activity in the Δ guaA mutant, which results in a 5-fold increase in production of the antioxidant pigment staphyloxanthin. Under biologically relevant flow conditions, Δ guaA cells failed to form robust biofilms when limited for guanine or guanosine. RNA-seq analysis of S. aureus transcriptome during growth in guanosine-limited chemostats revealed substantial CodY-dependent and -independent alteration of gene expression profiles. Importantly, these changes increase production of proteases and δ-toxin, suggesting that S. aureus exhibits a more invasive lifestyle when limited for guanosine. Further, gene-products upregulated under GN limitation, including those necessary for lipoic acid biosynthesis and sugar transport, may prove to be useful drug targets for treating Gram-positive infections. Importance Staphylococcus aureus infections impose a serious economic burden on healthcare facilities and patients because of the emergence of strains resistant to last-line antibiotics. Understanding the physiological processes governing

Duplex Identification of Staphylococcus aureus by Aptamer and Gold Nanoparticles.

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Chang, Tianjun; Wang, Libo; Zhao, Kexu; Ge, Yu; He, Meng; Li, Gang

2016-06-01

Staphylococcus aureus is the top common pathogen causing infections and food poisoning. Identification of S. aureus is crucial for the disease diagnosis and regulation of food hygiene. Herein, we report an aptamer-AuNPs based method for duplex identification of S. aureus. Using AuNPs as an indicator, SA23, an aptamer against S. aureus, can well identify its target from Escherichia coli, Listeria monocytogenes and Pseudomonas aeruginosa. Furthermore, we find citrate-coated AuNPs can strongly bind to S. aureus, but not bind to Salmonella enterica and Proteus mirabilis, which leads to different color changes in salt solution. This colorimetric response is capable of distinguishing S. aureus from S. enteritidis and P. mirabilis. Thus, using the aptasensor and AuNPs together, S. aureus can be accurately identified from the common pathogens. This duplex identification system is a promising platform for simple visual identification of S. aureus. Additionally, in the aptasensing process, bacteria are incubated with aptamers and then be removed before the aptamers adding to AuNPs, which may avoid the interactions between bacteria and AuNPs. This strategy can be potentially applied in principle to detect other cells by AuNPs-based aptasensors.

Detection and Characterization of Staphylococcus aureus and Methicillin-Resistant S. aureus in Foods Confiscated in EU Borders

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David Rodríguez-Lázaro

2017-07-01

Full Text Available The aim of the study was to evaluate the potential role of the illegal entry of food in UE in the Methicillin-resistant S. aureus (MRSA spread. We studied the prevalence and characteristics of Staphylococcus aureus and MRSA isolated from foods of animal origin confiscated from passengers on flights from 45 non-EU countries from 2012 to 2015 by the Border Authorities at Bilbao International Airport (Spain and Vienna International Airport (Austria, as well as foods from open markets close to EU land borders. Of 868 food samples tested (diverse meat samples including antelope, duck, guinea pig, pork, rodents, turkey, dairy products, and eggs, 136 (15.7% were positive for S. aureus and 26 (3.0% for MRSA. All MRSA strains were mecA-positive. The prevalence of S. aureus-positive dairy samples among food confiscated at Bilbao International Airport was 64.6%, and this airport also had the highest value (11.8% for MRSA-positive samples. The predominant sequence type was ST5 (30.8%, followed by ST8, ST1649, ST1, and other lineages were found to a lesser extent (ST7, ST22, ST72, ST97, and ST398. Six isolates tested positive for luk-PVL genes (SCCmec IV subtypes IVc and IVe. Enterotoxin profiling revealed that 19 MRSA strains were enterotoxigenic, harboring one or more se genes. The MRSA isolates positive for luk-PVL genes were not enterotoxigenic, and none of the isolates tested positive for enterotoxin E. We found 14 resistance profiles, and more than 69% of the MRSA isolates were resistant to three or more types of antimicrobial agents. This finding reveals both the wide diversity of the antimicrobial resistance found in the strains and the capacity to resist not only to beta-lactam drugs. One MRSA strain showed unusual characteristics: it was oxacillin-susceptible, harbored SCCmec V, and was positive for sed, seg, and sej but negative for PVL virulence factors. This study shows the presence of enterotoxigenic HA-, CA-, and LA-MRSA in foods illegally

Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts.

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Shimamura, Yuko; Kidokoro, Shiho; Murata, Masatsune

2006-07-01

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.

Staphylococcus aureus: methicillin-susceptible S. aureus to methicillin-resistant S. aureus and vancomycin-resistant S. aureus.

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Rehm, Susan J; Tice, Alan

2010-09-15

The evolution of methicillin-resistant and vancomycin-resistant Staphylococcus aureus has demanded serious review of antimicrobial use and development of new agents and revised approaches to prevent and overcome drug resistance. Depending on local conditions and patient risk factors, empirical therapy of suspected S. aureus infection may require coverage of drug-resistant organisms with newer agents and novel antibiotic combinations. The question of treatment with inappropriate antibiotics raises grave concerns with regard to methicillin-resistant S. aureus selection, overgrowth, and increased virulence. Several strategies to reduce the nosocomial burden of resistance are suggested, including shortened hospital stays and outpatient parenteral antimicrobial therapy of the most serious infections.

Immunological role of nasal staphylococcus aureus carriage in patients with persistent allergic rhinitis

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Mohamed Yousif Atia

2008-10-01

Full Text Available Nasal carriage of staphylococcus aureus (S.aureus exerts immunomodulatory effect in patients with atopic dermatitis and it may contribute to airway inflammation and allergic response in patients with allergic rhinitis. We Aim to investigate the frequency of nasal S.aureus carriage in patients with persistent allergic rhinitis and its possible influence on their symptoms and immune markers. We chosed 20 non smoker patients with house dust mite (HDM allergy causing allergic rhinitis and 20 non smoker healthy subjects matched for age and sex. For all subjects rhinoscopy was done, skin prick test, nasal culture for S.aureus, nasal interleukin 4,nasal total IgE, serum total IgE and serum specific IgE(SSIgE for HDM. Nasal S.aureus was detected in 16/20 patients (80% and 5/20 (25% in healthy subjects with highly significant statistical difference plt0.01. Correlation of nasal staph.aureus count and different systemic and local immune markers revealed highly significant positive correlation between nasal S.aureus count and serum total IgE (r = 0.78, plt0.01 and significant positive correlation with SSIgE (HDM (r = 0.53, plt0.05, nasal total IgE (r = 0.39, plt0.05 and nasal IL-4 (r = 0.55, plt0.05. Nasal staph.aureus actively modulated the immune reaction in persistent allergic rhinitis patients by promoting local IgE production, so we recommend early detection and treatment of S.aureus carriage in patients

Tracing and inhibiting growth of Staphylococcus aureus in barbecue cheese production after product recall.

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Johler, S; Zurfluh, K; Stephan, R

2016-05-01

Staphylococcal food poisoning is one of the most prevalent causes of foodborne intoxication worldwide. It is caused by ingestion of enterotoxins formed by Staphylococcus aureus during growth in the food matrix. Following a recall of barbecue cheese due to the detection of staphylococcal enterotoxins in Switzerland in July 2015, we analyzed the production process of the respective dairy. Although most cheese-making processes involve acidification to inhibit the growth of pathogenic bacteria, barbecue cheese has to maintain a pH >6.0 to prevent undesired melting of the cheese. In addition, the dairy decided to retain the traditional manual production process of the barbecue cheese. In this study, therefore, we aimed to (1) trace Staph. aureus along the barbecue cheese production process, and (2) develop a sustainable strategy to inhibit growth of Staph. aureus and decrease the risk of staphylococcal food poisoning without changing the traditional production process. To this end, we traced Staph. aureus in a step-wise blinded process analysis on 4 different production days using spa (Staphylococcus protein A gene) typing, DNA microarray profiling, and pulsed-field gel electrophoresis analysis. We subsequently selected a new starter culture and used a model cheese production including a challenge test assay to assess its antagonistic effect on Staph. aureus growth, as well as its sensory and technological implications. We detected Staph. aureus in 30% (37/124) of the collected samples taken from the barbecue cheese production at the dairy. This included detection of Staph. aureus in the final product on all 4 production days, either after enrichment or using quantitative detection. We traced 2 enterotoxigenic Staph. aureus strains (t073/CC45 and t282/CC45) colonizing the nasal cavity and the forearms of the cheesemakers to the final product. In the challenge test assay, we were able to show that the new starter culture inhibited growth of Staph. aureus while meeting

Staphylococcus aureus CC398

DEFF Research Database (Denmark)

Price, Lance B.; Stegger, Marc; Hasman, Henrik

2012-01-01

Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection...... of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected...... among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock...

Morphology-Independent Virulence of Candida Species during Polymicrobial Intra-abdominal Infections with Staphylococcus aureus.

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Nash, Evelyn E; Peters, Brian M; Fidel, Paul L; Noverr, Mairi C

2016-01-01

Intra-abdominal polymicrobial infections cause significant morbidity and mortality. An experimental mouse model of Candida albicans-Staphylococcus aureus intra-abdominal infection (IAI) results in 100% mortality by 48 to 72 h postinoculation, while monomicrobial infections are avirulent. Mortality is associated with robust local and systemic inflammation without a requirement for C. albicans morphogenesis. However, the contribution of virulence factors coregulated during the yeast-to-hypha transition is unknown. This also raised the question of whether other Candida species that are unable to form hyphae are as virulent as C. albicans during polymicrobial IAI. Therefore, the purpose of this study was to evaluate the ability of non-albicans Candida (NAC) species with various morphologies and C. albicans transcription factor mutants (efg1/efg1 and cph1/cph1) to induce synergistic mortality and the accompanying inflammation. Results showed that S. aureus coinoculated with C. krusei or C. tropicalis was highly lethal, similar to C. albicans, while S. aureus-C. dubliniensis, S. aureus-C. parapsilosis, and S. aureus-C. glabrata coinoculations resulted in little to no mortality. Local and systemic interleukin-6 (IL-6) and prostaglandin E2 (PGE2) levels were significantly elevated during symptomatic and/or lethal coinfections, and hypothermia strongly correlated with mortality. Coinoculation with C. albicans strains deficient in the transcription factor Efg1 but not Cph1 reversed the lethal outcome. These results support previous findings and demonstrate that select Candida species, without reference to any morphological requirement, induce synergistic mortality, with IL-6 and PGE2 acting as key inflammatory factors. Mechanistically, signaling pathways controlled by Efg1 are critical for the ability of C. albicans to induce mortality from an intra-abdominal polymicrobial infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Staphylococcus aureus nasal carriage in Ukraine: antibacterial resistance and virulence factor encoding genes.

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Netsvyetayeva, Irina; Fraczek, Mariusz; Piskorska, Katarzyna; Golas, Marlena; Sikora, Magdalena; Mlynarczyk, Andrzej; Swoboda-Kopec, Ewa; Marusza, Wojciech; Palmieri, Beniamino; Iannitti, Tommaso

2014-03-05

The number of studies regarding the incidence of multidrug resistant strains and distribution of genes encoding virulence factors, which have colonized the post-Soviet states, is considerably limited. The aim of the study was (1) to assess the Staphylococcus (S.) aureus nasal carriage rate, including Methicillin Resistant S. aureus (MRSA) strains in adult Ukrainian population, (2) to determine antibiotic resistant pattern and (3) the occurrence of Panton Valentine Leukocidine (PVL)-, Fibronectin-Binding Protein A (FnBPA)- and Exfoliative Toxin (ET)-encoding genes. Nasal samples for S. aureus culture were obtained from 245 adults. The susceptibility pattern for several classes of antibiotics was determined by disk diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The virulence factor encoding genes, mecA, lukS-lukF, eta, etb, etd, fnbA, were detected by Polymerase Chain Reaction (PCR). The S. aureus nasal carriage rate was 40%. The prevalence of nasal MRSA carriage in adults was 3.7%. LukS-lukF genes were detected in over 58% of the strains. ET-encoding genes were detected in over 39% of the strains and the most prevalent was etd. The fnbA gene was detected in over 59% of the strains. All MRSA isolates tested were positive for the mecA gene. LukS-lukF genes and the etd gene were commonly co-present in MRSA, while lukS-lukF genes and the fnbA gene were commonly co-present in Methicillin Sensitive S. aureus (MSSA) isolates. No significant difference was detected between the occurrence of lukS-lukF genes (P > 0.05) and the etd gene (P > 0.05) when comparing MRSA and MSSA. The occurrence of the fnbA gene was significantly more frequent in MSSA strains (P aureus is a common cause of infection. The prevalence of S. aureus nasal carriage in our cohort of patients from Ukraine was 40.4%. We found that 9.1% of the strains were classified as MRSA and all MRSA isolates tested positive for the mecA gene

Methicillin-resistant staphylococcus aureus isolates in a hospital of shanghai.

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Wang, Xiaoguang; Ouyang, Lin; Luo, Lingfei; Liu, Jiqian; Song, Chiping; Li, Cuizhen; Yan, Hongjing; Wang, Ping

2017-01-24

Methicillin-resistant Staphylococcus aureus (MRSA) strains are now common both in the health care setting and in the community. Active surveillance is critical for MRSA control and prevention. Specimens of patients (200 patients with 1119 specimens) as well as medical staff and hospital setting (1000 specimens) were randomly sampled in a level 2 hospital in Shanghai from September 2011 to August 2012. Isolation, cultivation and identification of S. aureus were performed. Totally, 67 S. aureus strains were isolated. 32 S. aureus strains were isolated from patient samples; 13 (13/32, 40.6%) of the 32 S. aureus isolates were MRSA; sputum sample and patients in the department of general internal medicine were the most frequent specimen and patient group for S. aureus strains isolation. Remaining 35 S. aureus strains were isolated from the medical staff and hospital setting; 20 (20/35, 57.1%) of the 35 S. aureus isolates were MRSA; specimens sampled from doctors and nurses' hands and nose and hospital facilities were the most frequent samples to isolate S. aureus. Resistant and virulent genes detection showed that, all 33 MRSA strains were mecA positive which accounts for 49.3% of the 67 S. aureus strains; 38 isolates were Panton-Valentine leukocidin (PVL) gene positive which accounts for 56.7% of the 67 S. aureus strains; and 17 (17/67, 25.4%) isolates are mecA and PVL genes dual positive. Multidrug-resistant strains of MRSA and PVL positive S. aureus are common in patients, medical staff and hospital setting, the potential health threat is worthy of our attention.

Occurrence and antimicrobial resistance of Staphylococcus aureus in bulk tank milk and milk filters

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Kateřina Bogdanovičová

2014-02-01

Full Text Available This work is focused on the monitoring of Staphylococcus aureus prevalence in raw milk and milk filters, its antibiotic resistance and detection of methicillin resistant Staphylococcus aureus (MRSA. Samples of raw cow´s milk and milk filters were collected in the period from 2012 till 2014, from 50 dairy farms in the Czech Republic. The total of 261 samples (164 samples of raw milk and 97 milk filters were cultivated on Baird-Parker agar. Both the typical and atypical colonies were examined by plasmacoagulase test and PCR method was used for detection of species specific fragment SA442 and mecA gene. Standard disk diffusion method was used to determinate resistance to antimicrobial agents. The bacterium Staphylococcus aureus was detected on 25 farms (50%. The antimicrobial resistance showed differences between the farms. Total of 58 samples were positive for Staphylococcus aureus, of which were 37 (14.2% isolated from raw milk samples and 21 (8.1% from milk filters. From these samples we isolated 62 Staphylococcus aureus strains, 41 isolates bacteria S. aureus from raw milk (66.1% and 21 isolates S. aureus from milk filters (33.9%. The presence of antibiotic resistance in Staphylococcus aureus isolates was low, most of them were resistant to amoxicilin. According to the results obtained by the PCR method for the methicillin - resistant S. aureus (MRSA, the mecA gene was present in 6 strains (9.7%, 4 isolates obtained from milk samples (6.5% and 2 isolates from milk filters (3.2%.  These isolates can be considered as a possible source of resistance genes, which can be spread through the food chain. Nowadays, a globally unfavourable increasing trend of prevalence of methicillin resistant staphylococci strains especially Staphylococcus aureus is being observed worldwide. The improper hygiene and poor farm management practices contributed to the presence of S. aureus in the milk. This may have contributed to the high level of S. aureus isolated

Comparison of automated processing of flocked swabs with manual processing of fiber swabs for detection of nasal carriage of Staphylococcus aureus.

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Jones, Gillian; Matthews, Roger; Cunningham, Richard; Jenks, Peter

2011-07-01

The sensitivity of automated culture of Staphylococcus aureus from flocked swabs versus that of manual culture of fiber swabs was prospectively compared using nasal swabs from 867 patients. Automated culture from flocked swabs significantly increased the detection rate, by 13.1% for direct culture and 10.2% for enrichment culture.

Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus.

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Pragman, Alexa A; Yarwood, Jeremy M; Tripp, Timothy J; Schlievert, Patrick M

2004-04-01

Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

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Kellie Burnside

2010-06-01

Full Text Available Exotoxins, including the hemolysins known as the alpha (alpha and beta (beta toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1 were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1 increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU, serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE and a hypothetical protein (NWMN_1123 were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

Prevalence of methicillin resistant Staphylococcus aureus in Lumbini Medical College and Teaching Hospital, Palpa, Western Nepal.

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Raut, Shristi; Bajracharya, Kishor; Adhikari, Janak; Pant, Sushama Suresh; Adhikari, Bipin

2017-06-02

Multidrug resistant Staphylococcus aureus is common in both tertiary and primary health care settings. Emergence of methicillin resistance in S. aureus (MRSA) along with macrolide, lincosamide, streptogramin B (MLSB) has made treatment of Staphylococcal infection more challenging. The main objective of this study was to detect MRSA, MLSB (inducible; MLSBi and constitutive; MLSBc) resistant S. aureus using phenotypic methods and to determine their antibiogram. Various samples were collected from 1981 patients who attended Lumbini Medical College and Teaching Hospital (LMCTH) during the period of 6 months from September 2015 to February 2016. Out of a total of 1981 samples, 133 S. aureus were isolated. Cefoxitin was used to detect MRSA by the disk diffusion test. Inducible clindamycin resistance (MLSBi) was detected by the D-zone test. The antibiotic profile of all isolates was tested by a modified Kirby Bauer disk diffusion method. Among 133 S. aureus, there were 58 (43.6%) MRSA, 34 (25.6%) MLSBi and 30 (22.6%) MLSBc. Of a total of 64 MLSB, a significant proportion (62.5%) was MRSA (p aureus, MRSA showed significant resistance to 9 (p resistance to multiple antibiotics (p resistance profiles from this study can optimize the treatment of multi-drug resistant S. aureus.

Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus

OpenAIRE

Cao, Xiaoxiao; Li, Shaohua; Chen, Liucun; Ding, Hongmei; Xu, Hua; Huang, Yanping; Li, Jie; Liu, Nongle; Cao, Weihong; Zhu, Yanjun; Shen, Beifen; Shao, Ningsheng

2009-01-01

In this article, a panel of ssDNA aptamers specific to Staphylococcus aureus was obtained by a whole bacterium-based SELEX procedure and applied to probing S. aureus. After several rounds of selection with S. aureus as the target and Streptococcus and S. epidermidis as counter targets, the highly enriched oligonucleic acid pool was sequenced and then grouped under different families on the basis of the homology of the primary sequence and the similarity of the secondary structure. Eleven sequ...

Identification and characterization of sigma, a novel component of the Staphylococcus aureus stress and virulence responses.

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Lindsey N Shaw

Full Text Available S. aureus is a highly successful pathogen that is speculated to be the most common cause of human disease. The progression of disease in S. aureus is subject to multi-factorial regulation, in response to the environments encountered during growth. This adaptive nature is thought to be central to pathogenesis, and is the result of multiple regulatory mechanisms employed in gene regulation. In this work we describe the existence of a novel S. aureus regulator, an as yet uncharacterized ECF-sigma factor (sigma(S, that appears to be an important component of the stress and pathogenic responses of this organism. Using biochemical approaches we have shown that sigma(S is able to associates with core-RNAP, and initiate transcription from its own coding region. Using a mutant strain we determined that sigma(S is important for S. aureus survival during starvation, extended exposure to elevated growth temperatures, and Triton X-100 induced lysis. Coculture studies reveal that a sigma(S mutant is significantly outcompeted by its parental strain, which is only exacerbated during prolonged growth (7 days, or in the presence of stressor compounds. Interestingly, transcriptional analysis determined that under standard conditions, S. aureus SH1000 does not initiate expression of sigS. Assays performed hourly for 72 h revealed expression in typically background ranges. Analysis of a potential anti-sigma factor, encoded downstream of sigS, revealed it to have no obvious role in the upregulation of sigS expression. Using a murine model of septic arthritis, sigS-mutant infected animals lost significantly less weight, developed septic arthritis at significantly lower levels, and had increased survival rates. Studies of mounted immune responses reveal that sigS-mutant infected animals had significantly lower levels of IL-6, indicating only a weak immunological response. Finally, strains of S. aureus lacking sigS were far less able to undergo systemic dissemination

Pseudomonas aeruginosa induces pigment production and enhances virulence in a white phenotypic variant of Staphylococcus aureus

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Antonic V

2013-11-01

Full Text Available Vlado Antonic,1–3 Alexander Stojadinovic,3–5 Binxue Zhang,1–3 Mina J Izadjoo,1–3,5 Mohammad Alavi1–3 1Henry M Jackson Foundation for the Advancement of Military Medicine, Bethesda, MD, USA; 2Diagnostic and Translational Research Center, Gaithersburg, MD, USA; 3Combat Wound Initiative Program, Bethesda, MD, USA; 4Walter Reed National Military Medical Center, Bethesda, MD, USA; 5Uniformed Services University of the Health Sciences, Bethesda, MD, USA Abstract: Staphyloxanthin is a virulence factor which protects Staphylococcus aureus in stress conditions. We isolated two pigment variants of S. aureus and one strain of Pseudomonas aeruginosa from a single wound infection. S. aureus variants displayed white and yellow colony phenotypes. The sequence of the operons for staphyloxanthin synthesis indicated that coding and promoter regions were identical between the two pigment variants. Quorum sensing controls pigment synthesis in some bacteria. It is also shown that P. aeruginosa quorum-sensing molecules affect S. aureus transcription. We explored whether the co-infecting P. aeruginosa can affect pigment production in the white S. aureus variant. In co-culture experiments between the white variants and a selected number of Gram-positive and Gram-negative bacteria, only P. aeruginosa induced pigment production in the white variant. Gene expression analysis of the white variant did not indicate upregulation of the crtM and other genes known to be involved in pigment production (sigB, sarA, farnesyl pyrophosphate synthase gene [FPP-synthase], hfq. In contrast, transcription of the catalase gene was significantly upregulated after co-culture. P. aeruginosa-induced pigment synthesis and catalase upregulation correlated with increased resistance to polymyxin B, hydrogen peroxide, and the intracellular environment of macrophages. Our data indicate the presence of silent but functional staphyloxanthin synthesis machinery in a white phenotypic variant

Staphylococcus aureus in Some Brazilian Dairy Industries: Changes of Contamination and Diversity

DEFF Research Database (Denmark)

Dittmann, Karen Kiesbye; Chaul, Luiza T.; Lee, Sarah H. I.

2017-01-01

Staphylococcus aureus, a major food-poisoning pathogen, is a common contaminant in dairy industries worldwide, including in Brazil. We determined the occurrence of S. aureus in five dairies in Brazil over 8 months. Of 421 samples, 31 (7.4%) were positive for S. aureus and prevalence varied from 0.......8% of strains being sensitive to all antibiotic classes and no Methicillin-resistant S. aureus (MRSA) strains were found. The enterotoxin-encoding genes involved in food-poisoning, e.g., sea, sed, see, and seg were targeted by PCR. The two toxin-encoding genes, sed and see, were not detected. Only three strains...... contamination....

Expression of multidrug resistance efflux pump gene norA is iron responsive in Staphylococcus aureus.

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Deng, Xin; Sun, Fei; Ji, Quanjiang; Liang, Haihua; Missiakas, Dominique; Lan, Lefu; He, Chuan

2012-04-01

Staphylococcus aureus utilizes efflux transporter NorA to pump out a wide range of structurally dissimilar drugs, conferring low-level multidrug resistance. The regulation of norA expression has yet to be fully understood although past studies have revealed that this gene is under the control of the global transcriptional regulator MgrA and the two-component system ArlRS. To identify additional regulators of norA, we screened a transposon library in strain Newman expressing the transcriptional fusion norA-lacZ for altered β-galactosidase activity. We identify a transposon insertion in fhuB, a gene that encodes a ferric hydroxamate uptake system permease, and propose that the norA transcription is iron responsive. In agreement with this observation, addition of FeCl(3) repressed the induction of norA-lacZ, suggesting that bacterial iron uptake plays an important role in regulating norA transcription. In addition, a fur (ferric uptake regulator) deletion exhibited compromised norA transcription and reduced resistance to quinolone compared to the wild-type strain, indicating that fur functions as a positive regulator of norA. A putative Fur box identified in the promoter region of norA was confirmed by electrophoretic mobility shift and DNase I footprint assays. Finally, by employing a siderophore secretion assay, we reveal that NorA may contribute to the export of siderophores. Collectively, our experiments uncover some novel interactions between cellular iron level and norA regulation in S. aureus.

Methicillin-Resistant Staphylococcus aureus (MRSA) Detected at Four U.S. Wastewater Treatment Plants

OpenAIRE

Goldstein, Rachel E. Rosenberg; Micallef, Shirley A.; Gibbs, Shawn G.; Davis, Johnnie A.; He, Xin; George, Ashish; Kleinfelter, Lara M.; Schreiber, Nicole A.; Mukherjee, Sampa; Sapkota, Amir; Joseph, Sam W.; Sapkota, Amy R.

2012-01-01

Background: The incidence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infections is increasing in the United States, and it is possible that municipal wastewater could be a reservoir of this microorganism. To date, no U.S. studies have evaluated the occurrence of MRSA in wastewater. Objective: We examined the occurrence of MRSA and methicillin-susceptible S. aureus (MSSA) at U.S. wastewater treatment plants. Methods: We collected wastewater samples from two Mid...

Rapid detection of methicillin-resistant Staphylococcus aureus directly from clinical samples: methods, effectiveness and cost considerations

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Stürenburg, Enno

2009-07-01

Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA isolates is a serious public health problem whose ever-increasing rate is commensurate with the pressure it is exerting on the healthcare system. At present, more than 20% of clinical S. aureus isolates in German hospitals are methicillin resistant. Strategies from low-prevalence countries show that this development is not necessarily inevitable. In the Scandinavian countries and the Netherlands, thanks to a rigorous prevention programme, MRSA prevalence has been kept at an acceptably low level (<1–3%. Central to these ‘search and destroy’ control strategies is an admission screening using several MRSA swabs taken from mucocutaneous colonisation sites of high-risk patients (‘MRSA surveillance’. It has also been reported that the speed with which MRSA carriage is detected has an important role to play, as it is a key component of any effective strategy to prevent the pathogen from spreading. Since MRSA culturing involves a 2–3 day delay before the final results are available, rapid detection techniques (commonly referred to as ‘MRSA rapid tests’ using PCR methods and, most recently, rapid culturing methods have been developed. The implementation of rapid tests reduces the time of detection of MRSA carriers from 48–72 to 2–5 h. Clinical evaluation data have shown that MRSA can thus be detected with very high sensitivity. Specificity however is sometimes impaired due to false-positive PCR signals occurring in mixed flora specimens. In order to rule out any false-positive PCR results, a culture screen must always be carried out simultaneously.The data provide preliminary evidence that a PCR assay can reduce nosocomial MRSA transmission in high-risk patients or high-risk areas, whereas an approach that screens all patients admitted to the hospital is probably not effective. Information concerning the cost-effectiveness of rapid MRSA tests is still sparse and thus the issue remains

Detection of Oxacillin Resistance in Staphylococcus aureus Isolated from the Neonatal and Pediatric Units of a Brazilian Teaching Hospital

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Valéria Cataneli Pereira

2009-01-01

Full Text Available Objective: To determine, by phenotypic and genotypic methods, oxacillin susceptibility in Staphylococcus aureus strains isolated from pediatric and neonatal intensive care unit patients seen at the University Hospital of the Botucatu School of Medicine.Methods: A total of 100 S. aureus strains isolated from the following materials were studied: 25 blood cultures, 21 secretions, 12 catheters, 3 cannulae and one chest drain from 62 patients in the neonatal unit, and 36 blood cultures, one pleural fluid sample and one peritoneal fluid sample from 38 patients in the pediatric unit. Resistance of the S. aureus isolates to oxacillin was evaluated by the disk diffusion method with oxacillin (1 μg and cefoxitin (30 μg, agar screening test using Mueller-Hinton agar supplemented with 6 μg/ml oxacillin and 4% NaCl, and detection of the mecA gene by PCR. In addition, the isolates were tested for β-lactamase production using disks impregnated with Nitrocefin and hyperproduction of β-lactamase using amoxicillin (20 μg and clavulanic acid (10 μg disks.Results: Among the 100 S. aureus strains included in the study, 18.0% were resistant to oxacillin, with 16.1% MRSA being detected in the neonatal unit and 21.0% in the pediatric unit. The oxacillin (1 μg and cefoxitin (30 μg disk diffusion methods presented 94.4% and 100% sensitivity, respectively, and 98.8% specificity. The screening test showed 100% sensitivity and 98.8% specificity. All isolates produced β-lactamase and one of these strains was considered to be a hyperproducer.Conclusions: The 30 μg cefoxitin disk diffusion method presented the best result when compared to the 1 μg oxacillin disk. The sensitivity of the agar screening test was similar to that of the cefoxitin disk diffusion method and higher than that of the oxacillin disk diffusion method. We observed variations in the percentage of oxacillin-resistant isolates during the study period, with a decline over the last years which

Potential use of 68Ga-apo-transferrin as a PET imaging agent for detecting Staphylococcus aureus infection

International Nuclear Information System (INIS)

Kumar, Vijay; Boddeti, Dilip K.; Evans, Scott G.; Roesch, Frank; Howman-Giles, Robert

2011-01-01

Introduction: 67 Ga citrate has been extensively used to detect infection and inflammation since 1971. However, its clinical utility is compromised due to several limitations. The present project explored whether 68 Ga-apo-transferrin ( 68 Ga-TF), when prepared in vitro, is a useful agent for positron emission tomography (PET) imaging of bacterial infection. Methods: An infection was induced in male Wistar rats by injecting 5x10 5 CFU units of Staphyococcus aureus in the right thigh muscle. 68 Ga-TF was synthesized by mixing 68 GaCl 3 with apo-transferrin (TF, 2 mg) in sodium carbonate (0.1 M, pH 7.0) and incubating at 40 o C for 1 h. Animals were injected with 10-15 MBq of 68 Ga-TF containing approximately 0.2 mg TF and imaged at different time intervals using Siemens Biograph PET-CT. Results: When 68 Ga-TF were injected in the infected rats, the infection lesion was detectable within 20 min post injection. The biodistribution showed the uptake at the lesion increased with time as shown by significantly increased standard uptake values for up to 4 h post injection. There was a considerable decrease in the background activity during the same period of study, giving higher target-to-muscle ratios. Blood pool activity at 3 h post injection was insignificant. 68 GaCl 3 (when not conjugated to TF) did not localize at the infection lesion up to 120 min post injection. Conclusion: The preliminary results suggest that 68 Ga-TF is capable of detecting S. aureus infection in the rat model, within an hour after intravenous injection.

The Frequency of Staphylococcus aureus Isolated from Endocervix of Infertile Women in Northwest Iran

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Akhi Mohammad Taghi

2017-01-01

Full Text Available Background Infertility is one of the major social issues. Due to the asymptomatic cervical infection associated with Staphylococcus aureus (S. aureus, the majority of patients remain undiagnosed. The present study intended to assess the frequency of S. aureus isolated from infertile women’s endocervix in northwest Iran. Materials and Methods In a descriptive cross sectional study, specimens were randomly collected during vagina examination using a sterile speculum and swabbing. After performance of antibiotic susceptibility testing, polymerase chain reaction (PCR was used to identify methicillin-resistance S. aureus (MRSA and toxic shock syndrome toxin-1 (TSST-1. Results About 26 (26% and 9 (9% women’s urogenital tracts were colonized by S. aureus and Candida spp., respectively, of which three (11.5% patients were infected with fungi and S. aureus, simultaneously. Antibiotic susceptibility results showed high activity of vancomycin and co-trimoxazole on isolates. Regarding PCR results, mecA sequences were detected in 7 (26.9% strains, whilst the tst gene encoding TSST-1 was not detected in any of clinical strains. Conclusion The prevalence of S. aureus was very high in infertile women. Therefore, it demands all patients undergoing infertility treatment to be investigated thoroughly for this type of infection.

Breast Milk Is a Potential Reservoir for Livestock-Associated Staphylococcus aureus and Community-Associated Staphylococcus aureus in Shanghai, China

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Xiaoliang Li

2018-01-01

Full Text Available Breast milk is the first choice in feeding newborn infants and provides multiple benefits for their growth and development. Staphylococcus aureus usually exists in breast milk and is considered one of the most important causative infective agents. To be effective in preventing and controlling S. aureus infections among infants, the aim of this study was to determine the occurrence and molecular characteristics of S. aureus isolated from 1102 samples of breast milk between 2015 and 2016 in Shanghai, China. Out of 71 S. aureus strains isolated, 15 (21.1%, 15/71 were MRSA and all the strains were characterized by spa typing, Multi-Locus Sequence Typing, SCCmec typing, antibiotic resistance testing and virulence-associated genes. A total of 18 distinct sequence types (STs and 36 spa types were identified within the 71 isolates, among which the most frequently represented was ST398 (19.7%, 14/71, followed by ST7 (18.3%, 13/71, ST59 (16.9%, 12/71. The three predominant STs accounted for more than one half of all S. aureus isolates. The most prevalent spa types were t091 (12.7%, 9/71, followed by t571 (8.5%, 6/71, t189 (7.0%, 5/71, t034 (5.6%, 4/71, t437 (5.6%, 4/71, and t701 (4.2%, 3/71. All MRSA isolates belonged to SCCmec IV and V, accounting for 66.7 and 33.3% respectively. Notably, 23 (32.4% S. aureus strains were multidrug resistance (MDR, including 4 (5.6% MRSA and 19 (26.8% MSSA strains, and MDR isolates were mostly resistant to penicillin, erythromycin and clindamycin. All isolates exhibited simultaneous carriage of at least 5 of 33 possible virulence genes and the most prevalent genes detected were icaA (100%, clfA (100%, hla (100%, sdrC (94.4%, hlg2 (88.7%, lukE (57.8%. 39 (54.9%, 39/71 isolates, including 9 (12.7% of MRSA isolates, harbored ≥10 tested virulence genes evaluated in this study. The pvl gene was detected in 8 strains, which represented 5 different STs, with ST59 being the most one. Overall, our findings showed that S. aureus

Characterization of Foodborne Strains of Staphylococcus aureus by Shotgun Proteomics: Functional Networks, Virulence Factors and Species-Specific Peptide Biomarkers

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Carrera, Mónica; Böhme, Karola; Gallardo, José M.; Barros-Velázquez, Jorge; Cañas, Benito; Calo-Mata, Pilar

2017-01-01

In the present work, we applied a shotgun proteomics approach for the fast and easy characterization of 20 different foodborne strains of Staphylococcus aureus (S. aureus), one of the most recognized foodborne pathogenic bacteria. A total of 644 non-redundant proteins were identified and analyzed via an easy and rapid protein sample preparation procedure. The results allowed the differentiation of several proteome datasets from the different strains (common, accessory, and unique datasets), which were used to determine relevant functional pathways and differentiate the strains into different Euclidean hierarchical clusters. Moreover, a predicted protein-protein interaction network of the foodborne S. aureus strains was created. The whole confidence network contains 77 nodes and 769 interactions. Most of the identified proteins were surface-associated proteins that were related to pathways and networks of energy, lipid metabolism and virulence. Twenty-seven virulence factors were identified, and most of them corresponded to autolysins, N-acetylmuramoyl-L-alanine amidases, phenol-soluble modulins, extracellular fibrinogen-binding proteins and virulence factor EsxA. Potential species-specific peptide biomarkers were screened. Twenty-one species-specific peptide biomarkers, belonging to eight different proteins (nickel-ABC transporter, N-acetylmuramoyl-L-alanine amidase, autolysin, clumping factor A, gram-positive signal peptide YSIRK, cysteine protease/staphopain, transcriptional regulator MarR, and transcriptional regulator Sar-A), were proposed to identify S. aureus. These results constitute the first major dataset of peptides and proteins of foodborne S. aureus strains. This repository may be useful for further studies, for the development of new therapeutic treatments for S. aureus food intoxications and for microbial source-tracking in foodstuffs. PMID:29312172

Antimicrobial resistance and mecA characterization of methicillin-susceptible S. aureus and non-S. aureus of beef meat origin in Egypt

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Kamelia Mahmoud Osman

2016-02-01

Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA have been found in various farm animal species throughout the world. Yet, methicillin-susceptible S. aureus (MSSA, methicillin-susceptible non-S. aureus (MS-NSA and methicillin-resistant non-S. aureus (MR-NSA were not investigated. Therefore, we persued to determine the diversity in theirphenotypic virulence assay, phenotypic antimicrobial resistance profile and molecular characterisation in one of the food chains in Egypt. Samples were collected during 2013 from beef meat at retail. Twenty seven isolates comprising five species (S. hyicus, S. aureus, S. schleiferi subsp. coagulans, S. intermedius and S. lentus were characterized for their antibiotic resistance phenotypic profile and antibiotic resistance genes (mecA, cfr, gyrA, gyrB and grlA. Out of the 27 Staphylococcus isolates only one isolate was resistant to the 12 antibiotics representing nine classes. Raw beef meat sold across the Great Cairo zone, contains 66.7% of MRS, with highest prevalence was reported in S. aureus (66.7%, while the MRS non-S. aureus strains constituted 66.7% from which S. hyicus (60%, S. intermedius (33.3%, S. schleiferi subsp. coagulans (100% and S. lentus (100% were MRS. Seven S. aureus, six S. hyicus, four S. schleiferi subsp. coagulans, three S. intermedius and one S. lentus isolates although being resistant to oxacillin yet, 11/27 (40.7% carried the mecA gene. At the same time, the cfr gene was present in 2 of the nine S. aureus isolates, and totally undetectable in S. hyicus, S. schleiferi subsp. coagulans, S. intermedius and S. lentus. Although global researches largely focused into MRSA and MR-NSA in animals on pigs, the analysis of our results stipulates that buffaloes and cattle could be MRSA dispersers and that this theme is not specific to pigs. Detection of MSSA virulence determinants is a must, as although oxacillin resistance may be absent yet, the MSSA may carry the virulence determinants which could

Detection and Prevalence of Penicillin-Susceptible Staphylococcus aureus in the United States in 2013

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Doern, G. V.; Heilmann, K. P.; Miner, S.; Tendolkar, S.; Riahi, F.; Diekema, D. J.

2016-01-01

Using blaZ PCR as the “gold standard,” the sensitivities of CLSI penicillin zone edge and nitrocefin-based tests for β-lactamase production in Staphylococcus aureus were 64.5% and 35.5%, respectively, with specificity of 99.8% for both methods. In 2013, 13.5% of 3,083 S. aureus isolates from 31 U.S. centers were penicillin susceptible. PMID:26763960

Opposing roles of Toll-like receptor and cytosolic DNA-STING signaling pathways for Staphylococcus aureus cutaneous host defense.

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Philip O Scumpia

2017-07-01

Full Text Available Successful host defense against pathogens requires innate immune recognition of the correct pathogen associated molecular patterns (PAMPs by pathogen recognition receptors (PRRs to trigger the appropriate gene program tailored to the pathogen. While many PRR pathways contribute to the innate immune response to specific pathogens, the relative importance of each pathway for the complete transcriptional program elicited has not been examined in detail. Herein, we used RNA-sequencing with wildtype and mutant macrophages to delineate the innate immune pathways contributing to the early transcriptional response to Staphylococcus aureus, a ubiquitous microorganism that can activate a wide variety of PRRs. Unexpectedly, two PRR pathways-the Toll-like receptor (TLR and Stimulator of Interferon Gene (STING pathways-were identified as dominant regulators of approximately 95% of the genes that were potently induced within the first four hours of macrophage infection with live S. aureus. TLR signaling predominantly activated a pro-inflammatory program while STING signaling activated an antiviral/type I interferon response with live but not killed S. aureus. This STING response was largely dependent on the cytosolic DNA sensor cyclic guanosine-adenosine synthase (cGAS. Using a cutaneous infection model, we found that the TLR and STING pathways played opposite roles in host defense to S. aureus. TLR signaling was required for host defense, with its absence reducing interleukin (IL-1β production and neutrophil recruitment, resulting in increased bacterial growth. In contrast, absence of STING signaling had the opposite effect, enhancing the ability to restrict the infection. These results provide novel insights into the complex interplay of innate immune signaling pathways triggered by S. aureus and uncover opposing roles of TLR and STING in cutaneous host defense to S. aureus.

Detection and enumeration of Staphylococcus aureus and Streptococcus agalactiae in milk by Real-Time PCR

OpenAIRE

Aline Gerato Dibbern

2015-01-01

O presente trabalho foi organizado em dois estudos. O objetivo do primeiro estudo foi determinar o efeito da infecção intramamária (IIM) causada por S. aureus e S. agalactiae na contagem de células somáticas (CCS) e na composição do leite (gordura, proteína, lactose, sólidos totais e extrato seco desengordurado), o efeito da contagem de S. aureus e S. agalactiae sobre a composição do leite e a estimativa da frequência de vacas e de quartos infectados com S. aureus e S. agalactiae por meio da ...

Detecting novel low-abundant transcripts in Drosophila

DEFF Research Database (Denmark)

Lee, Sanggyu; Bao, Jingyue; Zhou, Guolin

2005-01-01

Increasing evidence suggests that low-abundant transcripts may play fundamental roles in biological processes. In an attempt to estimate the prevalence of low-abundant transcripts in eukaryotic genomes, we performed a transcriptome analysis in Drosophila using the SAGE technique. We collected 244......,313 SAGE tags from transcripts expressed in Drosophila embryonic, larval, pupae, adult, and testicular tissue. From these SAGE tags, we identified 40,823 unique SAGE tags. Our analysis showed that 55% of the 40,823 unique SAGE tags are novel without matches in currently known Drosophila transcripts...... in the Drosophila genome. Our study reveals the presence of a significant number of novel low-abundant transcripts in Drosophila, and highlights the need to isolate these novel low-abundant transcripts for further biological studies. Udgivelsesdato: 2005-Jun...

MRI Visualization of Staphyloccocus aureus-Induced Infective Endocarditis in Mice

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Ring, Janine; Hoerr, Verena; Tuchscherr, Lorena; Kuhlmann, Michael T.; Löffler, Bettina; Faber, Cornelius

2014-01-01

Infective endocarditis (IE) is a severe and often fatal disease, lacking a fast and reliable diagnostic procedure. The purpose of this study was to establish a mouse model of Staphylococcus aureus-induced IE and to develop a MRI technology to characterize and diagnose IE. To establish the mouse model of hematogenous IE, aortic valve damage was induced by placing a permanent catheter into right carotid artery. 24 h after surgery, mice were injected intravenously with either iron particle-labeled or unlabeled S. aureus (strain 6850). To distinguish the effect of IE from mere tissue injury or recruited macrophages, subgroups of mice received sham surgery prior to infection (n = 17), received surgery without infection (n = 8), or obtained additionally injection of free iron particles to label macrophages (n = 17). Cardiac MRI was performed 48 h after surgery using a self-gated ultra-short echo time (UTE) sequence (TR/TE, 5/0.31 ms; in-plane/slice, 0.125/1 mm; duration, 12∶08 min) to obtain high-resolution, artifact-free cinematographic images of the valves. After MRI, valves were either homogenized and plated on blood agar plates for determination of bacterial titers, or sectioned and stained for histology. In the animal model, both severity of the disease and mortality increased with bacterial numbers. Infection with 105 S. aureus bacteria reliably caused endocarditis with vegetations on the valves. Cinematographic UTE MRI visualised the aortic valve over the cardiac cycle and allowed for detection of bacterial vegetations, while mere tissue trauma or labeled macrophages were not detected. Iron labeling of S. aureus was not required for detection. MRI results were consistent with histology and microbial assessment. These data showed that S. aureus-induced IE in mice can be detected by MRI. The established mouse model allows for investigation of the pathophysiology of IE, testing of novel drugs and may serve for the development of a clinical diagnostic

Mechanisms of antibiotic resistance in Staphylococcus aureus.

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Pantosti, Annalisa; Sanchini, Andrea; Monaco, Monica

2007-06-01

Staphylococcus aureus can exemplify better than any other human pathogen the adaptive evolution of bacteria in the antibiotic era, as it has demonstrated a unique ability to quickly respond to each new antibiotic with the development of a resistance mechanism, starting with penicillin and methicillin, until the most recent, linezolid and daptomycin. Resistance mechanisms include enzymatic inactivation of the antibiotic (penicillinase and aminoglycoside-modification enzymes), alteration of the target with decreased affinity for the antibiotic (notable examples being penicillin-binding protein 2a of methicillin-resistant S. aureus and D-Ala-D-Lac of peptidoglycan precursors of vancomycin-resistant strains), trapping of the antibiotic (for vancomycin and possibly daptomycin) and efflux pumps (fluoroquinolones and tetracycline). Complex genetic arrays (staphylococcal chromosomal cassette mec elements or the vanA operon) have been acquired by S. aureus through horizontal gene transfer, while resistance to other antibiotics, including some of the most recent ones (e.g., fluoroquinolones, linezolid and daptomycin) have developed through spontaneous mutations and positive selection. Detection of the resistance mechanisms and their genetic basis is an important support to antibiotic susceptibility surveillance in S. aureus.

Intracellular Detection of Viral Transcription and Replication Using RNA FISH

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2016-05-26

Chapter 14. Intracellular detection of viral transcription and replication using RNA FISH i. Summary/Abstract Many hemorrhagic fever viruses...only allow entirely new investigations into the replication of these viruses, but also how this method can be applied to any virus with a known...localization, TurboFISH, hemorrhagic fever virus replication 1. Introduction RNA FISH was developed as a method to visualize cellular RNA by binding a

Deciphering Transcriptome and Complex Alternative Splicing Transcripts in Mammary Gland Tissues from Cows Naturally Infected with Staphylococcus aureus Mastitis

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Jiang, Qiang; Yang, Chun Hong; Zhang, Yan; Sun, Yan; Li, Rong Ling; Wang, Chang Fa; Zhong, Ji Feng; Huang, Jin Ming

2016-01-01

Alternative splicing (AS) contributes to the complexity of the mammalian proteome and plays an important role in diseases, including infectious diseases. The differential AS patterns of these transcript sequences between the healthy (HS3A) and mastitic (HS8A) cows naturally infected by Staphylococcus aureus were compared to understand the molecular mechanisms underlying mastitis resistance and susceptibility. In this study, using the Illumina paired-end RNA sequencing method, 1352 differentially expressed genes (DEGs) with higher than twofold changes were found in the HS3A and HS8A mammary gland tissues. Gene ontology and KEGG pathway analyses revealed that the cytokine–cytokine receptor interaction pathway is the most significantly enriched pathway. Approximately 16k annotated unigenes were respectively identified in two libraries, based on the bovine Bos taurus UMD3.1 sequence assembly and search. A total of 52.62% and 51.24% annotated unigenes were alternatively spliced in term of exon skipping, intron retention, alternative 5′ splicing and alternative 3ʹ splicing. Additionally, 1,317 AS unigenes were HS3A-specific, whereas 1,093 AS unigenes were HS8A-specific. Some immune-related genes, such as ITGB6, MYD88, ADA, ACKR1, and TNFRSF1B, and their potential relationships with mastitis were highlighted. From Chromosome 2, 4, 6, 7, 10, 13, 14, 17, and 20, 3.66% (HS3A) and 5.4% (HS8A) novel transcripts, which harbor known quantitative trait locus associated with clinical mastitis, were identified. Many DEGs in the healthy and mastitic mammary glands are involved in immune, defense, and inflammation responses. These DEGs, which exhibit diverse and specific splicing patterns and events, can endow dairy cattle with the potential complex genetic resistance against mastitis. PMID:27459697

Reversal of methicillin resistance in Staphylococcus aureus by thioridazine

DEFF Research Database (Denmark)

Klitgaard, Janne K; Skov, Marianne N; Kallipolitis, Birgitte H

2008-01-01

of thioridazine in the presence of a fixed amount of oxacillin. Furthermore, the protein level of PBP2a was reduced when bacteria were treated with the combination of oxacillin and thioridazine. The two drugs also affected the mRNA level of the beta-lactamase gene, blaZ. Conclusions The present study indicates......Objectives Thioridazine has been shown to reverse oxacillin resistance in methicillin-resistant Staphylococcus aureus (MRSA) in vitro. The aim of this study was to investigate whether thioridazine alone or in combination with oxacillin affects the transcription of the methicillin resistance gene...... blotting in the presence of thioridazine and oxacillin. Results We observed an increased susceptibility of MRSA towards oxacillin in the presence of thioridazine compared with bacteria grown with oxacillin or thioridazine alone. Transcription of mecA was reduced with increasing concentrations...

ω-Hydroxyemodin Limits Staphylococcus aureus Quorum Sensing-Mediated Pathogenesis and Inflammation

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Daly, Seth M.; Elmore, Bradley O.; Kavanaugh, Jeffrey S.; Triplett, Kathleen D.; Figueroa, Mario; Raja, Huzefa A.; El-Elimat, Tamam; Crosby, Heidi A.; Femling, Jon K.; Cech, Nadja B.; Horswill, Alexander R.; Oberlies, Nicholas H.

2015-01-01

Antibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing. Staphylococcus aureus is a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures of Penicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM prevented agr signaling by all four S. aureus agr alleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by the agr operon, preventing the interaction of AgrA with the agr P2 promoter. Importantly, OHM was efficacious in a mouse model of S. aureus SSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing of S. aureus in vitro in an agr-dependent manner. These data suggest that bacterial disarmament through the suppression of S. aureus QS may bolster the host innate immune response and limit inflammation. PMID:25645827

Identification of the Staphylococcus aureus vfrAB operon, a novel virulence factor regulatory locus.

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Bose, Jeffrey L; Daly, Seth M; Hall, Pamela R; Bayles, Kenneth W

2014-05-01

During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis.

The prevalence of methicillin-resistant Staph. aureus strains producing enterotoxin A and B

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Safiyeh Abbasi

2016-02-01

Full Text Available Background: Staphylococcus aureus is a gram positive coccus which is able to cause different kinds of infection in certain condition. The function of this bacteria is to provide the conditions for the invasion of it to the host with the secretion of different sorts of toxins such as Staphylococcus aureus enterotoxin, including important virulence factors that super antigens are all factors digestive inconvenience. Staphylococcus aureus enterotoxin-secreting toxins such conditions provides invasion of host genes. There are different types of SE, but type A enterotoxin (SEA and type B enterotoxin (SEB are the most important types. Therefore, in this study, the prevalence of Staphylococcus aureus toxin-producing enterotoxin genes (SEB, SEA in clinical strains isolated from patients in teaching hospitals of Shahrekord city, Iran, were studied. Methods: This cross-sectional and descriptive study, which was conducted from May 2014 to December 2014. A hundred and ten isolates of Staphylococcus aureus from patients collected over a period of 8 months and were first identified using standard biochemical methods and laboratory. Using standard methods and laboratory tests were identified and compared with the antibiotic oxacillin minimum inhibitory concentration were determined by broth micro dilution, and then they were assessed by polymerase chain reaction (PCR technique. Results: The results indicated that, 110 samples of dairy products infected by Staphylococcus aureus were detected. Two cases (1.8% of these infected samples were carrying both enterotoxin A and enterotoxin B genes. The frequencies of enterotoxin A genes were twenty-six cases (23/6% and The frequencies of enterotoxin B genes were two cases (1/8%, respectively. Conclusion: The detection of enterotoxin A and enterotoxin B genes, shows the most important role they have in bringing about superinfection. The detection of enterotoxin A and B genes, shows the most important role they have in

Contribution of the nos-pdt operon to virulence phenotypes in methicillin-sensitive Staphylococcus aureus.

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April M Sapp

Full Text Available Nitric oxide (NO is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and

The δ subunit of RNA polymerase guides promoter selectivity and virulence in Staphylococcus aureus.

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Weiss, Andy; Ibarra, J Antonio; Paoletti, Jessica; Carroll, Ronan K; Shaw, Lindsey N

2014-04-01

In Gram-positive bacteria, and particularly the Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for more than 30 years for various organisms, but its function is still not well understood. In this study, we investigated its role in the major human pathogen Staphylococcus aureus. We showed conservation of important structural regions of RpoE in S. aureus and other species and demonstrated binding to core RNAP that is mediated by the β and/or β' subunits. To identify the impact of the δ subunit on transcription, we performed transcriptome sequencing (RNA-seq) analysis and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, while several mobile genetic elements (SaPI5 and prophage SA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including alpha toxin, secreted proteases, and Panton-Valentine leukocidin (PVL). We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection.

Prevalence of Staphylococcus aureus and of methicillin-resistant S. aureus clonal complexes in bulk tank milk from dairy cattle herds in Lombardy Region (Northern Italy).

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Cortimiglia, C; Luini, M; Bianchini, V; Marzagalli, L; Vezzoli, F; Avisani, D; Bertoletti, M; Ianzano, A; Franco, A; Battisti, A

2016-10-01

Staphylococcus aureus is the most important causative agent of subclinical mastitis in cattle resulting in reduced milk production and quality. Methicillin-resistant S. aureus (MRSA) strains has a clear zoonotic relevance, especially in the case of occupational exposure. The aim of the study was to evaluate the prevalence of S. aureus and MRSA in bulk tank milk (BTM) from dairy cattle herds in the Lombardy Region (Northern Italy) and to identify the main MRSA circulating genotypes. MRSA strains were characterized by susceptibility testing, multi-locus sequence typing (MLST), spa typing and SCCmec typing. A total 844 BTM samples were analysed and S. aureus and MRSA were detected in 47·2% and 3·8% of dairy herds, respectively. MLST showed that the majority (28/32) of isolates belonged to the typical livestock-associated lineages: ST398, ST97 and ST1. Interestingly, in this study we report for the first time the new ST3211, a single locus variant of ST(CC)22, with the newly described 462 aroE allele. Our study indicates high diffusion of S. aureus mastitis and low, but not negligible, prevalence of MRSA in the considered area, suggesting the need for planning specific control programmes for bovine mastitis caused by S. aureus, especially when MRSA is implicated.

Rapid detection of Prunus necrotic ringspot virus using magnetic nanoparticle-assisted reverse transcription loop-mediated isothermal amplification.

Science.gov (United States)

Zong, Xiaojuan; Wang, Wenwen; Wei, Hairong; Wang, Jiawei; Chen, Xin; Xu, Li; Zhu, Dongzi; Tan, Yue; Liu, Qingzhong

2014-11-01

Prunus necrotic ringspot virus (PNRSV) has seriously reduced the yield of Prunus species worldwide. In this study, a highly efficient and specific two-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed to detect PNRSV. Total RNA was extracted from sweet cherry leaf samples using a commercial kit based on a magnetic nanoparticle technique. Transcripts were used as the templates for the assay. The results of this assay can be detected using agarose gel electrophoresis or by assessing in-tube fluorescence after adding SYBR Green I. The assay is highly specific for PNRSV, and it is more sensitive than reverse-transcription polymerase chain reaction (RT-PCR). Restriction enzyme digestion verified further the reliability of this RT-LAMP assay. To our knowledge, this is the first report of the application of RT-LAMP to PNRSV detection in Prunus species. Copyright © 2014 Elsevier B.V. All rights reserved.

Virulence regulation in Staphylococcus aureus: the need for in vivo analysis of virulence factor regulation.

Science.gov (United States)

Pragman, Alexa A; Schlievert, Patrick M

2004-10-01

Staphylococcus aureus is a pathogenic microorganism that is responsible for a wide variety of clinical infections. These infections can be relatively mild, but serious, life-threatening infections may result from the expression of staphylococcal virulence factors that are coordinated by virulence regulators. Much work has been done to characterize the actions of staphylococcal virulence regulators in broth culture. Recently, several laboratories showed that transcriptional analyses of virulence regulators in in vivo animal models or in human infection did not correlate with transcriptional analyses accomplished in vitro. In describing the differences between in vitro and in vivo transcription of staphylococcal virulence regulators, we hope to encourage investigators to study virulence regulators using infection models whenever possible.

Cyclic peptide inhibitors of the β-sliding clamp in Staphylococcus aureus

DEFF Research Database (Denmark)

Kjelstrup, Susanne; Hansen, Paula Melo Paulon; Thomsen, Line Elnif

2013-01-01

Interaction between pairs of Staphylococcus aureus replication proteins was detected in an Escherichia coli based two-hybrid analysis. A reverse two-hybrid system was constructed for selection of compounds that hindered interaction between interacting protein pairs. A number of cyclic peptides, f....... The minimum inhibitory concentration was ∼50 μg/ml for S. aureus cells. These compounds may serve as lead candidates for future development into novel classes of antibiotics as well as provide information on the function of the S. aureus replication process....

PRODUKSI IGY SPESIFIK STAPHYLOCOCCUS AUREUS DARI ISOLAT ASAL KASUS STAPHYLOCOCCOSIS PADA KELINCI

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Triwardhani Cahyaningsih

2017-04-01

Full Text Available This experiment aims to produce immunoglobulin Y (Ig-Y Staphylococcus aureus isolates origin staphylococcosis case in rabbits. Vaccination is done several times using Staphylococcus aureus active in 109 cfu/ml. Injecting the chicken is done four weeks in a row, the first week is done by intravenous injection of S. aureus antigens, the second week of the injection sub cutan S. aureus emulsified with Freund's complete adjuvant, followed by the third and fourth week of the injection of S. aureus antigen emulsified with incomplete Freund's adjuvant. Egg samples were taken after four weeks of the last injection for identification, purification, and determination of specific IgY against S. aureus in egg yolk. IgY specificity qualitatively tests performed by the AGP test (To Gel Precipitation. IgY extraction is done using PEG - Ammonium sulfate, purified IgY concentration calculated by the method of Bradford. The results showed that IgY began to be detected in egg yolk at week six after immunization, with the average levels of IgY is 1.7 mg/ml.

Relationship and susceptibility profile of Staphylococcus aureus infection diabetic foot ulcers with Staphylococcus aureus nasal carriage.

Science.gov (United States)

Taha, Aza Bahadeen

2013-03-01

Staphylococcus aureus is the main cause of diabetic foot infection with the patient's endogenous flora as the principal source. Nasal carriage of S. aureus has been identified as an important risk factor for the acquisition of diabetic foot infections. The study assessment the associations of S. aureus with methicillin resistant S. aureus were isolation from diabetic foot infection and nasal carriage of the same patients and their antibiotic susceptibility profile. Diagnosis of S. aureus and methicillin resistant S. aureus were carried out by using standard procedures. Antibiotic sensitivity profiles were determent by breakpoint dilution method. Out of 222 S. aureus isolation, 139 (62.61%) were isolated from the diabetic foot and 83 (37.39%) from the nasal carriage. Seventy one (30.87%) of the patients were S. aureus infection diabetic foot with nasal carriage. Among diabetic foot infection and nasal carriage patients, 40.85% of S. aureus were considered as methicillin resistant S. aureus. Rifampicin (96.40%) and Levofloxacin (91.44%) were active against S. aureus. Patients at strong risk for methicillin resistant S. aureus nasal carriage and subsequent diabetic foot infection with high resistance to antibiotics. Copyright © 2012 Elsevier Ltd. All rights reserved.

Identification of pathogenic factors potentially involved in Staphylococcus aureus keratitis using proteomics.

Science.gov (United States)

Khan, Shamila; Cole, Nerida; Hume, Emma B H; Garthwaite, Linda L; Nguyen-Khuong, Terry; Walsh, Bradley J; Willcox, Mark D P

2016-10-01

Staphylococcus is a leading cause of microbial keratitis, characterized by destruction of the cornea by bacterial exoproteins and host-associated factors. The aim of this study was to compare extracellular and cell-associated proteins produced by two different isolates of S. aureus, a virulent clinical isolate (Staph 38) and a laboratory strain (Staphylococcus aureus 8325-4) of weaker virulence in the mouse keratitis model. Proteins were analyzed using 2D polyacrylamide gel electrophoresis and identified by subsequent mass spectrometry. Activity of staphylococcal adhesins was assessed by allowing strains to bind to various proteins adsorbed onto polymethylmethacrylate squares. Thirteen proteins in the extracellular fraction and eight proteins in the cell-associated fractions after bacterial growth were produced in increased amounts in the clinical isolate Staph 38. Four of these proteins were S. aureus virulence factor adhesins, fibronectin binding protein A, staphopain, glyceraldehyde-3-phosphate dehydrogenase 2 and extracellular adherence protein. The clinical isolate Staph 38 adhered to a greater extent to all mammalian proteins tested, indicating the potential of the adhesins to be active on its surface. Other proteins with increased expression in Staph 38 included potential moonlighting proteins and proteins involved in transcription or translation. This is the first demonstration of the proteome of S. aureus isolates from keratitis. These results indicate that the virulent clinical isolate produces more potentially important virulence factors compared to the less virulent laboratory strain and these may be associated with the ability of a S. aureus strain to cause more severe keratitis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Toxigenic profile of methicillin-sensitive and resistant Staphylococcus aureus isolated from special groups.

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de Souza, Camila Sena Martins; Fortaleza, Carlos Magno Castelo Branco; Witzel, Claudia Lima; Silveira, Mônica; Bonesso, Mariana Fávero; Marques, Silvio Alencar; Cunha, Maria de Lourdes Ribeiro de Souza da

2016-02-16

Staphylococcus aureus is characterized by its pathogenicity and high prevalence, causing disease in both healthy and immunocompromised individuals due to its easy dissemination. This fact is aggravated by the widespread dissemination of S. aureus carrying toxigenic genes. The objective of this study was to determine the toxigenic profile of methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in patients with purulent skin and/or soft tissue infections seen at the Dermatology Department of the University Hospital of the Botucatu Medical School, asymptomatic adults older than 60 years living in nursing homes, and prison inmates of the Avaré Detention Center. PCR was used for the detection of the mecA gene, enterotoxin genes (sea, seb, and sec), exfoliative toxins A and B (eta and etb), toxic shock syndrome toxin 1 (tst), panton-valentine leukocidin (lukS-PV and lukF-PV), and alpha- and delta-hemolysins or cytotoxins (hla and hld). The results showed a significant prevalence of toxigenic genes among S. aureus isolates from asymptomatic individuals, with the observation of a higher prevalence of cytotoxin genes. However, the panton-valentine leukocidin gene was only detected in MSSA isolated from patients with skin infections and the tst gene was exclusively found in MSSA isolated from prison inmates. The present study demonstrated a significant prevalence of toxigenic genes in MSSA and MRSA strains isolated from asymptomatic S. aureus carriers. There was a higher prevalence of cytotoxin genes.

In silico detection of sequence variations modifying transcriptional regulation.

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Malin C Andersen

2008-01-01

Full Text Available Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers. The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation.

In Silico Detection of Sequence Variations Modifying Transcriptional Regulation

Science.gov (United States)

Andersen, Malin C; Engström, Pär G; Lithwick, Stuart; Arenillas, David; Eriksson, Per; Lenhard, Boris; Wasserman, Wyeth W; Odeberg, Jacob

2008-01-01

Identification of functional genetic variation associated with increased susceptibility to complex diseases can elucidate genes and underlying biochemical mechanisms linked to disease onset and progression. For genes linked to genetic diseases, most identified causal mutations alter an encoded protein sequence. Technological advances for measuring RNA abundance suggest that a significant number of undiscovered causal mutations may alter the regulation of gene transcription. However, it remains a challenge to separate causal genetic variations from linked neutral variations. Here we present an in silico driven approach to identify possible genetic variation in regulatory sequences. The approach combines phylogenetic footprinting and transcription factor binding site prediction to identify variation in candidate cis-regulatory elements. The bioinformatics approach has been tested on a set of SNPs that are reported to have a regulatory function, as well as background SNPs. In the absence of additional information about an analyzed gene, the poor specificity of binding site prediction is prohibitive to its application. However, when additional data is available that can give guidance on which transcription factor is involved in the regulation of the gene, the in silico binding site prediction improves the selection of candidate regulatory polymorphisms for further analyses. The bioinformatics software generated for the analysis has been implemented as a Web-based application system entitled RAVEN (regulatory analysis of variation in enhancers). The RAVEN system is available at http://www.cisreg.ca for all researchers interested in the detection and characterization of regulatory sequence variation. PMID:18208319

Calcium-chelating alizarin and other anthraquinones inhibit biofilm formation and the hemolytic activity of Staphylococcus aureus.

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Lee, Jin-Hyung; Kim, Yong-Guy; Yong Ryu, Shi; Lee, Jintae

2016-01-14

Staphylococcal biofilms are problematic and play a critical role in the persistence of chronic infections because of their abilities to tolerate antimicrobial agents. Thus, the inhibitions of biofilm formation and/or toxin production are viewed as alternative means of controlling Staphylococcus aureus infections. Here, the antibiofilm activities of 560 purified phytochemicals were examined. Alizarin at 10 μg/ml was found to efficiently inhibit biofilm formation by three S. aureus strains and a Staphylococcus epidermidis strain. In addition, two other anthraquinones purpurin and quinalizarin were found to have antibiofilm activity. Binding of Ca(2+) by alizarin decreased S. aureus biofilm formation and a calcium-specific chelating agent suppressed the effect of calcium. These three anthraquinones also markedly inhibited the hemolytic activity of S. aureus, and in-line with their antibiofilm activities, increased cell aggregation. A chemical structure-activity relationship study revealed that two hydroxyl units at the C-1 and C-2 positions of anthraquinone play important roles in antibiofilm and anti-hemolytic activities. Transcriptional analyses showed that alizarin repressed the α-hemolysin hla gene, biofilm-related genes (psmα, rbf, and spa), and modulated the expressions of cid/lrg genes (the holin/antiholin system). These findings suggest anthraquinones, especially alizarin, are potentially useful for controlling biofilm formation and the virulence of S. aureus.

Mild Staphylococcus aureus Skin Infection Improves the Course of Subsequent Endogenous S. aureus Bacteremia in Mice.

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Sanne van den Berg

Full Text Available Staphylococcus aureus carriers with S. aureus bacteremia may have a reduced mortality risk compared to non-carriers. A role for the immune system is suggested. Here, we study in mice the effect of mild S. aureus skin infection prior to endogenous or exogenous S. aureus bacteremia, and evaluate protection in relation to anti-staphylococcal antibody levels. Skin infections once or twice by a clinical S. aureus isolate (isolate P or S. aureus strain 8325-4 were induced in mice free of S. aureus and anti-staphylococcal antibodies. Five weeks later, immunoglobulin G (IgG levels in blood against 25 S. aureus antigens were determined, and LD50 or LD100 bacteremia caused by S. aureus isolate P was induced. S. aureus skin infections led to elevated levels of anti-staphylococcal IgG in blood. One skin infection improved the course of subsequent severe endogenous bacteremia only. A second skin infection further improved animal survival rate, which was associated with increased pre-bacteremia IgG levels against Efb, IsaA, LukD, LukE, Nuc, PrsA and WTA. In conclusion, S. aureus isolate P skin infection in mice reduces the severity of subsequent endogenous S. aureus bacteremia only. Although cellular immune effects cannot be rules out, anti-staphylococcal IgG against specified antigens may contribute to this effect.

Staphylococcus aureus resistente a la meticilina (SARM)

Centers for Disease Control (CDC) Podcasts

2007-10-22

Datos importantes sobre las infecciones por SARM en Estados Unidos, en las escuelas y los entornos médicos. (Title: Methicillin-resistant Staphylococcus aureus (MRSA)Created: 10/2007).  Created: 10/22/2007 by National Center for Preparedness, Detection, and Control of Infectious Diseases.   Date Released: 11/9/2007.

Genetic and Virulent Difference Between Pigmented and Non-pigmented Staphylococcus aureus.

Science.gov (United States)

Zhang, Jing; Suo, Yujuan; Zhang, Daofeng; Jin, Fangning; Zhao, Hang; Shi, Chunlei

2018-01-01

Staphyloxanthin (STX), a golden carotenoid pigment produced by Staphylococcus aureus , is suggested to act as an important virulence factor due to its antioxidant properties. Restraining biosynthesis of STX was considered as an indicator of virulence decline in pigmented S. aureus isolates. However, it is not clear whether natural non-pigmented S. aureus isolates have less virulence than pigmented ones. In this study, it is aimed to compare the pigmented and non-pigmented S. aureus isolates to clarify the genetic and virulent differences between the two groups. Here, 132 S. aureus isolates were divided into two phenotype groups depending on the absorbance (OD 450 ) of the extracted carotenoids. Then, all isolates were subjected to spa typing and multilocus sequence typing (MLST), and then the detection of presence of 30 virulence factors and the gene integrity of crtN and crtM . Furthermore, 24 typical S. aureus isolates and 4 S. argenteus strains were selected for the murine infection assay of in vivo virulence, in which the histological observation and enumeration of CFUs were carried out. These isolates were distributed in 26 sequence types (STs) and 49 spa types. The pigmented isolates were scattered in 25 STs, while the non-pigmented isolates were more centralized, which mainly belonged to ST20 (59%) and ST25 (13%). Among the 54 non-pigmented isolates, about 20% carried intact crtN and crtM genes. The in vivo assay suggested that comparing with pigmented S. aureus , non-pigmented S. aureus and S. argenteus strains did not show a reduced virulence in murine sepsis models. Therefore, it suggested that there were no significant genetic and virulent differences between pigmented and non-pigmented S. aureus .

Staphylococcus aureus Isolates from Bovine Mastitis in Eight Countries: Genotypes, Detection of Genes Encoding Different Toxins and Other Virulence Genes

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Valentina Monistero

2018-06-01

Full Text Available Staphylococcus aureus is recognized worldwide as one of the major agents of dairy cow intra-mammary infections. This microorganism can express a wide spectrum of pathogenic factors used to attach, colonize, invade and infect the host. The present study evaluated 120 isolates from eight different countries that were genotyped by RS-PCR and investigated for 26 different virulence factors to increase the knowledge on the circulating genetic lineages among the cow population with mastitis. New genotypes were observed for South African strains while for all the other countries new variants of existing genotypes were detected. For each country, a specific genotypic pattern was found. Among the virulence factors, fmtB, cna, clfA and leucocidins genes were the most frequent. The sea and sei genes were present in seven out of eight countries; seh showed high frequency in South American countries (Brazil, Colombia, Argentina, while sel was harboured especially in one Mediterranean country (Tunisia. The etb, seb and see genes were not detected in any of the isolates, while only two isolates were MRSA (Germany and Italy confirming the low diffusion of methicillin resistance microorganism among bovine mastitis isolates. This work demonstrated the wide variety of S. aureus genotypes found in dairy cattle worldwide. This condition suggests that considering the region of interest might help to formulate strategies for reducing the infection spreading.

Direct detection of methicillin resistance in Staphylococcus aureus in blood culture broth by use of a penicillin binding protein 2a latex agglutination test.

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Qian, Qinfang; Venkataraman, Lata; Kirby, James E; Gold, Howard S; Yamazumi, Toshiaki

2010-04-01

We studied the utility of performing a penicillin binding protein 2a latex agglutination (PBP-LA) assay directly on Bactec blood culture broth samples containing Staphylococcus aureus to rapidly detect methicillin resistance. The sensitivity, specificity, positive predictive value, and negative predictive value of this method were 94.1%, 97.5%, 98%, and 92.9%, respectively.

Direct Detection of Methicillin Resistance in Staphylococcus aureus in Blood Culture Broth by Use of a Penicillin Binding Protein 2a Latex Agglutination Test▿

OpenAIRE

Qian, Qinfang; Venkataraman, Lata; Kirby, James E.; Gold, Howard S.; Yamazumi, Toshiaki

2010-01-01

We studied the utility of performing a penicillin binding protein 2a latex agglutination (PBP-LA) assay directly on Bactec blood culture broth samples containing Staphylococcus aureus to rapidly detect methicillin resistance. The sensitivity, specificity, positive predictive value, and negative predictive value of this method were 94.1%, 97.5%, 98%, and 92.9%, respectively.

Crustin protein Amk1 from black tiger shrimp (Penaeus monodon inhibits Vibrio harveyi and Staphylococcus aureus

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Moltira Tonganunt1*

2008-05-01

Full Text Available A crustin gene (Amk1 was identified from a haemocyte library of the black tiger shrimp, Penaeus monodon. The full-length cDNA consists of 411 bp encoding a deduced precursor of 136 amino acids with a signal peptide of 17 aminoacids. Amk1 contains a hydrophobic and a Gly-rich region at the N-terminus and a 12 conserved cysteine domain (6-DSC at the C-terminus. Transcripts of Amk1 are mainly detected in haemocytes and gills by RT-PCR analysis. A recombinant Amk1was overexpressed and purified from Escherichia coli. This has a molecular mass of 43.66 kDa with a predicted pI of 8.23. Antibacterial assays demonstrated that recombinant Amk1 exhibited antibacterial activity against Gram-positive and Gramnegativebacteria with strong inhibition against Staphylococcus aureus and Vibrio harveyi.

Relationship between Vancomycin-Resistant Staphylococcus aureus, Vancomycin-Intermediate S. aureus, High Vancomycin MIC, and Outcome in Serious S. aureus Infections

OpenAIRE

Holmes, Natasha E.; Johnson, Paul D. R.; Howden, Benjamin P.

2012-01-01

Vancomycin has been used successfully for over 50 years for the treatment of Staphylococcus aureus infections, particularly those involving methicillin-resistant S. aureus. It has proven remarkably reliable, but its efficacy is now being questioned with the emergence of strains of S. aureus that display heteroresistance, intermediate resistance, and, occasionally, complete vancomycin resistance. More recently, an association has been established between poor outcome and infections with strain...

GAMMA Radiation Effect On Staphylococcus aureus (ATCC 19095) in Cheese MINAS FRESCALIRRADIATED

International Nuclear Information System (INIS)

Amaral Gurgel, M.S.C.C. DO; Spoto, M.H.F.; Domarco, R.E.

1999-01-01

Milk is an excellent medium of culture for development of Staphylococcus aureus. Gamma radiation can be an alternative method to guarantee the safety of the contaminated cheeses. The objective of this research was determine the effects of the gamma radiation on the resistance of S.aureus (ATCC 19095) in cheese M inas Frescal i rradiated. The cheeses elaborated in the Laboratory of Food Irradiation of CENA/USP, were contaminated during their production with 10 6 CFU/mL of culture of S.aureus (ATCC 19095). The cheeses were irradiated with 0; 1; 2; 3 and 4 kGy, maintained under refrigeration condition (5 0C ) and analyzed at 1, 7 and 14 days of storage. The evaluation microbiology was made through the S.aureus survival analysis using Baird Parker selective medium and confirmative test of coagulase, catalase and fermentation aerobics of the manitol. The capacity of enterotoxins production by irradiated S.aureus was detected by the method of Passive Reverse Agglutination Latex. Results showed that 3 kGy is enough to destroy the S.aureus and 2 kGy to inhibited its toxins production

An outbreak of food poisoning due to egg yolk reaction-negative Staphylococcus aureus.

Science.gov (United States)

Miwa, N; Kawamura, A; Masuda, T; Akiyama, M

2001-03-20

An outbreak of staphylococcal food poisoning due to an egg yolk (EY) reaction-negative strain occurred in Japan. Twenty-one of 53 dam construction workers who ate boxed lunches prepared at their company cafeteria became ill, and eight required hospital treatment. The outbreak showed a typical incubation time (1.5-4 h with a median time of 2.7 h) and symptoms (vomiting and diarrhea) of staphylococcal food poisoning. Staphylococcus aureus, which produces staphylococcal enterotoxin (SE) A, was isolated from four fecal specimens of eight patients tested. Scrambled egg in the boxed lunches contained 20-40 ng/g of SEA, and 3.0 x 10(9)/g of viable S. aureus cells that produced this toxin. All isolates from patients and the food were EY reaction-negative, coagulase type II, and showed the same restriction fragment length polymorphism (RFLP) pattern. We concluded that the outbreak was caused by scrambled egg contaminated with EY reaction-negative S. aureus. In Japan, outbreaks of staphylococcal food poisoning are mainly caused by EY reaction-positive S. aureus, and EY reaction-negative colonies grown on agar plates containing EY are usually not analyzed further for detection of S. aureus. The present outbreak suggested that EY reaction-negative isolates should be subjected to further analysis to detect the causative agents of staphylococcal food poisoning.

Lavandula angustifolia Mill. Essential Oil Exerts Antibacterial and Anti-Inflammatory Effect in Macrophage Mediated Immune Response to Staphylococcus aureus.

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Giovannini, D; Gismondi, A; Basso, A; Canuti, L; Braglia, R; Canini, A; Mariani, F; Cappelli, G

2016-01-01

Different studies described the antibacterial properties of Lavandula angustifolia (Mill.) essential oil and its anti-inflammatory effects. Besides, no data exist on its ability to activate human macrophages during the innate response against Staphylococcus aureus. The discovery of promising regulators of macrophage-mediated inflammatory response, without side effects, could be useful for the prevention of, or as therapeutic remedy for, various inflammation-mediated diseases. This study investigated, by transcriptional analysis, how a L. angustifolia essential oil treatment influences the macrophage response to Staphylococcus aureus infection. The results showed that the treatment increases the phagocytic rate and stimulates the containment of intracellular bacterial replication by macrophages. Our data showed that this stimulation is coupled with expression of genes involved in reactive oxygen species production (i.e., CYBB and NCF4). Moreover, the essential oil treatment balanced the inflammatory signaling induced by S. aureus by repressing the principal pro-inflammatory cytokines and their receptors and inducing the heme oxygenase-1 gene transcription. These data showed that the L. angustifolia essential oil can stimulate the human innate macrophage response to a bacterium which is responsible for one of the most important nosocomial infection and might suggest the potential development of this plant extract as an anti-inflammatory and immune regulatory coadjutant drug.

Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR.

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Benjamin P Howden

2011-11-01

Full Text Available Antimicrobial resistance in Staphylococcus aureus is a major public health threat, compounded by emergence of strains with resistance to vancomycin and daptomycin, both last line antimicrobials. Here we have performed high throughput DNA sequencing and comparative genomics for five clinical pairs of vancomycin-susceptible (VSSA and vancomycin-intermediate ST239 S. aureus (VISA; each pair isolated before and after vancomycin treatment failure. These comparisons revealed a frequent pattern of mutation among the VISA strains within the essential walKR two-component regulatory locus involved in control of cell wall metabolism. We then conducted bi-directional allelic exchange experiments in our clinical VSSA and VISA strains and showed that single nucleotide substitutions within either walK or walR lead to co-resistance to vancomycin and daptomycin, and caused the typical cell wall thickening observed in resistant clinical isolates. Ion Torrent genome sequencing confirmed no additional regulatory mutations had been introduced into either the walR or walK VISA mutants during the allelic exchange process. However, two potential compensatory mutations were detected within putative transport genes for the walK mutant. The minimal genetic changes in either walK or walR also attenuated virulence, reduced biofilm formation, and led to consistent transcriptional changes that suggest an important role for this regulator in control of central metabolism. This study highlights the dramatic impacts of single mutations that arise during persistent S. aureus infections and demonstrates the role played by walKR to increase drug resistance, control metabolism and alter the virulence potential of this pathogen.

The AbcA Transporter of Staphylococcus aureus Affects Cell Autolysis

Science.gov (United States)

Schrader-Fischer, Gesine; Berger-Bächi, Brigitte

2001-01-01

Increased production of penicillin-binding protein PBP 4 is known to increase peptidoglycan cross-linking and contributes to methicillin resistance in Staphylococcus aureus. The pbp4 gene shares a 400-nucleotide intercistronic region with the divergently transcribed abcA gene, encoding an ATP-binding cassette transporter of unknown function. Our study revealed that methicillin stimulated abcA transcription but had no effects on pbp4 transcription. Analysis of abcA expression in mutants defective for global regulators showed that abcA is under the control of agr. Insertional inactivation of abcA by an erythromycin resistance determinant did not influence pbp4 transcription, nor did it alter resistance to methicillin and other cell wall-directed antibiotics. However, abcA mutants showed spontaneous partial lysis on plates containing subinhibitory concentrations of methicillin due to increased spontaneous autolysis. Since the autolytic zymograms of cell extracts were identical in mutants and parental strains, we postulate an indirect role of AbcA in control of autolytic activities and in protection of the cells against methicillin. PMID:11158733

Methicillin-Resistant Staphylococcus aureus (MRSA Detection from the Hands of Jatinangor Community Health Center’s Health Care Providers

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Jeevanisha Patmanathan

2015-06-01

Full Text Available Background: Methicillin-resistant Staphylococcus aureus is a bacterium that is resistance towards β-lactam antibiotics, and it seems to be one of the leading causes of nosocomial infections. Hands of the health care workers are said to be the main source for the nosocomial transmission. Thus, the study aims to determine methicillin-resistant S. aureus from the hands of Jatinangor Community Health Center’s health care workers. Methods: Samples were taken from the hands of 30 Jatinangor Community Health Center’s staffs, including medical and paramedical; from October 2012 to November 2012. Then, these samples underwent further laboratory examinations, starting from culture, identification and susceptibility test towards cefoxitin, in identifying methicillin-resistant S. aureus. Results: Out of the 30 samples taken, 6 samples (20% were positive for S. aureus isolates. In which, 4 (13.33% of the samples were positive for methicillin-resistant S. aureus. Conclusions: Since, health care workers are the main people in contact with patients and maintaining proper hand hygiene makes a huge difference; hand hygiene should be given adequate attention for the benefit of all.

Application of a molecular beacon based real-time isothermal amplification (MBRTIA) technology for simultaneous detection of Bacillus cereus and Staphylococcus aureus.

Science.gov (United States)

Mandappa, I M; Joglekar, Prasanna; Manonmani, H K

2015-07-01

A multiplex real-time isothermal amplification assay was developed using molecular beacons for the detection of Bacillus cereus and Staphylococcus aureus by targeting four important virulence genes. A correlation between targeting highly accessible DNA sequences and isothermal amplification based molecular beacon efficiency and sensitivity was demonstrated using phi(Φ)29 DNA polymerase at a constant isothermal temperature of 30 °C. It was very selective and consistently detected down to 10(1) copies of DNA. The specificity and sensitivity of this assay, when tested with pure culture were high, surpassing those of currently used PCR assays for the detection of these organisms. The molecular beacon based real-time isothermal amplification (MBRTIA) assay could be carried out entirely in 96 well plates or well strips, enabling a rapid and high-throughput detection of food borne pathogens.

Nasal screening for Staphylococcus aureus--daily routine with improvement potentials.

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Philipp Warnke

Full Text Available OBJECTIVES: Staphylococcus aureus causes purulent bacterial infections with a considerable number of life-threatening complications and thus, is a serious cost factor in public health. Up to 50% of a given population could asymptomatically carry Staphylococcus aureus in their nares, thereby serving as a source for contact transmissions and endogenous infections. Nasal swab-based screening techniques are widely used to identify suchcarriers. This study investigated the skill of medical professionals in taking nasal swabs and the effect of teaching on improving bacterial recovery rates. METHODS: 364 persons with different medical educational background participated in this study. A novel anatomically correct artificial nose model was implemented and inoculated with a numerically defined mixture of Staphylococcus aureus and Staphylococcus epidermidis bacteria. Utilizing regular clinical swabs, participants performed screening of the inoculated nose models before and after standardized theoretical, visual, and practical teaching. Recovery of bacteria was measured by standard viable count techniques. Data were analyzed statistically by nonparametric tests. RESULTS: It could be demonstrated that combined theoretical and practical teaching improved bacterial recovery rates. Even experienced medical professionals increased their detection levels after training. Recovery rates of bacteria varied significantly between trained (158.1 CFU and untrained (47.5 CFU participants (Wilcoxon test, p<0.001; Kolmogorov-Smirnov test, p<0.001. CONCLUSIONS: Swabs are commonly used to detect nasal carriage of Staphylococcus aureus in patients. The present teaching algorithm combined with the novel nose model offers an excellent precondition to improve knowledge and performance of this technique. Increased detection rates may prevent from contact transmission due to suboptimum hygienic patient handling. Consecutively, this effect could reduce costs for patient care. This

A fluorescence resonance energy transfer (FRET) biosensor based on graphene quantum dots (GQDs) and gold nanoparticles (AuNPs) for the detection of mecA gene sequence of Staphylococcus aureus.

Science.gov (United States)

Shi, Jingyu; Chan, Chunyu; Pang, Yukting; Ye, Weiwei; Tian, Feng; Lyu, Jing; Zhang, Yu; Yang, Mo

2015-05-15

In this work, a novel fluorescence resonance energy transfer (FRET) biosensor based on graphene quantum dots (GQDs) and gold nanoparticles (AuNPs) pairs was developed for Staphylococcus aureus specific gene sequence detection. This FRET biosensor platform was realized by immobilization of capture probes on GQDs and conjugation of reporter probes on AuNPs. Target oligos then co-hybridized with capture probes and reporter probes to form a sandwich structure which brought GQDs and AuNPs to close proximity to trigger FRET effect. The fluorescence signals before and after addition of targets were measured and the fluorescence quenching efficiency could reach around 87% with 100 nM target oligo. The limit of detection (LOD) of this FRET biosensor was around 1 nM for S.aureus gene detection. Experiments with both single-base mismatched oligos and double-base mismatched oligos demonstrated the good sequence selectivity of this FRET biosensor. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic and Virulent Difference Between Pigmented and Non-pigmented Staphylococcus aureus

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Jing Zhang

2018-04-01

Full Text Available Staphyloxanthin (STX, a golden carotenoid pigment produced by Staphylococcus aureus, is suggested to act as an important virulence factor due to its antioxidant properties. Restraining biosynthesis of STX was considered as an indicator of virulence decline in pigmented S. aureus isolates. However, it is not clear whether natural non-pigmented S. aureus isolates have less virulence than pigmented ones. In this study, it is aimed to compare the pigmented and non-pigmented S. aureus isolates to clarify the genetic and virulent differences between the two groups. Here, 132 S. aureus isolates were divided into two phenotype groups depending on the absorbance (OD450 of the extracted carotenoids. Then, all isolates were subjected to spa typing and multilocus sequence typing (MLST, and then the detection of presence of 30 virulence factors and the gene integrity of crtN and crtM. Furthermore, 24 typical S. aureus isolates and 4 S. argenteus strains were selected for the murine infection assay of in vivo virulence, in which the histological observation and enumeration of CFUs were carried out. These isolates were distributed in 26 sequence types (STs and 49 spa types. The pigmented isolates were scattered in 25 STs, while the non-pigmented isolates were more centralized, which mainly belonged to ST20 (59% and ST25 (13%. Among the 54 non-pigmented isolates, about 20% carried intact crtN and crtM genes. The in vivo assay suggested that comparing with pigmented S. aureus, non-pigmented S. aureus and S. argenteus strains did not show a reduced virulence in murine sepsis models. Therefore, it suggested that there were no significant genetic and virulent differences between pigmented and non-pigmented S. aureus.

Poly-N-acetylglucosamine production in Staphylococcus aureus is essential for virulence in murine models of systemic infection.

Science.gov (United States)

Kropec, Andrea; Maira-Litran, Tomas; Jefferson, Kimberly K; Grout, Martha; Cramton, Sarah E; Götz, Friedrich; Goldmann, Donald A; Pier, Gerald B

2005-10-01

The contribution of the Staphylococcus aureus surface polysaccharide poly-N-acetylglucosamine (PNAG) to virulence was evaluated in three mouse models of systemic infection: bacteremia, renal abscess formation, and lethality following high-dose intraperitoneal (i.p.) infection. Deletion of the intercellular adhesin (ica) locus that encodes the biosynthetic enzymes for PNAG production in S. aureus strains Mn8, Newman, and NCTC 10833 resulted in mutant strains with significantly reduced abilities to maintain bacterial levels in blood following intravenous or i.p. injection, to spread systemically to the kidneys following i.p. injection, or to induce a moribund/lethal state following i.p. infection. In the bacteremia model, neither growth phase nor growth medium used to prepare the S. aureus inoculum affected the conclusion that PNAG production was needed for full virulence. As the SarA regulatory protein has been shown to affect ica transcription, PNAG synthesis, and biofilm formation, we also evaluated S. aureus strains Mn8 and 10833 deleted for the sarA gene in the renal infection model. A decrease in PNAG production was seen in sarA mutants using immunoblots of cell surface extracts but was insufficient to reduce the virulence of sarA-deleted strains in this model. S. aureus strains deleted for the ica genes were much more susceptible to antibody-independent opsonic killing involving human peripheral blood leukocytes and rabbit complement. Thus, PNAG confers on S. aureus resistance to killing mediated by these innate host immune mediators. Overall, PNAG production by S. aureus appears to be a critical virulence factor as assessed in murine models of systemic infection.

DNA aptamers as a novel approach to neutralize Staphylococcus aureus α-toxin.

Science.gov (United States)

Vivekananda, Jeevalatha; Salgado, Christi; Millenbaugh, Nancy J

2014-02-14

Staphylococcus aureus is a versatile pathogen capable of causing a broad spectrum of diseases ranging from superficial skin infections to life threatening conditions such as endocarditis, septicemia, pneumonia and toxic shock syndrome. In vitro and in vivo studies identified an exotoxin, α-toxin, as a major cause of S. aureus toxicity. Because S. aureus has rapidly evolved resistance to a number of antibiotics, including methicillin, it is important to identify new therapeutic strategies, other than antibiotics, for inhibiting the harmful effects of this pathogen. Aptamers are single-stranded DNA or RNA oligonucleotides with three-dimensional folded conformations that bind with high affinity and selectivity to targets and modulate their biological functions. The goal of this study was to isolate DNA aptamers that specifically inhibit the cytotoxic activity of α-toxin. After 10 rounds of Systematic Evolution of Ligands by EXponential Enrichment (SELEX), 49 potential anti-α-toxin aptamers were identified. In vitro neutralization assays demonstrated that 4 of these 49 aptamers, AT-27, AT-33, AT-36, and AT-49, significantly inhibited α-toxin-mediated cell death in Jurkat T cells. Furthermore, RT-PCR analysis revealed that α-toxin increased the transcription of the inflammatory cytokines TNF-α and IL-17 and that anti-α-toxin aptamers AT-33 and AT-36 inhibited the upregulation of these genes. Collectively, the data suggest the feasibility of generating functionally effective aptamers against α-toxin for treatment of S. aureus infections. Published by Elsevier Inc.

Prevalence of nasal portal of Staphylococcus aureus in disabled children.

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Clotilde Molin

2016-06-01

Full Text Available Introduction: Colonization of the nasal mucosa by Staphylococcus aureus set a carrier state. Which is recognized as a potential source of infection and a high risk factor for subsequent invasive infections. The prevalence of nasal carriage of this germ in disabled children in Paraguay is not known, thus contributing to the knowledge of their frequency and evaluate the profile of sensitivity to common antimicrobials was conducted this study, from May to July 2015.  Objective: to determine the prevalence of Staphylococcus aureus nasal carriage and profile of antimicrobial resistance in disabled children. Materials and Methods: A descriptive cross-sectional study in which 80 nasal swabs of children, who attended the service laboratory of SENADIS (Secretaria Nacional por los Derechos Humanos de las Personas con Discapacidad. The identification and sensitivity of germ was accomplished by conventional testing.  Results: 80 pediatric patients, 46 boys and 34 girls. 18 isolates of Staphylococcus aureus were obtained, corresponding to a prevalence of 22,5%. Susceptibility testing indicated that 14 strains were MSSA (Methicillin – Sensitive Staphylococcus aureus and 4 RMSA ( Methicillin- resistant Staphylococcus aureus. Conclusion: The prevalence of Staphylococcus aureus in a population with its own characteristics provides valuable data for the epidemiology, reflecting the need for continued vigilance and take steps to reduce associated infections. The detection of RMAR evidences their progress; it is important to evaluate the empirical treatment to primary care.

Portación y caracterización de Staphylococcus aureus en manipuladores de alimentos Carriage and characterization of Staphylococcus aureus in food handlers

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Graciela B Jordá

2012-06-01

Full Text Available Staphylococcus aureus es una causa de intoxicaciones alimentarias por su capacidad de producir enterotoxinas. Los manipuladores de alimentos que portan S. aureus productores de enterotoxinas pueden provocar intoxicaciones alimentarias. Se estudiaron muestras tomadas de fosas nasales de 88 manipuladores de alimentos en la provincia de Misiones. El 37,5 % de los individuos analizados eran portadores de S. aureus. Mediante técnicas de amplificación (PCR, se detectaron genes que codifican la producción de enterotoxinas en 13 de los 33 aislamientos obtenidos (39,4 % y en el 14,7 % de los manipuladores. De estos aislamientos, 10 portaban el gen sea y 3 el gen sec. El estudio de sensibilidad a los antibióticos mostró un 100 % de sensibilidad a teicoplanina, gentamiclna y rifampicina; 2 aislamientos fueron resistentes a clindamicina y a eritromicina y 4 resultaron resistentes a la meticilina. Estos resultados son un alerta e indicarían la necesidad de desarrollar medidas racionales para reducir el riesgo potencial de intoxicaciones alimentarias.Staphylococcus aureus causes food poisoning due to its ability to produce enterotoxins. Food handlers carrying enterotoxin-producing S. aureus can contaminate food, thus leading to food poisoning. Samples were obtained from 88 food handlers in the Province of Misiones, Argentina. S. aureus was isolated from nasal swaps and PCR amplification was performed for genes encoding staphylococcal enterotoxins. A total of 37.5 % food handlers were positive for S. aureus. Expression of enterotoxin genes was found in 13 of the 33 (39.4 % S. aureus isolates studied, accounting for 14.7 % of food handlers. Gene sea was detected in 10 isolates followed by gene sec in 3 isolates. All isolates were susceptible to teicoplanin, gentamicin and rifampicin. Four isolates were resistant to methicillin whereas 2 isolates were resistant to clindamycin and erythromycin. These results constitute a critical alert and indicate the need

spa typing and antimicrobial resistance of Staphylococcus aureus from healthy humans, pigs and dogs in Tanzania

DEFF Research Database (Denmark)

Katakweba, Abdul S.; Muhairwa, Amandus P.; Espinosa-Gongora, Carmen

2016-01-01

. aureus carrier frequencies in dogs and humans were within the expected range and low in pigs. The S. aureus spa types circulating in the community were generally not shared by different hosts and majority of types belonged to known clones. Besides ampicillin resistance, moderate levels of antimicrobial......Introduction: Staphylococcus aureus is an opportunistic pathogen causing infections in humans and animals. Here we report for the first time the prevalence of nasal carriage, spa typing and antimicrobial resistance of S. aureus in a Tanzanian livestock community. Methodology: Nasal swabs were taken...... from 100 humans, 100 pigs and 100 dogs in Morogoro Municipal. Each swab was enriched in Mueller Hinton broth with 6.5% NaCl and subcultured on chromogenic agar for S. aureus detection. Presumptive S. aureus colonies were confirmed to the species level by nuc PCR and analysed by spa typing...

Intracellular Biosynthesis of Fluorescent CdSe Quantum Dots in Bacillus subtilis: A Strategy to Construct Signaling Bacterial Probes for Visually Detecting Interaction Between Bacillus subtilis and Staphylococcus aureus.

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Yan, Zheng-Yu; Ai, Xiao-Xia; Su, Yi-Long; Liu, Xin-Ying; Shan, Xiao-Hui; Wu, Sheng-Mei

2016-02-01

In this work, fluorescent Bacillus subtilis (B. subtilis) cells were developed as probes for imaging applications and to explore behaviorial interaction between B. subtilis and Staphylococcus aureus (S. aureus). A novel biological strategy of coupling intracellular biochemical reactions for controllable biosynthesis of CdSe quantum dots by living B. subtilis cells was demonstrated, through which highly luminant and photostable fluorescent B. subtilis cells were achieved with good uniformity. With the help of the obtained fluorescent B. subtilis cells probes, S. aureus cells responded to co-cultured B. subtilis and to aggregate. The degree of aggregation was calculated and nonlinearly fitted to a polynomial model. Systematic investigations of their interactions implied that B. subtilis cells inhibit the growth of neighboring S. aureus cells, and this inhibition was affected by both the growth stage and the amount of surrounding B. subtilis cells. Compared to traditional methods of studying bacterial interaction between two species, such as solid culture medium colony observation and imaging mass spectrometry detection, the procedures were more simple, vivid, and photostable due to the efficient fluorescence intralabeling with less influence on the cells' surface, which might provide a new paradigm for future visualization of microbial behavior.

Risk factors assessment for nasal colonization of Staphylococcus aureus and its methicillin resistant strains among pre-clinical medical students of Nepal.

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Ansari, Shamshul; Gautam, Rajendra; Shrestha, Sony; Ansari, Safiur Rahman; Subedi, Shankar Nanda; Chhetri, Muni Raj

2016-04-12

Staphylococcus aureus (S. aureus), a normal flora of nasal cavity, can cause minor to life threatening invasive diseases and nosocomial infections. Methicillin resistant strains of S. aureus are causing a great challenge for treatment options. Therefore, the purpose of this study was to assess the nasal carriage rate of S. aureus, its methicillin resistant strains and risk factors in medical students prior to clinical exposure. The bacterial growth of S. aureus from nasal swab culture was identified by using standard microbiological methods recommended by American Society for Microbiology. Modified Kirby-Bauer disk diffusion method was used for antibiotic susceptibility testing and methicillin resistance was confirmed using cefoxitin and oxacillin disks. D-zone test method was used to determine the inducible clindamycin resistance. Among 200 participants, nasal carriage of S. aureus was detected from 30 (15%) subjects. Upper respiratory tract infections significantly (P aureus and their methicillin resistant strains. All of the isolates were reported to be susceptible to vancomycin and teicoplanin. S. aureus strains detected from 8 (4%) students were confirmed to be methicillin resistant. The result of our study demands for strict policy to screen all the students for nasal carriage of S. aureus and its MRSA strains to minimize the transmission of this organism from community to hospital settings.

Methicillin-resistant Staphylococcus aureus infection and hospitalization in high-risk patients in the year following detection.

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Susan S Huang

Full Text Available Many studies have evaluated methicillin-resistant Staphylococcus aureus (MRSA infections during single hospitalizations and subsequent readmissions to the same institution. None have assessed the comprehensive burden of MRSA infection in the period after hospital discharge while accounting for healthcare utilization across institutions.We conducted a retrospective cohort study of adult patients insured by Harvard Pilgrim Health Care who were newly-detected to harbor MRSA between January 1991 and December 2003 at a tertiary care medical center. We evaluated all MRSA-attributable infections associated with hospitalization in the year following new detection, regardless of hospital location. Data were collected on comorbidities, healthcare utilization, mortality and MRSA outcomes. Of 591 newly-detected MRSA carriers, 23% were colonized and 77% were infected upon detection. In the year following detection, 196 (33% patients developed 317 discrete and unrelated MRSA infections. The most common infections were pneumonia (34%, soft tissue (27%, and primary bloodstream (18% infections. Infections occurred a median of 56 days post-detection. Of all infections, 26% involved bacteremia, and 17% caused MRSA-attributable death. During the admission where MRSA was newly-detected, 14% (82/576 developed subsequent infection. Of those surviving to discharge, 24% (114/482 developed post-discharge infections in the year following detection. Half (99/185, 54% of post-discharge infections caused readmission, and most (104/185, 55% occurred over 90 days post-discharge.In high-risk tertiary care patients, newly-detected MRSA carriage confers large risks of infection and substantial attributable mortality in the year following acquisition. Most infections occur post-discharge, and 18% of infections associated with readmission occurred in hospitals other than the one where MRSA was newly-detected. Despite gains in reducing MRSA infections during hospitalization, the

[Change in drug resistance of Staphylococcus aureus].

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Lin, Yan; Liu, Yan; Luo, Yan-Ping; Liu, Chang-Ting

2013-11-01

To analyze the change in drug resistance of Staphylococcus aureus (SAU) in the PLA general hospital from January 2008 to December 2012, and to provide solid evidence to support the rational use of antibiotics for clinical applications. The SAU strains isolated from clinical samples in the hospital were collected and subjected to the Kirby-Bauer disk diffusion test. The results were assessed based on the 2002 American National Committee for Clinical Laboratory Standards (NCCLS) guidelines. SAU strains were mainly isolated from sputum, urine, blood and wound excreta and distributed in penology, neurology wards, orthopedics and surgery ICU wards. Except for glycopeptide drugs, methicillin-resistant Staphylococcus aureus (MRSA) had a higher drug resistance rate than those of the other drugs and had significantly more resistance than methicillin-sensitive Staphylococcus aureus (MSSA) (P resistance, we discovered a gradual increase in drug resistance to fourteen test drugs during the last five years. Drug resistance rate of SAU stayed at a higher level over the last five years; moreover, the detection ratio of MRSA keeps rising year by year. It is crucial for physicians to use antibiotics rationally and monitor the change in drug resistance in a dynamic way.

VfrB Is a Key Activator of the Staphylococcus aureus SaeRS Two-Component System.

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Krute, Christina N; Rice, Kelly C; Bose, Jeffrey L

2017-03-01

In previous studies, we identified the fatty acid kinase virulence factor regulator B (VfrB) as a potent regulator of α-hemolysin and other virulence factors in Staphylococcus aureus In this study, we demonstrated that VfrB is a positive activator of the SaeRS two-component regulatory system. Analysis of vfrB , saeR , and saeS mutant strains revealed that VfrB functions in the same pathway as SaeRS. At the transcriptional level, the promoter activities of SaeRS class I ( coa ) and class II ( hla ) target genes were downregulated during the exponential growth phase in the vfrB mutant, compared to the wild-type strain. In addition, saePQRS expression was decreased in the vfrB mutant strain, demonstrating a need for this protein in the autoregulation of SaeRS. The requirement for VfrB-mediated activation was circumvented when SaeS was constitutively active due to an SaeS (L18P) substitution. Furthermore, activation of SaeS via human neutrophil peptide 1 (HNP-1) overcame the dependence on VfrB for transcription from class I Sae promoters. Consistent with the role of VfrB in fatty acid metabolism, hla expression was decreased in the vfrB mutant with the addition of exogenous myristic acid. Lastly, we determined that aspartic acid residues D38 and D40, which are predicted to be key to VfrB enzymatic activity, were required for VfrB-mediated α-hemolysin production. Collectively, this study implicates VfrB as a novel accessory protein needed for the activation of SaeRS in S. aureus IMPORTANCE The SaeRS two-component system is a key regulator of virulence determinant production in Staphylococcus aureus Although the regulon of this two-component system is well characterized, the activation mechanisms, including the specific signaling molecules, remain elusive. Elucidating the complex regulatory circuit of SaeRS regulation is important for understanding how the system contributes to disease causation by this pathogen. To this end, we have identified the fatty acid kinase

Functional characterization of a cadmium resistance operon in Staphylococcus aureus ATCC12600: CadC does not function as a repressor.

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Hoogewerf, Arlene J; Dyk, Lisa A Van; Buit, Tyler S; Roukema, David; Resseguie, Emily; Plaisier, Christina; Le, Nga; Heeringa, Lee; Griend, Douglas A Vander

2015-02-01

Sequencing of a cadmium resistance operon from a Staphylococcus aureus ATCC12600 plasmid revealed that it is identical to a cadCA operon found in MRSA strains. Compared to plasmid-cured and cadC-mutant strains, cadC-positive ATCC12600 cells had increased resistance to cadmium (1 mg ml(-1) cadmium sulfate) and zinc (4 mg ml(-1) zinc sulfate), but not to other metal ions. After growth in media containing 20 µg ml(-1) cadmium sulfate, cadC-mutant cells contained more intracellular cadmium than cadC-positive ATCC12600 cells, suggesting that cadC absence results in impaired cadmium efflux. Electrophoretic mobility shift assays were performed with CadC proteins encoded by the S. aureus ATCC12600 plasmid and by the cadC gene of pI258, which is known to act as a transcriptional repressor and shares only 47% protein sequence identity with ATCC12600 CadC. Mobility shifts occurred when pI258 CadC protein was incubated with the promoter DNA-regions from the pI258 and S. aureus ATCC12600 cadCA operons, but did not occur with S. aureus ATCC12600 CadC protein, indicating that the ATCC12600 CadC protein does not interact with promoter region DNA. This cadCA operon, found in MRSA strains and previously functionally uncharacterized, increases resistance to cadmium and zinc by an efflux mechanism, and CadC does not function as a transcriptional repressor. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Method for Screening Compounds That Influence Virulence Gene Expression in Staphylococcus aureus

DEFF Research Database (Denmark)

Nielsen, A.; Nielsen, Kristian Fog; Frees, D.

2010-01-01

We present a simple assay to examine effects of compounds on virulence gene expression in the human pathogen Staphylococcus aureus. The assay employs transcriptional reporter strains carrying lacZ fused to central virulence genes. Compounds affecting virulence gene expression and activity...... of the agr locus are scored based on color change in the presence of a chromogenic beta-galactosidase substrate. The assay can be used to screen for novel antivirulence compounds from many different sources, such as fungi, as demonstrated here....

Detection of enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification.

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Wang, D; Wang, X; Geng, Y; An, C

2014-01-01

The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique. A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR) and real-time PCR. A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.

In Vitro Selection of Single-Stranded DNA Molecular Recognition Elements against S. aureus Alpha Toxin and Sensitive Detection in Human Serum

OpenAIRE

Hong, Ka L.; Battistella, Luisa; Salva, Alysia D.; Williams, Ryan M.; Sooter, Letha J.

2015-01-01

Alpha toxin is one of the major virulence factors secreted by Staphylococcus aureus, a bacterium that is responsible for a wide variety of infections in both community and hospital settings. Due to the prevalence of S. aureus related infections and the emergence of methicillin-resistant S. aureus, rapid and accurate diagnosis of S. aureus infections is crucial in benefiting patient health outcomes. In this study, a rigorous Systematic Evolution of Ligands by Exponential Enrichment (SELEX) var...

Interaction between the Staphylococcus aureus extracellular adherence protein Eap and its subdomains with platelets.

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Palankar, Raghavendra; Binsker, Ulrike; Haracska, Bianca; Wesche, Jan; Greinacher, Andreas; Hammerschmidt, Sven

2018-04-18

S. aureus associated bacteremia can lead to severe infections with high risk of mortality (e.g. sepsis, infective endocarditis). Many virulence factors and adhesins of S. aureus are known to directly interact with platelets. Extracellular adherence protein, Eap, one of the most important virulence factors in S. aureus mediated infections is a multi-tandem domain protein and has been shown to interact with almost all cell types in the human circulatory system. By using amine reactive fluorescent N-hydroxysuccinimidyl (NHS)-ester dyes and by direct detection with primary fluorescently conjugated anti-histidine (His-tag) antibodies against detect N-terminal His6, we show Eap subdomain Eap D 3 D 4 specifically interacts and rapidly activates human platelets. Furthermore, we validate our finding by using site directed directional immobilization of Eap D 3 D 4 through N-terminal His 6 on nickel (II)-nitrilotriacetic acid (Ni-NTA) functionalized bacteriomimetic microbead arrays to visualize real-time platelet activation through calcium release assay. These methods offer an easily adoptable protocols for screening of S.aureus derived virulence factors and adhesins with platelets. Copyright © 2018 Elsevier GmbH. All rights reserved.

Anti-biofilm, anti-hemolysis, and anti-virulence activities of black pepper, cananga, myrrh oils, and nerolidol against Staphylococcus aureus.

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Lee, Kayeon; Lee, Jin-Hyung; Kim, Soon-Il; Cho, Moo Hwan; Lee, Jintae

2014-11-01

The long-term usage of antibiotics has resulted in the evolution of multidrug-resistant bacteria. Unlike antibiotics, anti-virulence approaches target bacterial virulence without affecting cell viability, which may be less prone to develop drug resistance. Staphylococcus aureus is a major human pathogen that produces diverse virulence factors, such as α-toxin, which is hemolytic. Also, biofilm formation of S. aureus is one of the mechanisms of its drug resistance. In this study, anti-biofilm screening of 83 essential oils showed that black pepper, cananga, and myrrh oils and their common constituent cis-nerolidol at 0.01 % markedly inhibited S. aureus biofilm formation. Furthermore, the three essential oils and cis-nerolidol at below 0.005 % almost abolished the hemolytic activity of S. aureus. Transcriptional analyses showed that black pepper oil down-regulated the expressions of the α-toxin gene (hla), the nuclease genes, and the regulatory genes. In addition, black pepper, cananga, and myrrh oils and cis-nerolidol attenuated S. aureus virulence in the nematode Caenorhabditis elegans. This study is one of the most extensive on anti-virulence screening using diverse essential oils and provides comprehensive data on the subject. This finding implies other beneficial effects of essential oils and suggests that black pepper, cananga, and myrrh oils have potential use as anti-virulence strategies against persistent S. aureus infections.

Virulence Factors of Staphylococcus aureus Isolates in an Iranian Referral Children's Hospital.

Science.gov (United States)

Sabouni, Farah; Mahmoudi, Shima; Bahador, Abbas; Pourakbari, Babak; Sadeghi, Reihaneh Hosseinpour; Ashtiani, Mohammad Taghi Haghi; Nikmanesh, Bahram; Mamishi, Setareh

2014-04-01

The clinical importance of Staphylococcus aureus (S. aureus) is attributed to notable virulence factors, surface proteins, toxins, and enzymes as well as the rapid development of drug resistance. The aim of this study was to compare the occurrence of virulence factors produced by S. aureus strains isolated from children in an Iranian referral children's hospital. The presence of genes encoding for the enterotoxins A (sea), B (seb), C (sec), D (sed), TSST-1 (tsst), exfoliative toxin A (eta), and exfoliative toxin B (etb) were detected by Multiplex polymerase chain reaction (PCR) using specific primers. In addition, the standardized Kirby-Bauer disc-diffusion method was performed on Mueller-Hinton agar. In total, 133 S. aureus isolates were obtained from different patients. Of these S. aureus isolates, 64 (48%) were methicillin-resistant S. aureus (MRSA), and all of these tested positive for the mecA gene. Regarding the classical enterotoxin genes, sea gene (40.6%) was the most prevalent followed by seb (19.6%), tsst (12.8%), eta (11.3%), etb (9%), sed (4.5%), and sec (3%). Among methicillin-susceptible S. aureus (MSSA) isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates (p  0.05). In our study enterotoxin A was produced by 40.6% of the isolates (48% from MRSA and 33% from MSSA isolates) which was higher than in previous reports. According to our results, strict hygiene and preventative measures during food processing are highly recommended.

Methicillin resistance in Staphylococcus aureus : a review of the ...

African Journals Online (AJOL)

Methicillin resistance in Staphylococcus aureus : a review of the molecular epidemiology, clinical significance and laboratory detection methods. ... Added to this burden is the emergence of more virulent strains of community-associated MRSA (CAMRSA) which at the turn of the century, has been increasingly reported to ...

Short communication: Effects of lactose and milk on the expression of biofilm-associated genes in Staphylococcus aureus strains isolated from a dairy cow with mastitis.

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Xue, Ting; Chen, Xiaolin; Shang, Fei

2014-10-01

Staphylococcus aureus is the main etiological organism responsible for bovine mastitis. The ability of S. aureus to form biofilms plays an important role in the pathogenesis of mastitis. Biofilm formation in S. aureus is associated with the production of polysaccharide intercellular adhesin (PIA) protein and several other proteins. Several environmental factors, including glucose, osmolarity, oleic acid, temperature, and anaerobiosis, have been reported to affect biofilm formation in S. aureus. This study investigated the influence of lactose and milk on the biofilm formation capacity of 2 clinical bovine isolates of S. aureus. We found that lactose increased biofilm formation predominantly by inducing PIA production, whereas milk increased biofilm formation through PIA as well as by increasing the production of other biofilm-associated proteins, which might be mediated by the transcriptional regulators intercellular adhesion regulator (icaR) and repressor of biofilm (rbf). Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Quantitative Expression Analysis of SpA, FnbA and Rsp Genes in Staphylococcus aureus: Actively Associated in the Formation of Biofilms.

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Yeswanth, Sthanikam; Chaudhury, Abhijit; Sarma, Potukuchi Venkata Gurunadha Krishna

2017-12-01

In Staphylococcus aureus, adherence and secretory proteins play chief role in the formation of biofilms. This mode of growth exhibits resistance to a variety of antibiotics and spreads its infections. In the present study, secretary and adherence proteins, Protein-A, Fibronectin-binding protein-A (FnbA) and Rsp (a transcription regulator encoding proteolytic property) expression levels were evaluated at different stages of growth in S. aureus ATCC12600 a drug-sensitive strain and multidrug-resistant strains of S. aureus. Initially, the SpA, FnbA and Rsp genes of S. aureus ATCC12600 were cloned, sequenced, expressed and characterized. The proteolytic property of recombinant Rsp was conspicuously shown when this pathogen was grown in aerobic conditions correlating with reduced biofilm units. In anaerobic mode of growth, S. aureus exhibited a higher expression of SpA and FnbA in early and mid adherence phases and finally stabilized at 48 h of incubation. This expression was more pronounced in methicillin-resistant strains (LMV1-8 and D1-4) of S. aureus. In all these stages, Rsp gene expression was at the lowest level and these results concur with the increased biofilm units. The results of the present study explain proteins chiefly contribute in the formation of biofilms.

The microbiology of impetigo in indigenous children: associations between Streptococcus pyogenes, Staphylococcus aureus, scabies, and nasal carriage.

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Bowen, Asha C; Tong, Steven Y C; Chatfield, Mark D; Carapetis, Jonathan R

2014-12-31

Impetigo is caused by both Streptococcus pyogenes and Staphylococcus aureus; the relative contributions of each have been reported to fluctuate with time and region. While S. aureus is reportedly on the increase in most industrialised settings, S. pyogenes is still thought to drive impetigo in endemic, tropical regions. However, few studies have utilised high quality microbiological culture methods to confirm this assumption. We report the prevalence and antimicrobial resistance of impetigo pathogens recovered in a randomised, controlled trial of impetigo treatment conducted in remote Indigenous communities of northern Australia. Each child had one or two sores, and the anterior nares, swabbed. All swabs were transported in skim milk tryptone glucose glycogen broth and frozen at -70°C, until plated on horse blood agar. S. aureus and S. pyogenes were confirmed with latex agglutination. From 508 children, we collected 872 swabs of sores and 504 swabs from the anterior nares prior to commencement of antibiotic therapy. S. pyogenes and S. aureus were identified together in 503/872 (58%) of sores; with an additional 207/872 (24%) sores having S. pyogenes and 81/872 (9%) S. aureus, in isolation. Skin sore swabs taken during episodes with a concurrent diagnosis of scabies were more likely to culture S. pyogenes (OR 2.2, 95% CI 1.1 - 4.4, p = 0.03). Eighteen percent of children had nasal carriage of skin pathogens. There was no association between the presence of S. aureus in the nose and skin. Methicillin-resistance was detected in 15% of children who cultured S. aureus from either a sore or their nose. There was no association found between the severity of impetigo and the detection of a skin pathogen. S. pyogenes remains the principal pathogen in tropical impetigo; the relatively high contribution of S. aureus as a co-pathogen has also been confirmed. Children with scabies were more likely to have S. pyogenes detected. While clearance of S. pyogenes is the key

Rapid Differentiation between Livestock-Associated and Livestock-Independent Staphylococcus aureus CC398 Clades

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Larsen, Jesper; Soldanova, Katerina; Aziz, Maliha; Contente-Cuomo, Tania; Petersen, Andreas; Vandendriessche, Stien; Jiménez, Judy N.; Mammina, Caterina; van Belkum, Alex; Salmenlinna, Saara; Laurent, Frederic; Skov, Robert L.; Larsen, Anders R.; Andersen, Paal S.; Price, Lance B.

2013-01-01

Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies. PMID:24244535

Staphylococcus aureus bacteriuria as a prognosticator for outcome of Staphylococcus aureus bacteremia: a case-control study

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Weinstein Robert A

2010-07-01

Full Text Available Abstract Background When Staphylococcus aureus is isolated in urine, it is thought to usually represent hematogenous spread. Because such spread might have special clinical significance, we evaluated predictors and outcomes of S. aureus bacteriuria among patients with S. aureus bacteremia. Methods A case-control study was performed at John H. Stroger Jr. Hospital of Cook County among adult inpatients during January 2002-December 2006. Cases and controls had positive and negative urine cultures, respectively, for S. aureus, within 72 hours of positive blood culture for S. aureus. Controls were sampled randomly in a 1:4 ratio. Univariate and multivariable logistic regression analyses were done. Results Overall, 59% of patients were African-American, 12% died, 56% of infections had community-onset infections, and 58% were infected with methicillin-susceptible S. aureus (MSSA. Among 61 cases and 247 controls, predictors of S. aureus bacteriuria on multivariate analysis were urological surgery (OR = 3.4, p = 0.06 and genitourinary infection (OR = 9.2, p = 0.002. Among patients who died, there were significantly more patients with bacteriuria than among patients who survived (39% vs. 17%; p = 0.002. In multiple Cox regression analysis, death risks in bacteremic patients were bacteriuria (hazard ratio 2.9, CI 1.4-5.9, p = 0.004, bladder catheter use (2.0, 1.0-4.0, p = 0.06, and Charlson score (1.1, 1.1-1.3, p = 0.02. Neither length of stay nor methicillin-resistant Staphylococcus aureus (MRSA infection was a predictor of S. aureus bacteriuria or death. Conclusions Among patients with S. aureus bacteremia, those with S. aureus bacteriuria had 3-fold higher mortality than those without bacteriuria, even after adjustment for comorbidities. Bacteriuria may identify patients with more severe bacteremia, who are at risk of worse outcomes.

Structural characterization of Staphylococcus aureus biotin protein ligase and interaction partners: An antibiotic target

OpenAIRE

Pendini, Nicole R; Yap, Min Y; Polyak, Steven W; Cowieson, Nathan P; Abell, Andrew; Booker, Grant W; Wallace, John C; Wilce, Jacqueline A; Wilce, Matthew C J

2013-01-01

The essential metabolic enzyme biotin protein ligase (BPL) is a potential target for the development of new antibiotics required to combat drug-resistant pathogens. Staphylococcus aureus BPL (SaBPL) is a bifunctional protein, possessing both biotin ligase and transcription repressor activities. This positions BPL as a key regulator of several important metabolic pathways. Here, we report the structural analysis of both holo- and apo-forms of SaBPL using X-ray crystallography. We also present ...

Antibiotic resistance and molecular epidemiology of Staphylococcus aureus in Nigeria

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Oyedara Omotayo

2011-05-01

Full Text Available Abstract Background Staphylococcus aureus is an important pathogen causing a wide range of infections in the hospital and community setting. In order to have adequate information for treatment of S. aureus infections, it is crucial to understand the trends in the antibiotic-resistance patterns. In addition, the occurrence and changes in types of S. aureus, clonal identities, and their geographic spread is essential for the establishment of adequate infection control programmes. In this study, 68 S. aureus isolates obtained from clinical and non-clinical sources in Nigeria between January and April 2009 were characterized using phenotypic and molecular methods. Results All the S. aureus isolates were susceptible to teicoplanin, vancomycin, phosphomycin, fusidic acid, rifampicin, daptomycin, mupirocin, linezolid and tigecycline. Sixteen percent of the isolates were resistant to oxacillin, while 55% and 72% of isolates were resistant to tetracycline and trimethoprim/sulphamethoxazole (cotrimoxazole, respectively (Table 1. There was excellent correlation between the broth microdilution assay and detection of antibiotic resistance genes by the multiplex PCR, in the determination of S. aureus resistance to erythromycin, gentamicin, methicillin and tetracycline. A total of 28 spa types were identified in the study, and the predominant spa type among the methicillin-susceptible S. aureus (MSSA isolates was t084 (13 isolates. The t037-ST241-SCCmecIII type was the only clone identified in Maiduguri (North-East Nigeria while in South-West Nigeria, diversity among the MRSA isolates (t451-ST8-SCCmecV; t008-ST94-SCCmecIV; t002-ST5-SCCmecV; t064-ST8-SCCmecV was observed. The toxin genes seh and etd were detected in isolates affiliated with clonal complexes CC1, CC80 and sequence type ST25, respectively. The proportion of PVL-positive isolates among MSSA was high (40%. Most of the PVL-positive MSSA isolates were obtained from wound infections and associated

Combined analysis of DNA methylome and transcriptome reveal novel candidate genes with susceptibility to bovine Staphylococcus aureus subclinical mastitis.

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Song, Minyan; He, Yanghua; Zhou, Huangkai; Zhang, Yi; Li, Xizhi; Yu, Ying

2016-07-14

Subclinical mastitis is a widely spread disease of lactating cows. Its major pathogen is Staphylococcus aureus (S. aureus). In this study, we performed genome-wide integrative analysis of DNA methylation and transcriptional expression to identify candidate genes and pathways relevant to bovine S. aureus subclinical mastitis. The genome-scale DNA methylation profiles of peripheral blood lymphocytes in cows with S. aureus subclinical mastitis (SA group) and healthy controls (CK) were generated by methylated DNA immunoprecipitation combined with microarrays. We identified 1078 differentially methylated genes in SA cows compared with the controls. By integrating DNA methylation and transcriptome data, 58 differentially methylated genes were shared with differently expressed genes, in which 20.7% distinctly hypermethylated genes showed down-regulated expression in SA versus CK, whereas 14.3% dramatically hypomethylated genes showed up-regulated expression. Integrated pathway analysis suggested that these genes were related to inflammation, ErbB signalling pathway and mismatch repair. Further functional analysis revealed that three genes, NRG1, MST1 and NAT9, were strongly correlated with the progression of S. aureus subclinical mastitis and could be used as powerful biomarkers for the improvement of bovine mastitis resistance. Our studies lay the groundwork for epigenetic modification and mechanistic studies on susceptibility of bovine mastitis.

The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms.

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Athanasopoulos, Athanasios N; Economopoulou, Matina; Orlova, Valeria V; Sobke, Astrid; Schneider, Darius; Weber, Holger; Augustin, Hellmut G; Eming, Sabine A; Schubert, Uwe; Linn, Thomas; Nawroth, Peter P; Hussain, Muzaffar; Hammes, Hans-Peter; Herrmann, Mathias; Preissner, Klaus T; Chavakis, Triantafyllos

2006-04-01

Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

Because of nasal carriage of Staphylococcus aureus Bacteremia in Neonatal Intensive Care Unit

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Rezan Harman

2016-12-01

Full Text Available One of the significant risk factor in the development of nosocomial Staphylococcal infections is bacteria Staphylococcus aureus (S. aureuscolonized in the nose of health personnel. The meticilin resistant S. aureus (MRSA was detected in the repeated blood cultures of two babies who had been followed for about 20 days in neonatal intensive care due to premature birth. Because of the failure to find the source in the assesment of the infants of whom MRSA reproduction continue, despite the appropriate treatment according to the results of antibiograms, examinations were performed fort he environment and the healtyh personnel. Cultures were taken from the total parenteral nutrition (TPN solution given to the babies. S. aureus growth was detected in the received culture. Therefore cultures were obtained from the places where there will be source in the division where TPN was prepared and nsal cultures were taken from the personnel. Because of growth of S. aureus only in the nasal cultures of the personnel, considering that the source was the personnel, the personnel were given the treatment of mupirocin pomad for five days, and during this period the work of the staff were replaced. There was not any growth in the TPN received at the and of the treatment an in the blood cultures of the patients. These facts showed us the necessity of making the necessary screening by considering the health personnel can also be the source in case of any S. aureus growth. [J Contemp Med 2016; 6(4.000: 382-384

Staphylococcus aureus Enterotoxin A Gene Isolated From Raw Red Meat and Poultry in Tehran, Iran

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Mohammad Hossein Sarrafzadeh Zargar

2014-07-01

Full Text Available Background: Staphylococcus aureus is the most prevalent infectious agent of food materials. Enterotoxin producing types of S. aureus cause well-known food-borne disease. Staphylococcal Enterotoxin A (SEA is the most important agent of gastroenteritis. Objectives: The present study aimed to screen the raw meat samples collected from different regions of Tehran for S. aureus infection and type of encoding enterotoxin. Materials and Methods: Hundred and eighty six meat samples were collected randomly from city dealers and transferred to laboratory within screw cap containers. The samples were first cultured according to the standard bacteriological methods and then S. aureus isolates were identified using standard bacteriological tests. The isolates were subjected to Polymerase Chain Reaction (PCR to detect gene encoding SEA. Results: Staphylococcus aureus isolated from 29 (15.6% meat samples including beef 14.8%, raw lamb 15%, raw chicken 15.7% and raw turkey 16.6%. Using special primer sets proved that the species isolated from five samples (two raw chicken, two raw beef and one raw turkey encoded enterotoxin A. Conclusions: Although staphylococcal contamination within food material is more or less a routine, but detection of enterotoxin encoding species from raw meat samples is alarming for health authorities. These data highlight the importance of periodic surveillance of raw meat distributed among ordinary consumers.

Concomitant genotyping revealed diverse spreading between methicillin-resistant Staphylococcus aureus and methicillin-susceptible Staphylococcus aureus in central Taiwan.

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Ho, Cheng-Mao; Lin, Chien-Yu; Ho, Mao-Wang; Lin, Hsiao-Chuan; Peng, Ching-Tien; Lu, Jang-Jih

2016-06-01

Staphylococcus aureus is a versatile bacterium, which can lead to various infectious diseases. Various molecular typing methods are applied to the evolution and epidemiology surveys of S. aureus, mostly for methicillin-resistant S. aureus (MRSA). However, methicillin-susceptible S. aureus (MSSA) is still an important pathogen, but their molecular typing is evaluated infrequently. Pulsed-field gel electrophoresis (PFGE), spa typing, and detection of five virulent genes for 95 MRSA and 56 MSSA isolates (July-December 2008 and July 2008-December 2009, respectively) during an overlapping period were performed. More diversity was found in MSSA isolates (23 pulsotypes and 25 spa types, excluding 4 new-type and 1 nontypable isolates for spa typing) than in MRSA isolates (19 pulsotypes and 16 spa types, excluding 1 new-type and 1 nontypable isolates for spa typing). By spa typing, t002 (n = 30), t037 (n = 23), t437 (n = 21), t234 (n = 3), t1081 (n = 3), and t1094 (n = 3) were the six major MRSA clones. For MSSA isolates, t189 (n = 13), t437 (n = 4), t084 (n = 3), t213 (n = 3), t701 (n = 3), and t7200 (n = 3) were the six major types. Combining PFGE and spa typing, there were five combinations (pulsotype + spa type) that contained both MRSA and MSSA isolates (pulsotype 9-t437, pulsotype 15-t037, pulsotype 19-t002, pulsotype 21-t002, and pulsotype 28-t1081). For all 151 S. aureus or 95 MRSA isolates, the PFGE typing had more discrimination power, but spa typing had larger discrimination index for 56 MSSA isolates. In conclusion, there were different predominant MRSA and MSSA clones clinically. Continuing longitudinal tracking of molecular typing is necessary for elucidating the evolution of this important clinical pathogen. Copyright © 2014. Published by Elsevier B.V.

High diversity of Staphylococcus aureus and Staphylococcus pseudintermedius lineages and toxigenic traits in healthy pet-owning household members. Underestimating normal household contact?

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Gómez-Sanz, Elena; Torres, Carmen; Lozano, Carmen; Zarazaga, Myriam

2013-01-01

Forty-three unrelated pet-owning households were screened in Spain to study the Staphylococcus aureus and Staphylococcus pseudintermedius nasal carriage, their genetic lineages and virulence traits. Sixty-seven healthy owners and 66 healthy pets were investigated. Isolates characterization was performed and potential interspecies transmission was assessed. S. aureus was present in 51.2% of households studied while S. pseudintermedius in 30.2%. Twenty-eight owners (41.8%) carried S. aureus: one methicillin-resistant S. aureus (MRSA) [t5173-ST8-SCCmecIVa] and 27 methicillin-susceptible S. aureus (MSSA). Three owners (4.5%) were colonized by methicillin-susceptible S. pseudintermedius (MSSP). Fifteen pets (22.7%) carried S. pseudintermedius: two methicillin-resistant S. pseudintermedius (MRSP) [ST71-SCCmecII/III; ST92-SCCmecV] and 13 MSSP; in addition, 8 pets (12.1%) presented MSSA. High diversity of spa and sequence types (STs) was detected. Typical livestock-associated S. aureus lineages (CC398, CC9) were observed in humans and/or companion animals and hospital and/or community-acquired S. aureus lineages (CC45, CC121, CC5, CC8) were detected among pets. Almost 40% of S. pseudintermedius were multidrug-resistant. S. aureus isolates harboured a remarkable high number of virulence genes. The expA gene was detected in 3 S. pseudintermedius isolates. Identical strains from both owners and their pets were identified in 5 households (11.6%): (a) four MSSA (t073-ST45/CC45, t159-ST121/CC121, t209-ST109/CC9, t021-ST1654([new])/singleton) and (b) one multidrug-resistant MSSP (ST142([new])). Highly clonally diverse and toxigenic S. aureus and S. pseudintermedius are common colonizers of healthy humans and pets. The presence of these bacterial species, virulence genes, and interspecies transmission detected, points out to consider pet ownership as a risk factor to acquire, maintain and spread, potential pathogenic bacteria. Copyright © 2012 Elsevier Ltd. All rights reserved.

Staphylococcus aureus entrance into the dairy chain: Tracking S. aureus from dairy cow to cheese

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Judith Kümmel

2016-10-01

Full Text Available Staphylococcus aureus is one of the most important contagious mastitis pathogens in dairy cattle. Due to its zoonotic potential, control of S. aureus is not only of great economic importance in the dairy industry but also a significant public health concern. The aim of this study was to decipher the potential of bovine udder associated S. aureus as reservoir for S. aureus contamination in dairy production and processing. From 18 farms, delivering their milk to an alpine dairy plant for the production of smeared semi-hard and hard cheese. 1176 quarter milk (QM samples of all cows in lactation (n = 294 and representative samples form bulk tank milk (BTM of all farms were surveyed for coagulase positive (CPS and coagulase negative Staphylococci (CNS. Furthermore, samples from different steps of the cheese manufacturing process were tested for CPS and CNS. As revealed by chemometric-assisted FTIR spectroscopy and molecular subtyping (spa typing and multi locus sequence typing, dairy cattle represent indeed an important, yet underreported, entrance point of S. aureus into the dairy chain. Our data clearly show that certain S. aureus subtypes are present in primary production as well as in the cheese processing at the dairy plant. However, although a considerable diversity of S. aureus subtypes was observed in QM and BTM at the farms, only certain S. aureus subtypes were able to enter and persist in the cheese manufacturing at the dairy plant and could be isolated from cheese until day fourteen of ripening. Farm strains belonging to the FTIR cluster B1 and B3, which show genetic characteristics (t2953, ST8, enterotoxin profile: sea/sed/sej of the recently described S. aureus genotype B, most successfully contaminated the cheese production at the dairy plant. Thus our study fosters the hypothesis that genotype B S. aureus represent a specific challenge in control of S. aureus in the dairy chain that requires effective clearance strategies and hygienic

Detection of E6/E7 HPV oncogene transcripts as biomarker of cervical intaepithelial displasia

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Mauro Carcheri

2009-09-01

Full Text Available It is widely accepted that only persistent infection with high risk types of Human Papillomavirus (HPV HR is a significant risk factor for the development of an invasive squamous cervical cancer. The overexpression of viral oncogenes E6/E7 of HPV is considered a necessary process for incurring in a malignant phenotype.A HPV infection can be identified by detection of HPV DNA in biological samples, but the DNAbased tests cannot delineate between transient or persistent and potentially transforming infection. Instead there is many evidence to suggest that detection of HPV gene expression may constitute a more specific approach to highlight a clinically significant infection. Especially seems that the detection of E6/E7 transcripts can be usefully used for identify the women with a persistent HPV infection that will can induce a future cervical cancer. The aim of our study is to investigate if the detection of oncogenic viral gene activity by detecting transcripts of the E6 and E7 genes can be most usefull of HPV-DNA test in the triage of ASCUS or low grade cervical lesions. Our results confirm that HPV E6/E7 mRNA test can be considered a promising method to stratify HPV positive women for risk of future high-grade cervical lesions or cervical intaepithelial neoplasia.

Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification

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D Wang

2014-01-01

Full Text Available Purpose : The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD for an early treatment by using loop-mediated isothermal amplification (LAMP technique. Materials and Methods : A reverse-transcription loop-mediated isothermal amplification (RT-LAMP for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conventional reverse-transcription polymerase chain reaction (RT-PCR and real-time PCR. Results : A total of 116 clinical specimens from the suspected HFMD individual were detected with the RT-LAMP. The detection rate for EV71 was 56.89% by RT-LAMP, 41.38% by real-time PCR and 34.48% by RT-PCR. The minimum detection limit of RT-LAMP was 0.01 PFU, both of RT-PCR and real-time PCR was 0.1PFU. Non-cross-reactive amplification with other enteroviruses was detected in the survey reports. Conclusions : The effectiveness of RT-LAMP is higher than RT-PCR and real-time PCR. The protocol is easy to operate and time saving. It was not an expensive instrument, which was needed; it is an applicable method for rapid diagnosis of the disease, especially in resource-poor countries or in developing countries.

The effect of improved compliance with hygiene guidelines on transmission of Staphylococcus aureus to newborn infants: the Swedish Hygiene Intervention and Transmission of S aureus study.

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Mernelius, Sara; Löfgren, Sture; Lindgren, Per-Eric; Blomberg, Marie; Olhager, Elisabeth; Gunnervik, Christina; Lenrick, Raymond; Thrane, Malena Tiefenthal; Isaksson, Barbro; Matussek, Andreas

2013-07-01

Newborn infants are often colonized with Staphylococcus aureus originating from health care workers (HCWs). We therefore use colonization with S aureus of newborn infants to determine the effect of an improved compliance with hygiene guidelines on bacterial transmission. Compliance with hygiene guidelines was monitored prior to (baseline) and after (follow-up) a multimodal hygiene intervention in 4 departments of obstetrics and gynecology. spa typing was used to elucidate transmission routes of S aureus collected from newborn infants, mothers, fathers, staff members, and environment. The compliance with hygiene guidelines increased significantly from baseline to follow-up. The transmission of S aureus from HCWs to infants was however not affected. Fathers had the highest colonization rates. Persistent carriage was indicated in 18% of the HCWs. The most commonly isolated spa type was t084, which was not detected in a previous study from the same geographic area. It is possible to substantially improve the compliance with hygiene guidelines, by using multimodal hygiene intervention. The improved compliance did not decrease the transmission of S aureus from sources outside the own family to newborn infants. Furthermore, we show the establishment of a new spa type (t084), which now is very common in our region. Copyright © 2013 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection

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Denayer, Sarah; Nia, Yacine; Botteldoorn, Nadine

2017-01-01

Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented. PMID:29261162

Food-Borne Outbreak Investigation and Molecular Typing: High Diversity of Staphylococcus aureus Strains and Importance of Toxin Detection.

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Denayer, Sarah; Delbrassinne, Laurence; Nia, Yacine; Botteldoorn, Nadine

2017-12-20

Staphylococcus aureus is an important aetiological agent of food intoxications in the European Union as it can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Reported enterotoxin dose levels causing food-borne illness are scarce and varying. Three food poisoning outbreaks due to enterotoxin-producing S. aureus strains which occurred in 2013 in Belgium are described. The outbreaks occurred in an elderly home, at a barbecue event and in a kindergarten and involved 28, 18, and six cases, respectively. Various food leftovers contained coagulase positive staphylococci (CPS). Low levels of staphylococcal enterotoxins ranging between 0.015 ng/g and 0.019 ng/g for enterotoxin A (SEA), and corresponding to 0.132 ng/g for SEC were quantified in the food leftovers for two of the reported outbreaks. Molecular typing of human and food isolates using pulsed-field gel electrophoresis (PFGE) and enterotoxin gene typing, confirmed the link between patients and the suspected foodstuffs. This also demonstrated the high diversity of CPS isolates both in the cases and in healthy persons carrying enterotoxin genes encoding emetic SEs for which no detection methods currently exist. For one outbreak, the investigation pointed out to the food handler who transmitted the outbreak strain to the food. Tools to improve staphylococcal food poisoning (SFP) investigations are presented.

PCR assay with host specific internal control forStaphylococcus aureus from bovine milk samples

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Zafer Cantekin

2015-03-01

Full Text Available Staphylococcus aureus is considered as one of the most important and common pathogens of bovine mastitis. Polymerase Chain Reaction is frequently proposed in the diagnosis of S. aureus directly from milk samples instead of classical culture. However, false-negative results may occur in the polymerase chain reaction analysis performed directly from clinical material. For the purpose of disclosing the false negative results, the use of internal amplification controls can be beneficial. Therefore, in this study a new polymerase chain reaction technique with host specific internal amplification control was developed by optimizing S. aureus-specific primers in combination with bovine specific primers. The effectiveness of the developed technique in this study was attempted in milk samples from bovine subclinical mastitis. This technique has the potential to detect S. aureus from bovine milk samples or dairy products.

Distribution of mecA gene amongst Staphylococcus aureus isolates

African Journals Online (AJOL)

Dr Olaleye

Antibiotics susceptibility testing including methicillin sensitivity testing, beta lactamase testing, PCR for detection of mecA gene, and minimum inhibitory concentrations to methicillin were carried out on all the 194 isolates of S. aureus. Among the 194 strains, 40 (20.6%) were MRSA using 10 µg methicillin disc. PCR analysis.

PCR múltiple para la detección de los genes sea, seb, sec, sed y see de Staphylococcus aureus: Caracterización de aislamientos de origen alimentario Multiplex PCR for the detection of sea, seb, sec, sed and see genes of Staphylococcus aureus: Characterization of isolates from food

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E. A. Manfredi

2010-09-01

Full Text Available La presencia de Staphylococcus aureus en los alimentos representa un riesgo potencial para la salud pública; sus enterotoxinas son el principal factor de virulencia. La detección de las enterotoxinas de S. aureus puede realizarse por ELISA, aunque sólo es posible detectar el pool de enterotoxinas SEA, SEB, SEC, SED y SEE. Los objetivos del presente trabajo fueron optimizar dos técnicas de PCR múltiple para la detección de los genes sea, seb, sec, sed y see de S. aureus y caracterizar un conjunto de 115 aislamientos de Staphylococcus spp. asociados a intoxicaciones alimentarias provenientes de diferentes provincias de Argentina. La caracterización se realizó por pruebas bioquímicas, ELISA y PCR. Sesenta y ocho aislamientos (59,1% fueron positivos por ELISA, mientras que 61 (53% fueron positivos por PCR. De los aislamientos positivos por PCR, 34 (55,7% portaron el gen sea, 9 (14,8% el gen seb, 5 (8,1% el gen see, 4 (6,5% el gen sec, 6 (9,9% los genes sea y seb, 2 (3,3% los genes sea y sec, y 1 (1,7% los genes sea y sed. Este es el primer estudio de caracterización genotípica de aislamientos de S. aureus asociados con brotes de intoxicación alimentaria registrados en distintas provincias argentinas.The presence of Staphylococcus aureus in food represents a potential risk to public health, being its enterotoxins the major virulence factor. Enterotoxin detection can be determined by ELISA, but only for the pool of enterotoxins SEA, SEB, SEC, SED and SEE. The main aims of this study were to optimize two PCR techniques for detection of S. aureus sea, seb, sec, sed and see, and to characterize Staphylococcus spp. isolates associated with food intoxication. Two PCR techniques were optimized and 115 Staphylococcus spp. isolates from Ciudad Autónoma de Buenos Aires, and Buenos Aires, Córdoba, and Neuquén provinces were characterized. The characterization was performed by biochemical tests, ELISA and PCR. Sixty-eight isolates (59.1% were

A common variant of staphylococcal cassette chromosome mec type IVa in isolates from Copenhagen, Denmark, is not detected by the BD GeneOhm methicillin-resistant Staphylococcus aureus assay

DEFF Research Database (Denmark)

Bartels, Mette Damkjaer; Boye, Kit; Rohde, Susanne Mie

2009-01-01

Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Cop...

Mechanisms of methicillin resistance in Staphylococcus aureus and methods for laboratory detection.

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Jorgensen, J H

1991-01-01

Three distinctly different mechanisms of methicillin resistance have been described in Staphylococcus aureus. The best-documented and probably most important mechanism is production of a unique, low affinity penicillin-binding protein, PBP 2a. Strains possessing PBP 2a are resistant to methicillin, oxacillin, and probably all other currently available beta-lactam antibiotics. Two additional mechanisms of reduced susceptibility to methicillin have been described. Borderline resistance (BORSA) to the semi-synthetic penicillins has been attributed to the hyperproduction of normal staphylococcal beta-lactamase. A third mechanism has recently been advanced that describes an intermediate level of resistance to methicillin due to production of modified, normal PBPs with reduced affinity for beta-lactams (MODSA). Little is known regarding the prevalence or clinical significance of the BORSA and MODSA strains. The most reliable in vitro susceptibility test methods for detecting MRSA (strains possessing PBP 2a) include the microdilution minimum inhibitory concentration (MIC) test (with 2% NaCl supplemented broth), the oxacillin agar screen plate test (incorporating 6 micrograms/ml oxacillin in 4% NaCl supplemented agar), and the National Committee for Clinical Laboratory Standards (NCCLS) disk diffusion test with oxacillin. All three methods use direct inoculum preparation and incubation of tests at 35 degrees C for a full 24 hours.

Evaluación de cuatro métodos para la detección de Staphylococcus aureus meticilino-resistente de muestras clínicas en un hospital regional Evaluation of four methods for detecting methicillin-resistant Staphylococcus aureus isolates from clinical specimens at a regional hospital in Mexico

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Gabriel Acosta-Pérez

2012-02-01

Full Text Available OBJETIVO: Investigar la prevalencia de Staphylococcus aureus meticilino-resistente (MRSA en aislados clínicos y determinar la concordancia entre los métodos de detección de MRSA en un laboratorio con recursos y personal limitado. MATERIAL Y MÉTODOS: Se analizaron 140 cepas de Staphylococcus aureus aisladas de muestras clínicas de diferentes departamentos mediante pruebas convencionales: producción de β-lactamasa, sensibilidad a oxacilina con MIC-Vitek 2-XL, ChromID MRSA, difusión en agar para discos de 30 μg de cefoxitina, detección de PBP2a y PCR para el gen mecA. Se determinó el índice kappa de Cohen, para evaluar la concordancia entre los diferentes métodos utilizados. RESULTADOS: La prevalencia encontrada fue de 90.7%. La sensibilidad y especificidad para los diferentes métodos de detección fue: difusión en disco para cefoxitina 97 y 92% respectivamente, MIC Vitek 2-XL 97 y 69%, ChromoID MRSA 97 y 85% y detección de PBP2a 98 y 100%. CONCLUSIONES: Todos los métodos son muy buenos para la detección de MRSA; la elección en el uso de cada método dependerá de la infraestructura de cada laboratorio.OBJETIVE: To estimate the prevalence of methicillin-resistant Staphylococcus aureus (MRSA in clinical isolates and to compare different methods for detection of MRSA in a lab with limited available personnel and resources. MATERIAL AND METHODS: 140 Staphylococcus aureus strains isolated from patients in several departments were assayed for β-lactamase production, MIC-Vitek 2 oxacillin, ChromID MRSA, disk diffusion in agar for cefoxitin 30 μg and PBP2a detection. The results of conventional tests were compared with the "gold standard" PCR test for mecA gene. Cohen´s kappa index was also calculated in order to evaluate the intra assay agreement between the used methods. RESULTS: The found prevalence was 90.7%. Sensitivity and specificity were: disk diffusion for cefoxitin 97 and 92% respectively, MIC Vitek 2-XL 97 and 69%, Chromo

Prevalence and characterisation of Staphylococcus aureus causing community-acquired skin and soft tissue infections on Java and Bali, Indonesia.

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Santosaningsih, Dewi; Santoso, Sanarto; Setijowati, Nanik; Rasyid, Harun A; Budayanti, Nyoman S; Suata, Ketut; Widhyatmoko, Dicky B; Purwono, Priyo B; Kuntaman, Kuntaman; Damayanti, Damayanti; Prakoeswa, Cita R S; Laurens, Mitchell; van Nierop, Josephine W I; Nanninga, Geraldine L; Oudenes, Neline; de Regt, Michelle; Snijders, Susan V; Verbrugh, Henri A; Severin, Juliëtte A

2018-01-01

To define the role of Staphylococcus aureus in community settings among patients with skin and soft tissue infections (SSTI) in Indonesia. Staphylococcus aureus were cultured from anterior nares, throat and wounds of 567 ambulatory patients presenting with SSTI. The mecA gene and genes encoding Panton-Valentine leukocidin (PVL; lukF-PV and lukS-PV) and exfoliative toxin (ET; eta and etb) were determined by PCR. Clonal relatedness among methicillin-resistant S. aureus (MRSA) and PVL-positive S. aureus was analysed using multilocus variable-number tandem-repeat analysis (MLVA) typing, and multilocus sequence typing (MLST) for a subset of isolates. Staphylococcal cassette chromosome mec (SCCmec) was determined for all MRSA isolates. Moreover, determinants for S. aureus SSTI, and PVL/ET-positive vs PVL/ET-negative S. aureus were assessed. Staphylococcus aureus were isolated from SSTI wounds of 257 (45.3%) patients, eight (3.1%) of these were MRSA. Genes encoding PVL and ETs were detected in 21.8% and 17.5% of methicillin-susceptible S. aureus (MSSA), respectively. PVL-positive MRSA was not detected. Nasopharyngeal S. aureus carriage was an independent determinant for S. aureus SSTI (odds ratio [OR] 1.8). Primary skin infection (OR 5.4) and previous antibiotic therapy (OR 3.5) were associated with PVL-positive MSSA. Primary skin infection (OR 2.2) was the only factor associated with ET-positive MSSA. MLVA typing revealed two more prevalent MSSA clusters. One ST1-MRSA-SCCmec type IV isolate and a cluster of ST239-MRSA-SCCmec type III were found. Community-acquired SSTI in Indonesia was frequently caused by PVL-positive MSSA, and the hospital-associated ST239-MRSA may have spread from the hospital into the community. © 2017 John Wiley & Sons Ltd.

Effect of a glucose impulse on the CcpA regulon in Staphylococcus aureus

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Engelmann Susanne

2009-05-01

Full Text Available Abstract Background The catabolite control protein A (CcpA is a member of the LacI/GalR family of transcriptional regulators controlling carbon-metabolism pathways in low-GC Gram-positive bacteria. It functions as a catabolite repressor or activator, allowing the bacteria to utilize the preferred carbon source over secondary carbon sources. This study is the first CcpA-dependent transcriptome and proteome analysis in Staphylococcus aureus, focussing on short-time effects of glucose under stable pH conditions. Results The addition of glucose to exponentially growing S. aureus increased the expression of genes and enzymes of the glycolytic pathway, while genes and proteins of the tricarboxylic acid (TCA cycle, required for the complete oxidation of glucose, were repressed via CcpA. Phosphotransacetylase and acetate kinase, converting acetyl-CoA to acetate with a concomitant substrate-level phosphorylation, were neither regulated by glucose nor by CcpA. CcpA directly repressed genes involved in utilization of amino acids as secondary carbon sources. Interestingly, the expression of a larger number of genes was found to be affected by ccpA inactivation in the absence of glucose than after glucose addition, suggesting that glucose-independent effects due to CcpA may have a particular impact in S. aureus. In the presence of glucose, CcpA was found to regulate the expression of genes involved in metabolism, but also that of genes coding for virulence determinants. Conclusion This study describes the CcpA regulon of exponentially growing S. aureus cells. As in other bacteria, CcpA of S. aureus seems to control a large regulon that comprises metabolic genes as well as virulence determinants that are affected in their expression by CcpA in a glucose-dependent as well as -independent manner.

A Dual Filtration-Based Multiplex PCR Method for Simultaneous Detection of Viable Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus on Fresh-Cut Cantaloupe.

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Ke Feng

Full Text Available Fresh-cut cantaloupe is particularly susceptible to contamination with pathogenic bacteria, such as Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus. Therefore, development of rapid, yet accurate detection techniques is necessary to ensure food safety. In this study, a multiplex PCR system and propidium monoazide (PMA concentration were optimized to detect all viable pathogens in a single tube. A dual filtration system utilized a filtration membrane with different pore sizes to enrich pathogens found on fresh-cut cantaloupe. The results revealed that an optimized multiplex PCR system has the ability to effectively detect three pathogens in the same tube. The viable pathogens were simultaneously detected for PMA concentrations above 10 μg/ml. The combination of a nylon membrane (15 μm and a micro pore filtration membrane (0.22 μm formed the dual filtration system used to enrich pathogens. The achieved sensitivity of PMA-mPCR based on this dual filtration system was 2.6 × 103 cfu/g for L. monocytogenes, 4.3 × 10 cfu/g for E. coli O157:H7, and 3.1 × 102 cfu/g for S. aureus. Fresh-cut cantaloupe was inoculated with the three target pathogens using concentrations of 103, 102, 10, and 1 cfu/g. After 6-h of enrichment culture, assay sensitivity increased to 1 cfu/g for each of these pathogens. Thus, this technique represents an efficient and rapid detection tool for implementation on fresh-cut cantaloupe.

Diversity of Staphylococcus aureus Isolates in European Wildlife

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Monecke, Stefan; Gavier-Widén, Dolores; Hotzel, Helmut; Peters, Martin; Guenther, Sebastian; Lazaris, Alexandros; Loncaric, Igor; Müller, Elke; Reissig, Annett; Ruppelt-Lorz, Antje; Shore, Anna C.; Walter, Birgit; Coleman, David C.; Ehricht, Ralf

2016-01-01

Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST). The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963), some of which (ST2425, ST2691, ST2963) were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6). mecC-MRSA (n = 8) were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle-associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock-associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation. PMID:27992523

Influence of Host Genetics and Environment on Nasal Carriage of Staphylococcus aureus in Danish Middle-Aged and Elderly Twins

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Andersen, Paal Skytt; Pedersen, Jacob Krabbe; Fode, Peder

2012-01-01

Background. Nasal carriage is a major risk factor for Staphylococcus aureus infection. Approximately, one-quarter of adults carry S. aureus. However, the role of host genetics on S. aureus nasal carriage is unknown. Methods. Nasal swabs were obtained from a national cohort of middle-aged and elde......Background. Nasal carriage is a major risk factor for Staphylococcus aureus infection. Approximately, one-quarter of adults carry S. aureus. However, the role of host genetics on S. aureus nasal carriage is unknown. Methods. Nasal swabs were obtained from a national cohort of middle.......4%-34.5%), and opposite sex (21.4%; 95% CI, 12.0%-33.4%) dizygotic twins. Despite shared childhoods, only 1 of 617 pairs was concordant with respect to lineage. Although heritability increased for S. aureus and lineage persistency, no significant heritability was detected. Conclusion. In this study, host genetic factors...

The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

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John M Morrison

2012-03-01

Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

Virulence Genes of S. aureus from Dairy Cow Mastitis and Contagiousness Risk.

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Magro, Giada; Biffani, Stefano; Minozzi, Giulietta; Ehricht, Ralf; Monecke, Stefan; Luini, Mario; Piccinini, Renata

2017-06-21

Staphylococcus aureus ( S. aureus ) is a major agent of dairy cow intramammary infections: the different prevalences of mastitis reported might be related to a combination of S. aureus virulence factors beyond host factors. The present study considered 169 isolates from different Italian dairy herds that were classified into four groups based on the prevalence of S. aureus infection at the first testing: low prevalence (LP), medium-low (MLP), medium-high (MHP) and high (HP). We aimed to correlate the presence of virulence genes with the prevalence of intramammary infections in order to develop new strategies for the control of S. aureus mastitis. Microarray data were statistically evaluated using binary logistic regression and correspondence analysis to screen the risk factors and the relationship between prevalence group and gene. The analysis showed: (1) 24 genes at significant risk of being detected in all the herds with infection prevalence >5%, including genes belonging to microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), immune evasion and serine proteases; and (2) a significant correlation coefficient between the genes interacting with the host immune response and HP isolates against LP ones. These results support the hypothesis that virulence factors, in addition to cow management, could be related to strain contagiousness, offering new insights into vaccine development.

Virulence Genes of S. aureus from Dairy Cow Mastitis and Contagiousness Risk

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Giada Magro

2017-06-01

Full Text Available Staphylococcus aureus (S. aureus is a major agent of dairy cow intramammary infections: the different prevalences of mastitis reported might be related to a combination of S. aureus virulence factors beyond host factors. The present study considered 169 isolates from different Italian dairy herds that were classified into four groups based on the prevalence of S. aureus infection at the first testing: low prevalence (LP, medium–low (MLP, medium–high (MHP and high (HP. We aimed to correlate the presence of virulence genes with the prevalence of intramammary infections in order to develop new strategies for the control of S. aureus mastitis. Microarray data were statistically evaluated using binary logistic regression and correspondence analysis to screen the risk factors and the relationship between prevalence group and gene. The analysis showed: (1 24 genes at significant risk of being detected in all the herds with infection prevalence >5%, including genes belonging to microbial surface components recognizing adhesive matrix molecules (MSCRAMMs, immune evasion and serine proteases; and (2 a significant correlation coefficient between the genes interacting with the host immune response and HP isolates against LP ones. These results support the hypothesis that virulence factors, in addition to cow management, could be related to strain contagiousness, offering new insights into vaccine development.

Allele-dependent differences in quorum-sensing dynamics result in variant expression of virulence genes in Staphylococcus aureus.

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Geisinger, Edward; Chen, John; Novick, Richard P

2012-06-01

Agr is an autoinducing, quorum-sensing system that functions in many Gram-positive species and is best characterized in the pathogen Staphylococcus aureus, in which it is a global regulator of virulence gene expression. Allelic variations in the agr genes have resulted in the emergence of four quorum-sensing specificity groups in S. aureus, which correlate with different strain pathotypes. The basis for these predilections is unclear but is hypothesized to involve the phenomenon of quorum-sensing interference between strains of different agr groups, which may drive S. aureus strain isolation and divergence. Whether properties intrinsic to each agr allele directly influence virulence phenotypes within S. aureus is unknown. In this study, we examined group-specific differences in agr autoinduction and virulence gene regulation by utilizing congenic strains, each harboring a unique S. aureus agr allele, enabling a dissection of agr locus-dependent versus genotype-dependent effects on quorum-sensing dynamics and virulence factor production. Employing a reporter fusion to the principal agr promoter, P3, we observed allele-dependent differences in the timing and magnitude of agr activation. These differences were mediated by polymorphisms within the agrBDCA genes and translated to significant variations in the expression of a key transcriptional regulator, Rot, and of several important exoproteins and surface factors involved in pathogenesis. This work uncovers the contribution of divergent quorum-sensing alleles to variant expression of virulence determinants within a bacterial species.

Bats are rare reservoirs of Staphylococcus aureus complex in Gabon.

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Held, Jana; Gmeiner, Markus; Mordmüller, Benjamin; Matsiégui, Pierre-Blaise; Schaer, Juliane; Eckerle, Isabella; Weber, Natalie; Matuschewski, Kai; Bletz, Stefan; Schaumburg, Frieder

2017-01-01

The colonization of afro-tropical wildlife with Staphylococcus aureus and the derived clade Staphylococcus schweitzeri remains largely unknown. A reservoir in bats could be of importance since bats and humans share overlapping habitats. In addition, bats are food sources in some African regions and can be the cause of zoonotic diseases. Here, we present a cross-sectional survey employing pharyngeal swabs of captured and released bats (n=133) in a forest area of Gabon. We detected low colonization rates of S. aureus (4-6%) and S. schweitzeri (4%) in two out of four species of fruit bats, namely Rousettus aegyptiacus and Micropteropus pusillus, but not in insectivorous bats. Multilocus sequence typing showed that S. aureus from Gabonese bats (ST2984, ST3259, ST3301, ST3302) were distinct from major African human associated clones (ST15, ST121, ST152). S. schweitzeri from bats (ST1697, ST1700) clustered with S. schweitzeri from other species (bats, monkeys) from Nigeria and Côte d'Ivoire. In conclusion, colonization rates of bats with S. aureus and S. schweitzeri were low in our study. Phylogenetic analysis supports an intense geographical dispersal of S. schweitzeri among different mammalian wildlife hosts. Copyright © 2016 Elsevier B.V. All rights reserved.

Microarray analysis of toxicogenomic effects of Ortho-phenylphenol in Staphylococcus aureus

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Toghrol Freshteh

2008-09-01

Full Text Available Abstract Background Staphylococcus aureus (S. aureus, is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome. Ortho-phenylphenol (OPP is an antimicrobial agent and an active ingredient of EPA-registered disinfectants with wide human exposure in various agricultural, hospital and veterinary disinfectant products. Despite many uses, an understanding of a cellular response to OPP and it's mechanism of action, targeted genes, and the connectivity between targeted genes and the rest of cell metabolism remains obscure. Results Herein, we performed a genome-wide transcriptome analysis of the cellular responses of S. aureus when exposed to 0.82 mM of OPP for 20 and 60 min. Our data indicated that OPP downregulated the biosynthesis of many amino acids, which are required for protein synthesis. In particular, the genes encoding the enzymes of the diaminopimelate (DAP pathway which results in lysine biosynthesis were significantly downregualted. Intriguingly, we revealed that the transcription of genes encoding ribosomal proteins was upregulated by OPP and at the same time, the genes encoding iron acquisition and transport were downregulated. The genes encoding virulence factors were upregulated and genes encoding phospholipids were downregulated upon 20 min exposure to OPP. Conclusion By using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we have revealed novel information regarding the mode of action of OPP on Staphylococcus: OPP inhibits anabolism of many amino acids and highly downregulates the genes that encode the enzymes involved in the DAP pathway. Lysine and DAP are essential for building up the peptidoglycan cell wall. It was concluded that the mode of action of OPP is similar to the mechanism of action of some antibiotics. The discovery of this phenomenon provides useful

Phenotypic and molecular characteristics of methicillin-resistant Staphylococcus aureus isolates from Ekiti State, Nigeria

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Olowe OA

2013-08-01

Full Text Available Olugbenga Adekunle Olowe,1 Olayinka Oluwatoyin Kukoyi,2 Samuel Sunday Taiwo,1 Olusola Ojurongbe,1 Oluyinka Oladele Opaleye,1 Oloyede Samuel Bolaji,1 Abiodun Adebimpe Adegoke,1 Olufunmilola Bamidele Makanjuola,1 David Olusoga Ogbolu,3 Oyebode Terry Alli31Department of Medical Microbiology and Parasitology, College of Health Sciences, Ladoke Akintola University of Technology, Ogbomoso, Nigeria; 2Department of Microbiology, College of Sciences, Afe Babalola University, Ado-Ekiti, Nigeria; 3Department of Biomedical Sciences, College of Health Sciences, Lautech, Osogbo, NigeriaIntroduction: The characteristics and antimicrobial resistance profiles of Staphylococcus aureus differs according to geographical regions and in relation to antibiotic usage. The aim of this study was to determine the biochemical characteristics of the prevalent S. aureus from Ekiti State, Nigeria, and to evaluate three commonly used disk diffusion methods (cefoxitin, oxacillin, and methicillin for the detection of methicillin resistance in comparison with mecA gene detection by polymerase chain reaction.Materials and methods: A total of 208 isolates of S. aureus recovered from clinical specimens were included in this study. Standard microbiological procedures were employed in isolating the strains. Susceptibility of each isolate to methicillin (5 µg, oxacillin (1 µg, and cefoxitin (30 µg was carried out using the modified Kirby–Bauer/Clinical and Laboratory Standard Institute disk diffusion technique. They were also tested against panels of antibiotics including vancomycin. The conventional polymerase chain reaction method was used to detect the presence of the mecA gene.Results: Phenotypic resistance to methicillin, oxacillin, and cefoxitin were 32.7%, 40.3%, and 46.5%, respectively. The mecA gene was detected in 40 isolates, giving a methicillin-resistant S. aureus (MRSA prevalence of 19.2%. The S. aureus isolates were resistant to penicillin (82.7% and tetracycline

Identification of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from burn patients by multiplex PCR.

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Montazeri, Effat Abbasi; Khosravi, Azar Dokht; Jolodar, Abbas; Ghaderpanah, Mozhgan; Azarpira, Samireh

2015-05-01

Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MRCoNS) as important human pathogens are causes of nosocomial infections worldwide. Burn patients are at a higher risk of local and systemic infections with these microorganisms. A screening method for MRSA by using a multiplex polymerase chain reaction (PCR) targeting the 16S ribosomal RNA (rRNA), mecA, and nuc genes was developed. The aim of the present study was to investigate the potential of this PCR assay for the detection of MRSA strains in samples from burn patients. During an 11-month period, 230 isolates (53.11%) of Staphylococcus spp. were collected from burn patients. The isolates were identified as S. aureus by using standard culture and biochemical tests. DNA was extracted from bacterial colonies and multiplex PCR was used to detect MRSA and MRCoNS strains. Of the staphylococci isolates, 149 (64.9%) were identified as S. aureus and 81 (35.21%) were described as CoNS. Among the latter, 51 (62.97%) were reported to be MRCoNS. From the total S. aureus isolates, 132 (88.6%) were detected as MRSA and 17 (11.4%) were methicillin-susceptible S. aureus (MSSA). The presence of the mecA gene in all isolates was confirmed by using multiplex PCR as a gold standard method. This study presented a high MRSA rate in the region under investigation. The 16S rRNA-mecA-nuc multiplex PCR is a good tool for the rapid characterization of MRSA strains. This paper emphasizes the need for preventive measures and choosing effective antimicrobials against MRSA and MRCoNS infections in the burn units. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Thioridazine potentiates the effect of a beta-lactam antibiotic against Staphylococcus aureus independently of mecA expression

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Poulsen, Marianne Østergaard; Jacobsen, Kirstine; Bonde, Mette

2013-01-01

The neuroleptic antipsychotic derivate thioridazine has been shown to increase the susceptibility of a methicillin-resistant Staphylococcus aureus (MRSA) isolate towards dicloxacillin. The aim of this study was to investigate the combinatorial effect of the two drugs on a broad selection of staph......The neuroleptic antipsychotic derivate thioridazine has been shown to increase the susceptibility of a methicillin-resistant Staphylococcus aureus (MRSA) isolate towards dicloxacillin. The aim of this study was to investigate the combinatorial effect of the two drugs on a broad selection...... of staphylococcal strains by analyzing a large collection of MRSA strains carrying different types of SCCmec, as well as MSSA strains. Transcription and translation of the resistance marker PBP2a encoded by mecA within the SCCmec cassette were analyzed by primer extension and western blotting. We observed increased...... susceptibility to dicloxacillin in the presence of thioridazine in all tested MRSA isolates. In contrast to previously published results, the synergistic effect was also applicable to methicillin-susceptible S. aureus (MSSA). We conclude that the combination of dicloxacillin and thioridazine potentiates...

Methicillin resistant Staphylococcus aureus nasal colonization among secondary school students at Duhok City-Iraq

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Ary Habeeb

2014-06-01

Full Text Available Objective:Methicillin-resistant Staphylococcus aureus (MRSA widely distributed in hospitals around the world. There is strong relationship between disease development and S. aureus nasal carriage. The aim of this study was to evaluate the prevalence and epidemiology of nasal colonization with S. aureus and MRSA in the community of Duhok city, Iraq. Methods: 489 students aged 16 to18 years were included. Nasal swab samples were collected followed by antimicrobial susceptibility test. MRSA isolates were selected and investigated for the mecA gene. Also the prevalence of PantonValentine Leukocidin (PVL gene was also studied. Results: A total of 90 (18.4% out of 489 (18.4% of the students were found to be colonized by S. aureus . Only 10 (2.04% of the students were found to be MRSA carrier. All MRSA isolates were sensitive to Vancomycin. PLV gene was detected in one MRSA strain. Conclusion: This is the first study investigating S. aureus colonization in students in the Duhok city. Nasal carriage of S. aureus and MRSA is comparable with reports from elsewhere. Fortunately, all trains included in our study were sensitive to vancomycin. Further research is needed to examine the SCCmec elements and the evolution of MRSA over the time. J Microbiol Infect Dis 2014;4(2: 59-63

Poor survival of Methicillin-resistant Staphylococcus aureus on inanimate objects in the public spaces.

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Domon, Hisanori; Uehara, Yoshio; Oda, Masataka; Seo, Hiromi; Kubota, Noriko; Terao, Yutaka

2016-02-01

We investigated the prevalence of Staphylococcus aureus on shopping baskets in Osaka Prefecture, Japan. Multilocus sequence typing was performed to determine the genotypes of S. aureus isolates, and then a polymerase chain reaction method was used to detect staphylococcal enterotoxins and antibiotic resistance genes. In addition, desiccation tolerance of S. aureus isolates was evaluated in vitro. Forty-six (6.2%) S. aureus isolates were collected from 740 shopping baskets, though only one MRSA strain was identified. In multilocus sequence typing findings, ten sequence types and 24 singletons were classified, which were divided into ten clonal complexes and six singletons. The most frequent staphylococcal enterotoxin gene was seg (30.4%). Our in vitro findings demonstrated that 70% of the S. aureus isolates, including the MRSA strain, became undetectable at 12 h after desiccation at an appropriate cell density, while the others remained viable for up to 24 h. Thus, it is difficult for MRSA organisms to survive on dry surfaces found in public areas. We speculated that inanimate objects in the community are unlikely to be a potential source for transmission of MRSA and that S. aureus on such objects outside of hospital settings is not a public health threat. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Staphylococcus aureus Nasal Colonization Differs among Pig Lineages and Is Associated with the Presence of Other Staphylococcal Species.

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Verstappen, Koen M; Willems, Eveline; Fluit, Ad C; Duim, Birgitta; Martens, Marc; Wagenaar, Jaap A

2017-01-01

Staphylococcus aureus is a common colonizer in pigs, with methicillin-resistant S. aureus (MRSA) in particular being a potential health risk to humans. To reduce the exposure to humans, the colonization in pigs should be reduced. The aim of this study was to quantitatively compare the susceptibility of pig lineages for S. aureus colonization, and if the absence of S. aureus could be associated with the presence or absence of other staphylococcal species. Nasal samples ( n  = 129) were obtained from seven different pig lineages in the Netherlands, France, and Germany. S. aureus and other staphylococci were enumerated from these samples by real-time (RT)-PCR and culture. Associations were explored between the presence of S. aureus and other staphylococci. S. aureus was detected by RT-PCR on all farms and in samples from pigs of all lineages. Twenty-five percent of the pigs from lineage F (from two farms) were colonized with S. aureus , while in all other lineages it was more than 50% ( p  Staphylococcus sciuri, Staphylococcus cohnii , and Staphylococcus saprophyticus were usually not found in combination with S. aureus in these samples. (i) pigs from different genetic lineages have different susceptibilities for colonization with S. aureus . These pigs might contain a genetic factor influencing nasal colonization. (ii) Colonization of S. aureus is also associated with the absence of S. sciuri, S. cohnii , or S. saprophyticus . (iii) The farm environment seems to influence the presence of S. aureus in pigs.

Detection of Intracellular Adhesion (ica and Biofilm Formation Genes in Staphylococcus aureus Isolates from Clinical Samples

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Khadije Rezaie Keikhaie

2017-02-01

Full Text Available Introduction: Nosocomial infections that result in the formation of biofilms on the surfaces of biomedical implants are a leading cause of sepsis and are often associated with colonization of the implants by Staphylococcus epidermidis. Biofilm formation is thought to require two sequential steps: adhesion of cells to a solid substrate followed by cell-cell adhesion, creating multiple layers of cells. Intercellular adhesion requires the polysaccharide intercellular adhesion (PIA, which is composed of linear β-1, 6-linked glucosaminylglycans and can be synthesized in vitro from UDP-N-acetylglucosamine by products of the intercellular adhesion (ica locus. We have investigated a variety of Staphylococcus aureus strains and find that all strains tested contain the ica locus and that several can form biofilms in vitro. Material and Method: A total of 31 clinical S. aureus isolates were collected from Zabol, Iran. In vitro biofilm formation ability was determined by microliter tissue culture plates. All clinical isolates were examined for determination the ica locus by using PCR method. Result: The results of this study showed that 40 strains of Staphylococcus aureus, 12 strains carrying the gene Cocos icaA (30% and 8 strains carrying the gene icaD (20% and the number of five strains (12.5% containing both genes ica A and has been ica D. Conclusions:  S. aureus clinical isolates have different ability to form biofilm. This may be caused by the differences in the expression of biofilm related genes, genetic make-up and physiological conditions.

Molecular Studies on Methicillin-Resistant Staphylococcus aureus

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Tawfik, Z.S.; Mohamed, H.A.; Abu Shady, H.M.; Abu Shady, M.R.

2011-01-01

The present study of the MecA gene in our clinical isolates has been detected and verified by antibiotic disc diffusion test and nested Polymerase Chain Reaction (PCR). Part of the product of the second PCR was also sequenced. The results indicated 97.7% similarity between the sequences of the mecA gene isolated from an Egyptian Staphylococcus aureus strain and that compared from Staphylococcus aureus strain no. GI46628 cited at the European Molecular Biology Laboratory (EMBL) database under accession number Y00688 in the region from nucleotide 467 to 875. The effect of gamma-radiation on these isolates was observed by plotting the dose survival curves of these isolates and determining their D10 values. Their D10 values were found to be ranged from 0.44 to 0.66 kGy. Antibiotic sensitivity tests were also carried out after exposure of Oxacillin-susceptible isolate to sub -lethal doses of γ-radiation.Results indicated that Staphylococcus aureus isolates which were sensitive to oxacillin discs were found by PCR to harbor the mecA gene in their genomes. Also, exposure of a sensitive isolate to sublethal doses of gamma radiation led to the emergence of a oxacillin-resistant variant which could be a serious problem in case of using sub-lethal doses of radiation for the sterilization of medical products

Antibiofilm activity of cashew juice pulp against Staphylococcus aureus, high performance liquid chromatography/diode array detection and gas chromatography-mass spectrometry analyses, and interference on antimicrobial drugs

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Marcus V. Dias-Souza

2017-07-01

Full Text Available The epidemiology of Staphylococcus aureus infections has evolved in recent years, as this species is a major Gram-positive pathogen associated with healthcare services. The antimicrobial resistance of this species raises an urgent need for new treatment strategies. Fruits play important nutritional and economic roles in society, but their biological and pharmacological features are poorly explored when compared to nonedible parts of plants such as barks and leaves. In this study, we show that the cashew apple juice [cashew juice pulp (CJP] extract is active against the planktonic cells of S. aureus strains, and for the first time, we show that CJP is also active against S. aureus biofilms. High performance liquid chromatography and gas chromatography-mass spectrometry analyses were conducted to prospect for polyphenols and free carbohydrates, respectively. Cashew apple juice, which is rich in nutrients, is widely consumed in Brazil; therefore, the quality attributes of CJPs were investigated. Samples were evaluated for pH, total titratable acidity, vitamin C levels, and total soluble solids. We also detected an antagonistic interference of CJP when it was combined with different antimicrobial drugs.

Cytokine responses to Staphylococcus aureus bloodstream infection differ between patient cohorts that have different clinical courses of infection.

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McNicholas, Sinead; Talento, Alida Fe; O'Gorman, Joanne; Hannan, Margaret M; Lynch, Maureen; Greene, Catherine M; Humphreys, Hilary; Fitzgerald-Hughes, Deirdre

2014-11-15

The clinical course of Staphylococcus aureus bloodstream infection is unpredictable and bacterial virulence, host immune response and patient characteristics are among the factors that contribute to the clinical course of infection. To investigate the relationship between cytokine response and clinical outcome, circulating cytokine levels were investigated in response to S. aureus bloodstream infection in patients with different clinical courses of infection. A prospective study was carried out in 61 patients with S. aureus bloodstream infection and circulating levels of IL-6, GRO-γ, RANTES and leptin were assessed over the course of the infection. Levels were compared in patients with complicated courses of infection (e.g. infective endocarditis) versus uncomplicated courses of S. aureus bloodstream infection and methicillin-resistant S. aureus Vs methicillin-susceptible S. aureus infection. Significantly lower leptin levels (p < 0.05) and significantly higher IL-6 levels (p < 0.05) were detected at laboratory diagnosis in patients with complicated compared to uncomplicated S. aureus bloodstream infection. Significantly higher levels of GRO-γ were associated with MRSA infection compared to MSSA infection. IL-6 may be an early inflammatory marker of complicated S. aureus bloodstream infection. Leptin may be protective against the development of a complicated S. aureus bloodstream infection.

Comparison of air samples, nasal swabs, ear-skin swabs and environmental dust samples for detection of Methicillin Resistant Staphylococcus aureus (MRSA) in pig herds

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Agersø, Yvonne; Vigre, Håkan; Cavaco, Lina

2014-01-01

To identify a cost-effective and practical method for detection of methicillin-resistant Staphylococcus aureus (MRSA) in pig herds, the relative sensitivity of four sample types: nasal swabs, ear-skin (skin behind the ears) swabs, environmental dust swabs and air was compared. Moreover, dependency......-herd prevalence ⩾25%]. The results indicate that taking swabs of skin behind the ears (ten pools of five) was even more sensitive than taking nasal swabs (ten pools of five) at the herd level and detected significantly more positive samples. spa types t011, t034 and t4208 were observed. In conclusion, MRSA...... detection by air sampling is easy to perform, reduces costs and analytical time compared to existing methods, and is recommended for initial testing of herds. Ear-skin swab sampling may be more sensitive for MRSA detection than air sampling or nasal swab sampling....

Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates.

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Galán-Sánchez, Fátima; Pérez-Eslava, Maria; Machuca, Jesús; Trujillo-Soto, Teresa; Arca-Suarez, Jorge; Rodríguez-Iglesias, Manuel

2018-06-20

The epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. A cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton-Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. A total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. A low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA. Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac

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2011-01-01

Background Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. Methods Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. Results Growth of S. aureus strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. Conclusions The results of

Rapid detection and semi-quantification of IgG-accessible Staphylococcus aureus surface-associated antigens using a multiplex competitive Luminex assay

NARCIS (Netherlands)

Hansenova Manaskova, S.; Bikker, F.J.; Veerman, E.C.I.; van Belkum, A.; van Wamel, W.J.B.

2013-01-01

The surface characterization of Staphylococcus aureus is currently labor intensive and time consuming. Therefore, we developed a novel method for the rapid yet comprehensive characterization of S. aureus cell-surface-associated proteins and carbohydrates, based on a competitive Luminex assay. In

Staphylococcus aureus Nasal Colonization Differs among Pig Lineages and Is Associated with the Presence of Other Staphylococcal Species

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Koen M. Verstappen

2017-06-01

Full Text Available Staphylococcus aureus is a common colonizer in pigs, with methicillin-resistant S. aureus (MRSA in particular being a potential health risk to humans. To reduce the exposure to humans, the colonization in pigs should be reduced. The aim of this study was to quantitatively compare the susceptibility of pig lineages for S. aureus colonization, and if the absence of S. aureus could be associated with the presence or absence of other staphylococcal species. Nasal samples (n = 129 were obtained from seven different pig lineages in the Netherlands, France, and Germany. S. aureus and other staphylococci were enumerated from these samples by real-time (RT-PCR and culture. Associations were explored between the presence of S. aureus and other staphylococci. S. aureus was detected by RT-PCR on all farms and in samples from pigs of all lineages. Twenty-five percent of the pigs from lineage F (from two farms were colonized with S. aureus, while in all other lineages it was more than 50% (p < 0.01. Moreover, in S. aureus-positive samples from pigs of lineage F smaller amounts of S. aureus were found than in other lineages. Staphylococcus sciuri, Staphylococcus cohnii, and Staphylococcus saprophyticus were usually not found in combination with S. aureus in these samples. In conclusion: (i pigs from different genetic lineages have different susceptibilities for colonization with S. aureus. These pigs might contain a genetic factor influencing nasal colonization. (ii Colonization of S. aureus is also associated with the absence of S. sciuri, S. cohnii, or S. saprophyticus. (iii The farm environment seems to influence the presence of S. aureus in pigs.

The MazEF Toxin-Antitoxin System Alters the β-Lactam Susceptibility of Staphylococcus aureus.

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Christopher F Schuster

Full Text Available Toxin-antitoxin (TA systems are genetic elements of prokaryotes which encode a stable toxin and an unstable antitoxin that can counteract toxicity. TA systems residing on plasmids are often involved in episomal maintenance whereas those on chromosomes can have multiple functions. The opportunistic pathogen Staphylococcus aureus possesses at least four different families of TA systems but their physiological roles are elusive. The chromosomal mazEF system encodes the RNase toxin MazF and the antitoxin MazE. In the light of ambiguity regarding the cleavage activity, we here verify that MazF specifically targets UACAU sequences in S. aureus in vivo. In a native strain background and under non-stress conditions, cleavage was observed in the absence or presence of mazE. Transcripts of spa (staphylococcal protein A and rsbW (anti-σB factor were cut, but translational reporter fusions indicated that protein levels of the encoded products were unaffected. Despite a comparable growth rate as the wild-type, an S. aureus mazEF deletion mutant was more susceptible to β-lactam antibiotics, which suggests that further genes, putatively involved in the antibiotic stress response or cell wall synthesis or turnover, are controlled by this TA system.

Staphylococcus aureus α-toxin modulates skin host response to viral infection.

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Bin, Lianghua; Kim, Byung Eui; Brauweiler, Anne; Goleva, Elena; Streib, Joanne; Ji, Yinduo; Schlievert, Patrick M; Leung, Donald Y M

2012-09-01

Patients with atopic dermatitis (AD) with a history of eczema herpeticum have increased staphylococcal colonization and infections. However, whether Staphylococcus aureus alters the outcome of skin viral infection has not been determined. We investigated whether S aureus toxins modulated host response to herpes simplex virus (HSV) 1 and vaccinia virus (VV) infections in normal human keratinocytes (NHKs) and in murine infection models. NHKs were treated with S aureus toxins before incubation of viruses. BALB/c mice were inoculated with S aureus 2 days before VV scarification. Viral loads of HSV-1 and VV were evaluated by using real-time PCR, a viral plaque-forming assay, and immunofluorescence staining. Small interfering RNA duplexes were used to knockdown the gene expression of the cellular receptor of α-toxin, a disintegrin and metalloprotease 10 (ADAM10). ADAM10 protein and α-toxin heptamers were detected by using Western blot assays. We demonstrate that sublytic staphylococcal α-toxin increases viral loads of HSV-1 and VV in NHKs. Furthermore, we demonstrate in vivo that the VV load is significantly greater (P skin inoculated with an α-toxin-producing S aureus strain compared with murine skin inoculated with the isogenic α-toxin-deleted strain. The viral enhancing effect of α-toxin is mediated by ADAM10 and is associated with its pore-forming property. Moreover, we demonstrate that α-toxin promotes viral entry in NHKs. The current study introduces the novel concept that staphylococcal α-toxin promotes viral skin infection and provides a mechanism by which S aureus infection might predispose the host toward disseminated viral infections. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci.

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McClure, Jo-Ann; Zaal DeLongchamp, Johanna; Conly, John M; Zhang, Kunyan

2017-06-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screening Staphylococcus isolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminating S. aureus from coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including the Staphylococcus 16S rRNA gene (specifically detecting Staphylococcus spp.), nuc (distinguishing S. aureus from CoNS), mecA (distinguishing MRSA from methicillin-susceptible S. aureus ), mupA and mupB (identifying high-level mupirocin resistance), and qac and smr (identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistant Staphylococcus monitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments. Copyright © 2017 American Society for Microbiology.

Methicillin-Resistant Staphylococcus aureus Infections: A Comprehensive Review and a Plastic Surgeon's Approach to the Occult Sites.

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Hunter, Cedric; Rosenfield, Lorne; Silverstein, Elena; Petrou-Zeniou, Panayiota

2016-08-01

Up to 20 percent of the general population is persistently colonized with Staphylococcus aureus, and 1 to 3 percent of the population is colonized with community-acquired methicillin-resistant S. aureus. Currently, the knowledge of methicillin-resistant Staphylococcus aureus carriage sites other than the nose, and their effect on surgical site infections in cosmetic surgery, is lacking. A comprehensive literature review using the PubMed database to analyze prevalence, anatomical carrier sites, current screening and decontamination protocols and guidelines, and methicillin-resistant S. aureus in cosmetic surgery was performed. The senior author's (L.R.) methicillin-resistant S. aureus infection experience and prevention protocols were also reviewed. Nasal swabs detect only 50.5 percent of methicillin-resistant S. aureus colonization, and broad screening has noted the presence of methicillin-resistant S. aureus in the ear canal and umbilicus. Decolonization protocols within the orthopedic and cardiothoracic surgery literature have reduced rates of methicillin-resistant S. aureus surgical-site infections. There are no decolonization guidelines for plastic surgeons. Since instituting their decolonization protocol, the authors have had no cases of methicillin-resistant S. aureus infection in nearly 1000 cosmetic surgery procedures. There are very limited, if any, Level I or II data regarding methicillin-resistant S. aureus screening and decolonization. As the sequelae of a surgical-site infection can be disastrous, expert opinions recommend that plastic surgeons vigorously address methicillin-resistant S. aureus colonization and infection. The authors have developed and recommend a simple decolonization protocol that includes treatment of the umbilicus, ear canal, and nares to limit surgical-site infection and improve surgical outcomes.

A ChIP-Seq benchmark shows that sequence conservation mainly improves detection of strong transcription factor binding sites.

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Tony Håndstad

Full Text Available BACKGROUND: Transcription factors are important controllers of gene expression and mapping transcription factor binding sites (TFBS is key to inferring transcription factor regulatory networks. Several methods for predicting TFBS exist, but there are no standard genome-wide datasets on which to assess the performance of these prediction methods. Also, it is believed that information about sequence conservation across different genomes can generally improve accuracy of motif-based predictors, but it is not clear under what circumstances use of conservation is most beneficial. RESULTS: Here we use published ChIP-seq data and an improved peak detection method to create comprehensive benchmark datasets for prediction methods which use known descriptors or binding motifs to detect TFBS in genomic sequences. We use this benchmark to assess the performance of five different prediction methods and find that the methods that use information about sequence conservation generally perform better than simpler motif-scanning methods. The difference is greater on high-affinity peaks and when using short and information-poor motifs. However, if the motifs are specific and information-rich, we find that simple motif-scanning methods can perform better than conservation-based methods. CONCLUSIONS: Our benchmark provides a comprehensive test that can be used to rank the relative performance of transcription factor binding site prediction methods. Moreover, our results show that, contrary to previous reports, sequence conservation is better suited for predicting strong than weak transcription factor binding sites.

Organizational requirements of the SaeR binding sites for a functional P1 promoter of the sae operon in Staphylococcus aureus.

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Cho, Hoonsik; Jeong, Do-Won; Li, Chunling; Bae, Taeok

2012-06-01

In Staphylococcus aureus, the SaeRS two-component system controls the expression of multiple virulence factors. Of the two promoters in the sae operon, P1 is autoinduced and has two binding sites for the response regulator SaeR. In this study, we examined the organizational requirements of the SaeR binding sites in P1 for transcription activation. Mutational studies showed that both binding sites are essential for binding to phosphorylated SaeR (P-SaeR) and transcription activation. When the 21-bp distance between the centers of the two SaeR binding sites was altered to 26 bp, 31 bp, 36 bp, or 41 bp, only the 31-bp mutant retained approximately 40% of the original promoter activity. When the -1-bp spacing (i.e.,1-bp overlap) between the primary SaeR binding site and the -35 promoter region was altered, all mutant P1 promoters failed to initiate transcription; however, when the first nucleotide of the -35 region was changed from A to T, the mutants with 0-bp or 22-bp spacing showed detectable promoter activity. Although P-SaeR was essential for the binding of RNA polymerase to P1, it was not essential for the binding of the enzyme to the alpha-hemolysin promoter. When the nonoptimal spacing between promoter elements in P1 or the coagulase promoter was altered to the optimal spacing of 17 bp, both promoters failed to initiate transcription. These results suggest that SaeR binding sites are under rather strict organizational restrictions and provide clues for understanding the molecular mechanism of sae-mediated transcription activation.

Virulence Factor Genes in Staphylococcus aureus Isolated From Diabetic Foot Soft Tissue and Bone Infections.

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Víquez-Molina, Gerardo; Aragón-Sánchez, Javier; Pérez-Corrales, Cristian; Murillo-Vargas, Christian; López-Valverde, María Eugenia; Lipsky, Benjamin A

2018-03-01

The aim of this study is to describe the presence of genes encoding for 4 virulence factors (pvl, eta, etb, and tsst), as well as the mecA gene conferring resistance to beta-lactam antibiotics, in patients with diabetes and a staphylococcal foot infection. We have also analyzed whether isolates of Staphylococcus aureus from bone infections have a different profile for these genes compared with those from exclusively soft tissue infections. In this cross-sectional study of a prospectively recruited series of patients admitted to the Diabetic Foot Unit, San Juan de Dios Hospital, San José, Costa Rica with a moderate or severe diabetic foot infection (DFI), we collected samples from infected soft tissue and from bone during debridement. During the study period (June 1, 2014 to May 31, 2016), we treated 379 patients for a DFI. S aureus was isolated from 101 wound samples, of which 43 were polymicrobial infections; we only included the 58 infections that were monomicrobial S aureus for this study. Infections were exclusively soft tissue in 17 patients (29.3%) while 41 (70.7%) had bone involvement (osteomyelitis). The mecA gene was detected in 35 cases (60.3%), pvl gene in 4 cases (6.9%), and tsst gene in 3 (5.2%). We did not detect etA and etB in any of the cases. There were no differences in the profile of S aureus genes encoding for virulence factors (pvl, etA, etB, and tsst) recovered from DFIs between those with just soft tissue compared to those with osteomyelitis. However, we found a significantly higher prevalence of pvl+ strains of S aureus associated with soft tissue compared with bone infections. Furthermore, we observed a significantly longer time to healing among patients infected with mecA+ (methicillin-resistant) S aureus (MRSA).

RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus.

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Lei, Mei G; Lee, Chia Y

2015-12-01

Staphylococcus aureus capsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of the cap operon. A 10-bp inverted repeat (IR) located 13 bp upstream of the -35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of the cap promoter. To search for potential proteins which directly interact with the cap promoter region (Pcap), we directly analyzed the proteins interacting with the Pcap DNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulate cap gene expression by specifically binding to the cap promoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed that rbsR was directly controlled by SigB and that RbsR was a repressor of the rbsUDK operon, involved in ribose uptake and phosphorylation. The repression of rbsUDK by RbsR could be derepressed by D-ribose. However, D-ribose did not affect RbsR activation of capsule. Staphylococcus aureus is an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression of rbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits the rbs operon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence in S. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation

Prevalence and characterization of methicillin-resistant Staphylococcus aureus carrying mecA or mecC and methicillin-susceptible Staphylococcus aureus in dairy sheep farms in central Italy.

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Giacinti, G; Carfora, V; Caprioli, A; Sagrafoli, D; Marri, N; Giangolini, G; Amoruso, R; Iurescia, M; Stravino, F; Dottarelli, S; Feltrin, F; Franco, A; Amatiste, S; Battisti, A

2017-10-01

Between January and May 2012, a total of 286 bulk tank milk samples from dairy sheep farms located in central Italy were tested for the presence of Staphylococcus aureus. One hundred fifty-three samples were positive for S. aureus (53.5%), with an average count of 2.53 log cfu/mL. A total of 679 S. aureus colonies were screened for methicillin resistance by the cefoxitin disk diffusion test, and 104 selected cefoxitin-susceptible isolates were also tested for their susceptibility to other antimicrobials representative of the most relevant classes active against Staphylococcus spp. by using the Kirby-Bauer disk diffusion method. Two methicillin-resistant Staphylococcus aureus (MRSA) isolates, carrying respectively the mecA and the mecC genes, were detected in 2 samples from 2 different farms (prevalence 0.7%). The mecA-positive MRSA isolate was blaZ positive, belonged to spa type t127, sequence type (ST)1, clonal complex (CC)1, carried a staphylococcal cassette chromosome mec (SCCmec) type IVa, and was phenotypically resistant to all the β-lactams tested and to erythromycin, streptomycin, kanamycin, and tetracycline. The mecC-positive MRSA isolate was negative for the chromosomally or plasmid-associated blaZ gene but positive for the blaZ allotype associated with SCCmec XI (blaZ-SCCmecXI), belonged to spa type 843, ST(CC)130, carried a SCCmec type XI, and was resistant only to β-lactams. Both MRSA were negative for the presence of specific immune-evasion and virulence genes such as those coding for the Panton-Valentine leucocidin, the toxic shock syndrome toxin 1, and the immune evasion cluster genes. Regarding the presence of the major S. aureus enterotoxin genes, the mecC-positive MRSA tested negative, whereas the ST (CC)1 mecA-positive MRSA harbored the seh gene. Among the 104 methicillin-susceptible S. aureus isolates examined for antimicrobial susceptibility, 63 (60.58%) were susceptible to all the antimicrobials tested, and 41 (39.42%) were resistant to at

Role of Rot in bacterial autolysis regulation of Staphylococcus aureus NCTC8325.

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Chu, Xinmin; Xia, Rui; He, Nianan; Fang, Yuting

2013-09-01

Autolysis is an important process in cell wall turnover in Staphylococcus aureus, performed by several peptidoglycan hydrolases or so-called autolysins and controlled by many regulators. Rot is a global regulator that regulates numerous virulence genes, including genes encoding lipase, hemolysins, proteases and genes related to cell surface adhesion. The aim of our study was to determine whether Rot has the ability to regulate autolysis. We compared Triton-X-100-induced autolysis of S. aureus NCTC8325 and its rot knock-out mutant. We found that the rot mutant showed increased autolysis rates. By examining the transcript level of several autolysins and some known regulators responsible for regulating autolysis using real-time RT-PCR assays, we found that transcription of two autolysins (lytM, lytN) and one regulatory operon (lrgAB) was changed in the rot mutant. An in vitro approach was undertaken to determine which of these genes are directly controlled by Rot. Rot proteins were overproduced in Escherichia coli and purified. Gel mobility shift DNA binding assays were used and showed that in-vitro-purified Rot can directly bind to the promoter region of lytM, lytN, lrgA and lytS. We also tested biofilm formation of the rot mutant, and it showed enhancement in biofilm formation. Taken together, our results reveal that Rot affects autolysis by directly regulating autolysins LytM and LytN, and, via a regulatory system, LrgAB. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk.

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Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-06-01

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk

Science.gov (United States)

Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A.; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-01-01

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene. PMID:26273271

Methicillin-resistant Staphylococcus aureus (MRSA)

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Methicillin-resistant Staphylococcus aureus; Hospital-acquired MRSA (HA-MRSA); Staph - MRSA; Staphylococcal - MRSA ... Centers for Disease Control and Prevention website. Methicillin-resistant Staphylococcus aureus (MRSA). www.cdc.gov/mrsa/index.html . Updated ...

Genetic diversity of Staphylococcus aureus in Buruli ulcer.

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Nana Ama Amissah

2015-02-01

Full Text Available Buruli ulcer (BU is a necrotizing skin disease caused by Mycobacterium ulcerans. Previous studies have shown that wounds of BU patients are colonized with M. ulcerans and several other microorganisms, including Staphylococcus aureus, which may interfere with wound healing. The present study was therefore aimed at investigating the diversity and topography of S. aureus colonizing BU patients during treatment.We investigated the presence, diversity, and spatio-temporal distribution of S. aureus in 30 confirmed BU patients from Ghana during treatment. S. aureus was isolated from nose and wound swabs, and by replica plating of wound dressings collected bi-weekly from patients. S. aureus isolates were characterized by multiple-locus variable number tandem repeat fingerprinting (MLVF and spa-typing, and antibiotic susceptibility was tested.Nineteen (63% of the 30 BU patients tested positive for S. aureus at least once during the sampling period, yielding 407 S. aureus isolates. Detailed analysis of 91 isolates grouped these isolates into 13 MLVF clusters and 13 spa-types. Five (26% S. aureus-positive BU patients carried the same S. aureus genotype in their anterior nares and wounds. S. aureus isolates from the wounds of seven (37% patients were distributed over two different MLVF clusters. Wounds of three (16% patients were colonized with isolates belonging to two different genotypes at the same time, and five (26% patients were colonized with different S. aureus types over time. Five (17% of the 30 included BU patients tested positive for methicillin-resistant S. aureus (MRSA.The present study showed that the wounds of many BU patients were contaminated with S. aureus, and that many BU patients from the different communities carried the same S. aureus genotype during treatment. This calls for improved wound care and hygiene.

Genetic diversity of Staphylococcus aureus in Buruli ulcer.

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Amissah, Nana Ama; Glasner, Corinna; Ablordey, Anthony; Tetteh, Caitlin S; Kotey, Nana Konama; Prah, Isaac; van der Werf, Tjip S; Rossen, John W; van Dijl, Jan Maarten; Stienstra, Ymkje

2015-02-01

Buruli ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Previous studies have shown that wounds of BU patients are colonized with M. ulcerans and several other microorganisms, including Staphylococcus aureus, which may interfere with wound healing. The present study was therefore aimed at investigating the diversity and topography of S. aureus colonizing BU patients during treatment. We investigated the presence, diversity, and spatio-temporal distribution of S. aureus in 30 confirmed BU patients from Ghana during treatment. S. aureus was isolated from nose and wound swabs, and by replica plating of wound dressings collected bi-weekly from patients. S. aureus isolates were characterized by multiple-locus variable number tandem repeat fingerprinting (MLVF) and spa-typing, and antibiotic susceptibility was tested. Nineteen (63%) of the 30 BU patients tested positive for S. aureus at least once during the sampling period, yielding 407 S. aureus isolates. Detailed analysis of 91 isolates grouped these isolates into 13 MLVF clusters and 13 spa-types. Five (26%) S. aureus-positive BU patients carried the same S. aureus genotype in their anterior nares and wounds. S. aureus isolates from the wounds of seven (37%) patients were distributed over two different MLVF clusters. Wounds of three (16%) patients were colonized with isolates belonging to two different genotypes at the same time, and five (26%) patients were colonized with different S. aureus types over time. Five (17%) of the 30 included BU patients tested positive for methicillin-resistant S. aureus (MRSA). The present study showed that the wounds of many BU patients were contaminated with S. aureus, and that many BU patients from the different communities carried the same S. aureus genotype during treatment. This calls for improved wound care and hygiene.

A Case of Childhood Lichen Aureus

OpenAIRE

Kim, Min Ji; Kim, Byung Yoon; Park, Kyung Chan; Youn, Sang Woong

2009-01-01

Lichen aureus is a rare type of chronic pigmented purpuric dermatosis. The eruptions consist of discrete or confluent golden to brownish lichenoid macules and papules, and are usually asymptomatic. Lichen aureus commonly occurs in young adults, but less frequently in children. We report the first case of multiple lichen aureus occurring in a Korean child.

PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk.

Science.gov (United States)

Rall, V L M; Vieira, F P; Rall, R; Vieitis, R L; Fernandes, A; Candeias, J M G; Cardoso, K F G; Araújo, J P

2008-12-10

Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 x 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. Of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent (16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%), seb (3 strains, 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains, 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of staphylococci may be higher than previously thought; however, further studies are required to assess the expression of these new SEs by S. aureus, and their impact in foodborne disease. The quality of Brazilian milk is still low, and efforts from the government and the entire productive chain are required to attain consumer safety.

Evaluation of antimicrobial susceptibilities and virulence factors of Staphylococcus aureus strains isolated from community-acquired and health-care associated pediatric infections.

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Karbuz, Adem; Karahan, Zeynep Ceren; Aldemir-Kocabaş, Bilge; Tekeli, Alper; Özdemir, Halil; Güriz, Haluk; Gökdemir, Refik; İnce, Erdal; Çiftçi, Ergin

2017-01-01

Karbuz A, Karahan ZC, Aldemir-Kocabaş B, Tekeli A, Özdemir H, Güriz H, Gökdemir R, İnce E, Çiftçi E. Evaluation of antimicrobial susceptibilities and virulence factors of Staphylococcus aureus strains isolated from community-acquired and health-care associated pediatric infections. Turk J Pediatr 2017; 59: 395-403. The aim of this study was to investigate the enterotoxins and Panton-Valentine leukocidin (PVL) gene as virulence factor, identification if antimicrobial sensitivity patterns, agr (accessory gene regulator) types and sequence types and in resistant cases to obtain SCCmec (staphylococcal cassette chromosome mec) gene types which will be helpful to decide empirical therapy and future health politics for S. aureus species. Total of 150 isolates of S. aureus were isolated from the cultures of the child patients in January 2011 and December 2012. In this study, the penicillin resistance was observed as 93.8%. PVL and mecA was detected positive in 8.7% and in 6% of all S. aureus strains, respectively. Two MRSA (methicillin resistant S.aureus) strains were detected as SCCmec type III and SCCmec type V and five MRSA strains were detected as SCCmec type IV. SET-I and SET-G were the most common detected enterotoxins. In both community-associated and healthcare-associated MRSA strains, agr type 1 was detected most commonly. The most common sequence types were ST737 in 13 patients than ST22 in eight patients and ST121 in six patients. This study highlights a necessity to review the cause of small changes in the structural genes in order to determine whether it is a cause or outcome; community-acquired and healthcare associated strains overlap.

Trapping and proteomic identification of cellular substrates of the ClpP protease in Staphylococcus aureus

DEFF Research Database (Denmark)

Feng, Jingyuan; Michalik, Stephan; Varming, Anders Nissen

2013-01-01

In the important human pathogen Staphylococcus aureus the cytoplasmic ClpP protease is essential for mounting cellular stress responses and for virulence. To directly identify substrates of the ClpP protease, we expressed in vivo a proteolytic inactive form of ClpP (ClpP(trap)) that will retain...... but not degrade substrates translocated into its proteolytic chamber. Substrates captured inside the proteolytic barrel were co-purified along with the His-tagged ClpP complex and identified by mass spectrometry. In total, approximately 70 proteins were trapped in both of the two S. aureus strains NCTC8325......A, and the cell division protein FtsZ. Newly identified ClpP substrates include the global transcriptional regulators PerR and HrcA, proteins involved in DNA damage repair (RecA, UvrA, UvrB), and proteins essential for protein synthesis (RpoB and Tuf). Our study hence underscores the central role of Clp...

Development of a Rapid Detection Method for Potato virus X by Reverse Transcription Loop-Mediated Isothermal Amplification

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Joojin Jeong

2015-09-01

Full Text Available The primary step for efficient control of viral diseases is the development of simple, rapid, and sensitive virus detection. Reverse transcription loop-mediated isothermal amplification (RT-LAMP has been used to detect viral RNA molecules because of its simplicity and high sensitivity for a number of viruses. RT-LAMP for the detection of Potato virus X (PVX was developed and compared with conventional reverse transcription polymerase chain reaction (RT-PCR to demonstrate its advantages over RT-PCR. RT-LAMP reactions were conducted with or without a set of loop primers since one out of six primers showed PVX specificity. Based on real-time monitoring, RT-LAMP detected PVX around 30 min, compared to 120 min for RT-PCR. By adding a fluorescent reagent during the reaction, the extra step of visualization by gel electrophoresis was not necessary. RT-LAMP was conducted using simple inexpensive instruments and a regular incubator to evaluate whether RNA could be amplified at a constant temperature instead of using an expensive thermal cycler. This study shows the potential of RT-LAMP for the diagnosis of viral diseases and PVX epidemiology because of its simplicity and rapidness compared to RT-PCR.

Induction of virulence gene expression in Staphylococcus aureus by pulmonary surfactant.

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Ishii, Kenichi; Adachi, Tatsuo; Yasukawa, Jyunichiro; Suzuki, Yutaka; Hamamoto, Hiroshi; Sekimizu, Kazuhisa

2014-04-01

We performed a genomewide analysis using a next-generation sequencer to investigate the effect of pulmonary surfactant on gene expression in Staphylococcus aureus, a clinically important opportunistic pathogen. RNA sequence (RNA-seq) analysis of bacterial transcripts at late log phase revealed 142 genes that were upregulated >2-fold following the addition of pulmonary surfactant to the culture medium. Among these genes, we confirmed by quantitative reverse transcription-PCR analysis that mRNA amounts for genes encoding ESAT-6 secretion system C (EssC), an unknown hypothetical protein (NWMN_0246; also called pulmonary surfactant-inducible factor A [PsiA] in this study), and hemolysin gamma subunit B (HlgB) were increased 3- to 10-fold by the surfactant treatment. Among the major constituents of pulmonary surfactant, i.e., phospholipids and palmitate, only palmitate, which is the most abundant fatty acid in the pulmonary surfactant and a known antibacterial substance, stimulated the expression of these three genes. Moreover, these genes were also induced by supplementing the culture with detergents. The induction of gene expression by surfactant or palmitate was not observed in a disruption mutant of the sigB gene, which encodes an alternative sigma factor involved in bacterial stress responses. Furthermore, each disruption mutant of the essC, psiA, and hlgB genes showed attenuation of both survival in the lung and host-killing ability in a murine pneumonia model. These findings suggest that S. aureus resists membrane stress caused by free fatty acids present in the pulmonary surfactant through the regulation of virulence gene expression, which contributes to its pathogenesis within the lungs of the host animal.

Relationships among superantigen toxin gene profiles, genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis.

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Wang, Dong; Zhang, Limei; Yong, Changfu; Shen, Mingliang; Ali, Tariq; Shahid, Muhammad; Han, Kun; Zhou, Xuezhang; Han, Bo

2017-06-01

Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes

Prevalence and Genetic Characteristics of Staphylococcus aureus and Staphylococcus argenteus Isolates Harboring Panton-Valentine Leukocidin, Enterotoxins, and TSST-1 Genes from Food Handlers in Myanmar.

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Aung, Meiji Soe; San, Thida; Aye, Mya Mya; Mya, San; Maw, Win Win; Zan, Khin Nyein; Htut, Wut Hmone Win; Kawaguchiya, Mitsuyo; Urushibara, Noriko; Kobayashi, Nobumichi

2017-08-04

Asymptomatic carriers of toxigenic Staphylococcus aureus are potential source of diseases, including food poisoning. Toxigenic potential and genetic traits of colonizing S. aureus were investigated for 563 healthy food handlers in Myanmar. Carriage of S. aureus was found in 110 individuals (19.5%), and a total of 144 S. aureus isolates were recovered from nasal cavities (110 isolates) and hands (34 isolates). Panton-Valentine leucocidin genes ( pvl ) were detected in 18 isolates (12.5%), among which 11 isolates were classified into coa -VIa, agr type III, and ST1930 (CC96) that had been also detected in pvl -positive clinical isolates in Myanmar. A pvl -positive, ST2250 nasal isolate was identified as S. argenteus , a novel coagulase-positive staphylococcus species. Toxic shock syndrome toxin-1 (TSST-1) gene was detected in five pvl -negative isolates. All of the 144 isolates harbored at least one of the 21 enterotoxin(-like) gene(s). The most prevalent enterotoxin(-like) gene was selw (98%), followed by selx (97%), sei (28%), sely (28%), sem (26%), sel (24%), and sea and sec (22% each). Considerable genetic diversity with five groups was detected for selw . The present study revealed the relatively high rate of pvl , as well as the wide distribution of enterotoxin(-like) genes among colonizing S. aureus in Myanmar.

Methicillin-resistant and -susceptible Staphylococcus aureus from retail meat in Denmark

DEFF Research Database (Denmark)

Tang, Yuanyue; Larsen, Jesper; Kjeldgaard, Jette

2017-01-01

belonged to CC398 (spa t034, t011, t2582, t108), and hereof one isolate derived from chicken (1%). Further findings were spa type t1430 (CC9) in turkey samples (16%) and the human-associated t008 (CC8) in chicken samples (16%). In conclusion, S. aureus was readily detected in Danish retail meat......-MRSA lineage causing human infections, and although pigs are the major source of CC398 worldwide, poultry and other animals are also reservoirs. This raises concern for transmission of MRSA via meat. In this study, the occurrence and characteristics of S. aureus isolated from Danish retail meat were examined...... in MRSA prevalence of 4% of chicken, 52% of turkey, and 15% of pork. Three MRSA positive samples were obtained by direct plating (Brilliance MRSA2), whereas 16 MRSA positive samples were detected only after enrichment (TSB + 6.5% NaCl and Brilliance MRSA2). Based on spa typing, 68% of MRSA isolates...

Diversity of Staphylococcus aureus Isolates in European Wildlife.

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Stefan Monecke

Full Text Available Staphylococcus aureus is a well-known colonizer and cause of infection among animals and it has been described from numerous domestic and wild animal species. The aim of the present study was to investigate the molecular epidemiology of S. aureus in a convenience sample of European wildlife and to review what previously has been observed in the subject field. 124 S. aureus isolates were collected from wildlife in Germany, Austria and Sweden; they were characterized by DNA microarray hybridization and, for isolates with novel hybridization patterns, by multilocus sequence typing (MLST. The isolates were assigned to 29 clonal complexes and singleton sequence types (CC1, CC5, CC6, CC7, CC8, CC9, CC12, CC15, CC22, CC25, CC30, CC49, CC59, CC88, CC97, CC130, CC133, CC398, ST425, CC599, CC692, CC707, ST890, CC1956, ST2425, CC2671, ST2691, CC2767 and ST2963, some of which (ST2425, ST2691, ST2963 were not described previously. Resistance rates in wildlife strains were rather low and mecA-MRSA isolates were rare (n = 6. mecC-MRSA (n = 8 were identified from a fox, a fallow deer, hares and hedgehogs. The common cattle-associated lineages CC479 and CC705 were not detected in wildlife in the present study while, in contrast, a third common cattle lineage, CC97, was found to be common among cervids. No Staphylococcus argenteus or Staphylococcus schweitzeri-like isolates were found. Systematic studies are required to monitor the possible transmission of human- and livestock-associated S. aureus/MRSA to wildlife and vice versa as well as the possible transmission, by unprotected contact to animals. The prevalence of S. aureus/MRSA in wildlife as well as its population structures in different wildlife host species warrants further investigation.

Evaluación de estrategias de control frente a biopelículas de "Staphylococcus aureus" potencialmente presentes en la industria pesquera

OpenAIRE

Vázquez Sánchez, Daniel

2014-01-01

Staphylococcus aureus is one of the major bacterial agents causing foodborne diseases in humans worldwide, due to the ingestion of food containing staphylococcal enterotoxins. Spain is one of the largest producers and consumers of fishery products in the European Union. However, S. aureus is repeatedly detected in these products as a consequence of cross-contamination from food handlers and food contact surfaces. Biofilm formation also provides S. aureus a high tolerance to biocides allowing ...

1H, 13C and 15N backbone resonance assignment of the arsenate reductase from Staphylococcus aureus in its reduced state

NARCIS (Netherlands)

Jacobs, D.M.; Messens, J.; Wechselberger, R.W.|info:eu-repo/dai/nl/304829005; Brosens, E.; Willem, R.; Wyns, L.; Martins, J.C.

2001-01-01

In S. aureus, resistance to the metal(III)oxyanions arsenite As(III)O− 2 and antimonite Sb(III)O− 2 is mediated by two proteins, ArsB and ArsR, encoded in the ars operon of plasmid pI258 (Silver, 1999). ArsR acts as the transcription repressor, which is de-repressed in the presence of intracellular

Antibiotic susceptibility pattern of staphylococcus aureus and methicillin-resistant staphylococcus aureus in a tertiary care hospital

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CP Bhatt

2014-04-01

Full Text Available Background: Methicillin resistant Staphylococcus aureushas emerged as one of the most important nosocomial pathogens. It invokes a tremendous financial burden and enhanced morbidity and mortality due to difficult to treat systemic infections.Aim of this study was to determine antibiotic susceptibility pattern of Staphylococcus aureus and Methicillin resistant Staphylococcus aureus. Materials and Methods: Different clinical specimens were collected and processed for routine culture and antibiotic sensitivity test by standard microbiology techniques. Results: Out of 1173 samples received for microbiological examination, 100 were found to be S. aureus with 19% cases were Methicillin resistant Staphylococcus aureus (MRSA. Fourteen MRSA were found from inpatient and 5 were from outpatient. MRSA was found higher in female than male and maximum number (31.5% was found in age group 0-10 years. Staphylococcus aureus was 100% sensitive to Vancomycin followed by Amikacin (90%, Gentamycin (83%, and tetracycline (81%. On urine isolates Nitrofurantoin(91.6% was drug of choice. All the isolates were resistant to Penicillin G. In case of Methicillin resistant Staphylococcus aureus showed 100% sensitive to Vancomycin followed by Amikacin (84.2%, Tetracycline (63.1%, Ciprofloxacin (42% and Gentamycin (36.8%. Among urine isolates Nitrofutantoin showed 87.5% sensitive followed by Norfloxacin (75%. Conclusion: Methicillin resistant Staphylococcus aureus was found 19% of Staphylococcus aureus isolates. It was most common in females, hospitalized patients and young age group. Vancomycin seems to be drug of choice followed by Amikacin. It would be helpful to formulating and monitoring the antibiotic policy and ensure proper empiric treatment. DOI: http://dx.doi.org/10.3126/jpn.v4i7.10297 Journal of Pathology of Nepal (2014 Vol. 4, 548-551   

Prevalence and molecular characterization of Staphylococcus aureus in commercially available meat over a one-year period in Iowa, USA.

Science.gov (United States)

Thapaliya, Dipendra; Forshey, Brett M; Kadariya, Jhalka; Quick, Megan K; Farina, Sarah; O' Brien, Ashley; Nair, Rajeshwari; Nworie, Amos; Hanson, Blake; Kates, Ashley; Wardyn, Shylo; Smith, Tara C

2017-08-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of infectious disease morbidity and mortality. Previous studies have confirmed the presence of S. aureus, including MRSA, on raw meat products. We investigated the prevalence and molecular epidemiology of S. aureus and MRSA in commercially-distributed antibiotic-free and conventional raw meat products (n = 3290) purchased in 8 Iowa retail stores weekly for a period of one year. Isolates were characterized using spa typing, and PCR was used to detect the presence of the Panton-Valentine leukocidin (PVL) and mecA genes. Quantitation of S. aureus on meat products was carried out one week per month. The prevalence of S. aureus on meat samples was 27.8% (913/3290). Compared to antibiotic-free meat samples, higher prevalence of both MRSA and methicillin-susceptible S. aureus (MSSA) were found in conventional meat samples. Among the S. aureus isolates, 18 were PVL-positive (1.9%) and 41 (4.5%) carried mecA. Phenotypic oxacillin resistance was observed for 17.1% (41/239) of the isolates tested, while 23% (55/239) were multi-drug resistant. A total of 132 spa types were detected from 913 contaminated meat samples. Overall, t002 was the most common spa type identified (137; 15.0%). The number of colony-forming units (CFU) per 10 g meat ranged from 2 to 517 (median: 8 CFU per 10 g of meat; mean: 28) with the highest bacterial load observed on turkey samples. These data reinforce the need to consider meat products as potential vehicles of S. aureus transmission from farm into human households, and the potential need for public health intervention programs pre and post-slaughter in meat processing facilities. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genomic investigation of Danish Staphylococcus aureus isolates from bulk tank milk and dairy cows with clinical mastitis

DEFF Research Database (Denmark)

Ronco, Troels; Klaas, Ilka C.; Stegger, Marc

2018-01-01

Staphylococcus aureus is one of the most common pathogens that cause mastitis in dairy cows. Various subtypes, virulence genes and pathogenicity islands have been associated with isolates from bulk tank milk and clinical mastitis. So far, no Danish cattle associated S. aureus isolates have been...... and clinical mastitis were of similar genetic background. This suggests that dairy cows are natural carriers of the S. aureus subtypes that cause clinical mastitis if the right conditions are present and that a broad range of subtypes cause mastitis. A phylogenetic cluster that mostly consisted of ST151...... isolates carried three pathogenicity islands that were primarily found in this group. The prevalence of resistance genes was generally low. However, the first ST398 methicillin resistant S. aureus isolate from a Danish dairy cow with clinical mastitis was detected....

Methicillin-resistant Staphylococcus aureus in North-east Croatia

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Tajana Pastuović

2015-05-01

Full Text Available Objective. The aim of this 5-year study was to determine the frequency and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA-related infections at Osijek Clinical Hospital. Materials and methods. A total of 1987 staphylococci-infected clinical isolates were collected and analysed at the Microbiology Department of the Public Health Institute of Osijek-Baranja County. Results. Between 2008 and 2012, the average rate of MRSA-related infections in staphylococci-infected patients was 27.4%. The proportion of MRSArelated infections on all Staphylococcus aureus (S. aureus isolates from clinical specimens showed a decreasing trend, from 32.6% in 2008 to 25.5% in 2012. MRSA-related infections were mostly detected in wound swabs (50.6% and aspirates (28.8% of patients hospitalized in the surgical (49.8% and intensive care units (27.9%. MRSA-related infection showed an increase compared to S. aureus-infections in samples of wounds and aspirates in 2011 and 2012 (57.9%/34.9% and 35.2%/16.3%, respectively. The majority of strains of MRSA-related infections were resistant to several antibiotics, including erythromycin and clindamycin, where susceptibility were less than 10%. All MRSA isolates were susceptible to vancomycin, teicoplanin and linezolid. Therefore, antibiotic therapies for MRSA infections include vancomycin, teicoplanin and linezolid, but microbiological diagnostics need to be performed in order to know when the use of glycopeptides and oxazolidinones is indicated. Conclusion. Our results suggest that appropriate prevention measures, combined with the more rational use of antibiotics are crucial to reduce the spread of MRSA-related infection in healthcare settings. Further monitoring is necessary of the incidence and antibiotic susceptibility of MRSA-related infections in our community.

Contribution of hydrogen peroxide to the inhibition of Staphylococcus aureus by Lactococcus garvieae in interaction with raw milk microbial community

OpenAIRE

Delbes, Céline; Dorchies, Géraud; Chaabna, Zineddine; Callon, Cecile; Montel, Marie-Christine

2010-01-01

The response of Staphylococcus aureus growth inhibition by Lactococcus garvieae to catalase and milk lactoperoxidase, and its efficiency in raw milk cheese were evaluated. S. aureus and L. garvieae were co-cultivated in broth buffered at pH 6.8, and in raw, pasteurized and microfiltered milk, in presence and absence of catalase. Although H(2)O(2) production by L garvieae was detected only in agitated broth, the inhibition of S. aureus by L garvieae was reduced by catalase both in stati...

Staphylococcus aureus and hand eczema severity

DEFF Research Database (Denmark)

Haslund, P; Bangsgaard, N; Jarløv, J O

2009-01-01

BACKGROUND: The role of bacterial infections in hand eczema (HE) remains to be assessed. OBJECTIVES: To determine the prevalence of Staphylococcus aureus in patients with HE compared with controls, and to relate presence of S. aureus, subtypes and toxin production to severity of HE. METHODS......: Bacterial swabs were taken at three different visits from the hand and nose in 50 patients with HE and 50 controls. Staphylococcus aureus was subtyped by spa typing and assigned to clonal complexes (CCs), and isolates were tested for exotoxin-producing S. aureus strains. The Hand Eczema Severity Index...... and in the nose in all cases, and between visits in 90% of cases. Ten different CC types were identified, no association with severity was found, and toxin-producing strains were not found more frequently in patients with HE than in controls. CONCLUSIONS: Staphylococcus aureus was present on hands in almost half...

Phenotypic and molecular characteristics of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in slaughterhouse pig-related workers and control workers in Guangdong Province, China.

Science.gov (United States)

Wang, X L; Li, L; Li, S M; Huang, J Y; Fan, Y P; Yao, Z J; Ye, X H; Chen, S D

2017-07-01

Pig farmers and veterinarians have high prevalence of methicillin-resistant Staphylococcus aureus (MRSA) due to the occupational livestock exposure, while few reported this association on slaughterhouse workers. We conducted this cross-sectional study to explore the phenotypic and molecular characteristics of S. aureus and MRSA in slaughterhouse pig-related workers and control workers in Guangdong Province, China. Participants were interviewed and provided two nasal swabs. Swabs were tested for S. aureus, and isolates were further tested for antimicrobial susceptibility, virulence genes and multi-locus sequence typing. Compared with control workers, pig-related workers have significantly higher prevalence of MRSA carriage (adjusted odd ratio (aOR) 3·70, 95% CI 1·63-8·40). The proportions of MRSA resistant to clindamycin, erythromycin, tetracycline or chloromycetin were significantly higher in pig-related workers than in control workers. The predominant phenotypes of S. aureus were resistant to penicillin, clindamycin, erythromycin and tetracycline. Three MRSA CC9 isolates with livestock-associated characteristics (resistance to tetracycline and absence of immune evasion cluster (IEC) genes) were detected in pig-related workers but not in control workers. For human-associated CCs (CC7, CC59, CC6, and CC188), there was no significant difference in IEC profile or antimicrobial resistance between the groups. These findings reveal that there may be a potential risk for livestock-to-human transmission of LA-MRSA and human-to-human transmission of human-associated MRSA.

Improved Detection of Lassa Virus by Reverse Transcription-PCR Targeting the 5′ Region of S RNA▿

OpenAIRE

Ölschläger, Stephan; Lelke, Michaela; Emmerich, Petra; Panning, Marcus; Drosten, Christian; Hass, Meike; Asogun, Danny; Ehichioya, Deborah; Omilabu, Sunday; Günther, Stephan

2010-01-01

The method of choice for the detection of Lassa virus is reverse transcription (RT)-PCR. However, the high degree of genetic variability of the virus poses a problem with the design of RT-PCR assays that will reliably detect all strains. Recently, we encountered difficulties in detecting some strains from Liberia and Nigeria in a commonly used glycoprotein precursor (GPC) gene-specific RT-PCR assay (A. H. Demby, J. Chamberlain, D. W. Brown, and C. S. Clegg, J. Clin. Microbiol. 32:2898-2903, 1...

Detecting Differential Transcription Factor Activity from ATAC-Seq Data

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Ignacio J. Tripodi

2018-05-01

Full Text Available Transcription factors are managers of the cellular factory, and key components to many diseases. Many non-coding single nucleotide polymorphisms affect transcription factors, either by directly altering the protein or its functional activity at individual binding sites. Here we first briefly summarize high-throughput approaches to studying transcription factor activity. We then demonstrate, using published chromatin accessibility data (specifically ATAC-seq, that the genome-wide profile of TF recognition motifs relative to regions of open chromatin can determine the key transcription factor altered by a perturbation. Our method of determining which TFs are altered by a perturbation is simple, is quick to implement, and can be used when biological samples are limited. In the future, we envision that this method could be applied to determine which TFs show altered activity in response to a wide variety of drugs and diseases.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk

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Katheryne Benini Martins

2015-06-01

Full Text Available The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT, somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst, biofilm (icaA, icaC, icaD, bap, leukocidin (luk-PV oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics. Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Staphylococcus aureus regulates the expression and production of the staphylococcal superantigen-like secreted proteins in a Rot-dependent manner.

Science.gov (United States)

Benson, Meredith A; Lilo, Sarit; Wasserman, Gregory A; Thoendel, Matthew; Smith, Amanda; Horswill, Alexander R; Fraser, John; Novick, Richard P; Shopsin, Bo; Torres, Victor J

2011-08-01

Staphylococcus aureus overproduces a subset of immunomodulatory proteins known as the staphylococcal superantigen-like proteins (Ssls) under conditions of pore-mediated membrane stress. In this study we demonstrate that overproduction of Ssls during membrane stress is due to the impaired activation of the two-component module of the quorum-sensing accessory gene regulator (Agr) system. Agr-dependent repression of ssl expression is indirect and mediated by the transcription factor repressor of toxins (Rot). Surprisingly, we observed that Rot directly interacts with and activates the ssl promoters. The role of Agr and Rot as regulators of ssl expression was observed across several clinically relevant strains, suggesting that overproduction of immunomodulatory proteins benefits agr-defective strains. In support of this notion, we demonstrate that Ssls contribute to the residual virulence of S. aureus lacking agr in a murine model of systemic infection. Altogether, these results suggest that S. aureus compensates for the inactivation of Agr by producing immunomodulatory exoproteins that could protect the bacterium from host-mediated clearance. © 2011 Blackwell Publishing Ltd.

[Enterotoxin genes occurance among S. aureus strains isolated from inpatients and carriers].

Science.gov (United States)

Lawrynowicz-Paciorek, Maja; Kochman, Maria; Piekarska, Katarzyna; Wyrebiak, Agata; Potracka, Ewa; Leniak-Chmiel, Urszula; Magdziak, Agnieszka

2006-01-01

We examined 44 inpatients and 66 carriers Staphylococcus aureus strains, isolated in years 2002-2005, for the presence of 18 enterotoxin genes (se/sel) (by PCR), the ability for A-D enterotoxin production (by SET-RPLA) and antibiotic resistance distribution (by disc diffusion method). se/sel genes were detected in 90,9% of all strains, sea (70,5%) and selk and selq (52,3%) - among inpatients strains and egc (65,2%) - among carriers strains were the most frequently se/sel genes found. Positive results of SET-RPLA were consistent with PCR results. There was no correlation observed between antibiotic resistance and se/sel genes distribution among tested S. aureus strains.

Detecção da toxina-1 da síndrome do choque tóxico em amostras de Staphylococcus aureus isoladas de mastite bovina Detection of toxic shock syndrome toxin by Staphylococcus aureus strains isolated from bovine mastitis

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H.F.T. Cardoso

2000-02-01

Full Text Available Este trabalho teve por objetivo caracterizar a produção da toxina da síndrome do choque tóxico (TSST-1 e de enterotoxinas estafilocócicas (SE A, B, C e D em 127 amostras de S. aureus, isoladas de amostras de leite proveniente de vacas com mastite no Estado de Minas Gerais, entre 1994 e 1997. A verificação da produção de toxinas foi feita pela técnica de sensibilidade ótima em placa. Das 127 amostras testadas, 60 (47% eram produtoras de TSST-1 e 54 (43% produtoras de SE, 38 amostras produziram SED (30%, 24 SEB (19%, 8 SEC (6% e 4 SEA (3%. Estes resultados trazem preocupações quanto à saúde pública pela alta prevalência de amostras de S. aureus produtoras de TSST-1 e de enterotoxinas em isolamentos a partir de leite de vacas com mastite.A total of 127 strains of Staphylococcus aureus were examined for the production of toxic shock syndrome toxin (TSST-1 and staphylococcal enterotoxins (SE A, B, C and D. The strains were isolated from milk samples from cows with mastitis in dairy herds of Minas Gerais State, Brazil, from 1994 to 1997. The toxins were detected using the optimum-sensitivity plate method. Of 127 isolates, 60 (47% produced TSST-1 and 54 (43% produced SE, 38 (30% produced SED, 24 (19% SEB, 8 (6% SEC and 4 (3% enterotoxin A..

Occurrence of Staphylococcus aureus in cattle, sheep, goat, and pig rearing in the Czech Republic

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Marcela Klimešová

2017-01-01

Full Text Available The study focused on the prevalence of Staphylococcus aureus and resistant strains in livestock. In this study, 114 different samples from three cattle farms (84 from two farms of dairy cows and 30 from one farm of suckler cows, 132 samples from one sheep farm, 120 samples from one goat farm, and 82 samples from three pig farms were examined. Strains identified as Staphylococcus aureus were further analysed by the polymerase chain reaction method for detection of the mecA gene and for confirmation of the sequence type 398. Positive incidence of Staphylococcus aureus was confirmed in farms of suckler cows, sheep, goats and pigs. The incidence of methicilin-resistant Staphylococcus aureus was confirmed at a goat farm, with all strains belonging to the sequence type 398. Repetitive element palindromic-polymerase chain reaction analysis was performed to compare the relatedness of selected human and animal S. aureus strains at the goat and sheep farms. The obtained data from repetitive element-polymerase chain reaction analysis showed significant clonal similarity among the tested isolates and indicated the possibility of mutual transmission between animals or animal and human and possible transfer in the food chain.

Investigation of the antibiotic resistance and biofilm-forming ability of Staphylococcus aureus from subclinical bovine mastitis cases.

Science.gov (United States)

Aslantaş, Özkan; Demir, Cemil

2016-11-01

A total of 112 Staphylococcus aureus isolates obtained from subclinical bovine mastitis cases were examined for antibiotic susceptibility and biofilm-forming ability as well as genes responsible for antibiotic resistance, biofilm-forming ability, and adhesin. Antimicrobial susceptibility of the isolates were determined by disk diffusion method. Biofilm forming ability of the isolates were investigated by Congo red agar method, standard tube method, and microplate method. The genes responsible for antibiotic resistance, biofilm-forming ability, and adhesion were examined by PCR. Five isolates (4.5%) were identified as methicillin-resistant Staph. aureus by antibiotic susceptibility testing and confirmed by mecA detection. The resistance rates to penicillin, ampicillin, tetracycline, erythromycin, trimethoprim-sulfamethoxazole, enrofloxacin, and amoxicillin-clavulanic acid were 45.5, 39.3, 33, 26.8, 5.4, 0.9, and 0.9%, respectively. All isolates were susceptible against vancomycin and gentamicin. The blaZ (100%), tetK (67.6%), and ermA (70%) genes were the most common antibiotic-resistance genes. Using Congo red agar, microplate, and standard tube methods, 70.5, 67, and 62.5% of the isolates were found to be biofilm producers, respectively. The percentage rate of icaA, icaD, and bap genes in Staph. aureus isolates were 86.6, 86.6, and 13.4%, respectively. The adhesion molecules fnbA, can, and clfA were detected in 87 (77.7%), 98 (87.5%), and 75 (70%) isolates, respectively. The results indicated that Staph. aureus from sublinical bovine mastitis cases were mainly resistant to β-lactams and, to a lesser extent, to tetracycline and erythromycin. Also, biofilm- and adhesion-related genes, which are increasingly accepted as an important virulence factor in the pathogenesis of Staph. aureus infections, were detected at a high rate. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Molecular characterization of methicillin resistant Staphylococcus aureus isolated from hospitals environments and patients in Northern Palestine

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Ghaleb Adwan

2015-09-01

Full Text Available BACKGROUND: Staphylococcus aureus (S. aureus is considered one of the most common pathogen to humans. Infections caused by this mocroorganism can be acquired through both hospital and community settings. This study was carried out to investigate molecular characterization of MRSA strains isolated from the patients and their environment in two hospitals (Rafidia hospital and Thabet hospital inNorthern Palestine, and to determine the clonal identity between these strains and their possible contribution to nosocomial infections.METHODS: Two hundred sixty five swabbed samples were collected from these hospitals, S. aureus was isolated,  antibiotic resistant genes were Panton–Valentin leukocidin (PVL gene were detected and SCCmec and spA were typed by PCR and/or sequencing.RESULTS: The prevalence of MRSA among S. aureus isolates was 29% and 8.2% in Rafidia hospital and Thabet hospital, respectively. All strains resistant to oxacilllin disk were carried mecA gene. Majority of strains (84.6% carried SCCmec type II (n = 11, type IVa and non-typeable were also detected. In addition, PVL was detected in 2 (14.3% clinical strains. ERIC PCR patterns revealed that 2 strains recovered from patient bed and nasal swab isolated from Thabet Hospital were nontypeable, spA typing showed that they belonged to type t386 and have identical DNA sequences. Other 2 clinical isolates were spa typed, one belonged to clone t044, while the other is new clone not exist in database.CONCLUSIONS: Results may give evidence that environmental contamination possibly contributing to nosocomial infections.

Methicillin-Resistant Staphylococcus aureus from Brazilian Dairy Farms and Identification of Novel Sequence Types.

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Oliveira, C J B; Tiao, N; de Sousa, F G C; de Moura, J F P; Santos Filho, L; Gebreyes, W A

2016-03-01

The aim of this study was to investigate the phenotypic and genotypic diversity and anti-microbial resistance among staphylococci of dairy herds that originated from Paraiba State, north-eastern Brazil, a region where such studies are rare. Milk samples (n = 552) were collected from 15 dairy farms. Isolates were evaluated for anti-microbial susceptibility by Kirby-Bauer disc diffusion method. Confirmation of methicillin-resistant Staphylococcus aureus (MRSA) was performed using multiplex PCR targeting mecA and nuc genes in addition to phenotypic assay based on PBP-2a latex agglutination. Clonal relatedness of isolates was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) genotyping. Staphylococci were detected in 269 (49%) of the samples. Among these, 65 (24%) were S. aureus. The remaining 204 isolates were either coagulase-negative staphylococci (n = 188; 70%) or coagulase positive other than S. aureus (n = 16; 6%). Staphylococci were cultured in seven (35%) of the 20 hand swab samples, from which five isolates were S. aureus. The isolates were most commonly resistant against penicillin (43%), ampicillin (38%) and oxacillin (27%). The gene mecA was detected in 21 S. aureus from milk and in one isolate from a milker's hand. None of the isolates were resistant to vancomycin. PFGE findings showed high clonal diversity among the isolates. Based on MLST, we identified a total of 11 different sequence types (STs 1, 5, 6, 83, 97, 126, 1583, 1622, 1623, 1624 and 1625) with four novel STs (ST1622-ST1625). The findings show that MRSA is prevalent in milk from semi-extensive dairy cows in north-eastern Brazil, and further investigation on its extent in various types of milk production systems and the farm-to-table continuum is warranted. © 2015 Blackwell Verlag GmbH.

Persistence of livestock-associated antibiotic-resistant Staphylococcus aureus among industrial hog operation workers in North Carolina over 14 days.

Science.gov (United States)

Nadimpalli, Maya; Rinsky, Jessica L; Wing, Steve; Hall, Devon; Stewart, Jill; Larsen, Jesper; Nachman, Keeve E; Love, Dave C; Pierce, Elizabeth; Pisanic, Nora; Strelitz, Jean; Harduar-Morano, Laurel; Heaney, Christopher D

2015-02-01

This study aimed to evaluate the persistence of nasal carriage of Staphylococcus aureus, methicillin-resistant S. aureus and multidrug-resistant S. aureus over 14 days of follow-up among industrial hog operation workers in North Carolina. Workers anticipating at least 24 h away from work were enrolled June-August 2012. Participants self-collected a nasal swab and completed a study journal on the evening of day 1, and each morning and evening on days 2-7 and 14 of the study. S. aureus isolated from nasal swabs were assessed for antibiotic susceptibility, spa type and absence of the scn gene. Livestock association was defined by absence of scn. Twenty-two workers provided 327 samples. S. aureus carriage end points did not change with time away from work (mean 49 h; range >0-96 h). Ten workers were persistent and six were intermittent carriers of livestock-associated S. aureus. Six workers were persistent and three intermittent carriers of livestock-associated multidrug-resistant S. aureus. One worker persistently carried livestock-associated methicillin-resistant S. aureus. Six workers were non-carriers of livestock-associated S. aureus. Eighty-two per cent of livestock-associated S. aureus demonstrated resistance to tetracycline. A majority of livestock-associated S. aureus isolates (n=169) were CC398 (68%) while 31% were CC9. No CC398 and one CC9 isolate was detected among scn-positive isolates. Nasal carriage of livestock-associated S. aureus, multidrug-resistant S. aureus and methicillin-resistant S. aureus can persist among industrial hog operation workers over a 14-day period, which included up to 96 h away from work. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Complete genome analysis of two new bacteriophages isolated from impetigo strains of Staphylococcus aureus.

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Botka, Tibor; Růžičková, Vladislava; Konečná, Hana; Pantůček, Roman; Rychlík, Ivan; Zdráhal, Zbyněk; Petráš, Petr; Doškař, Jiří

2015-08-01

Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated ϕB166 and ϕB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

Detection of canine cytokine gene expression by reverse transcription-polymerase chain reaction.

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Pinelli, E; van der Kaaij, S Y; Slappendel, R; Fragio, C; Ruitenberg, E J; Bernadina, W; Rutten, V P

1999-08-02

Further characterization of the canine immune system will greatly benefit from the availability of tools to detect canine cytokines. Our interest concerns the study on the role of cytokines in canine visceral leishmaniasis. For this purpose, we have designed specific primers using previously published sequences for the detection of canine IL-2, IFN-gamma and IL10 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). For IL-4, we have cloned and sequenced this cytokine gene, and developed canine-specific primers. To control for sample-to-sample variation in the quantity of mRNA and variation in the RT and PCR reactions, the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), a housekeeping gene, were determined in parallel. Primers to amplify G3PDH were designed from consensus sequences obtained from the Genbank database. The mRNA levels of the cytokines mentioned here were detected from ConA-stimulated peripheral mononuclear cells derived from Leishmania-infected dogs. A different pattern of cytokine production among infected animals was found.

Enterotoxigenicity and Antimicrobial Resistance of Staphylococcus aureus Isolated from Retail Food in China

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Wang, Wei; Baloch, Zulqarnain; Jiang, Tao; Zhang, Cunshan; Peng, Zixin; Li, Fengqin; Fanning, Séamus; Ma, Aiguo; Xu, Jin

2017-01-01

Staphylococcus aureus is one of the most common causes of zoonotic agent in the world, which are attributable to the contamination of food with enterotoxins. In this study, a total of 1,150 S. aureus isolates were cultured from 27,000 retail foods items from 203 cities of 24 provinces in China in 2015 and were test for antimicrobial susceptibility. Additionally, the role of the genes responsible for the staphylococcal enterotoxins (SEA to SEE), methicillin resistance (mecA) and the toxigenic capabilities were also assessed. The results showed that 4.3% retail foods were contaminated with S. aureus, and 7.9% retail foods isolates were mecA positive. Some 97.6% of S. aureus isolates were resistant to at least one antimicrobial compound, and 57.5% of these were multi drug resistant (MDR). Resistance to penicillin (83.7%, 963/1,150), was common, followed by linezolid (67.7%, 778/1,150) and erythromycin (52.1%, 599/1,150). The isolates cultured from raw meats showed high levels of resistant to tetracycline (42.8%), ciprofloxacin (17.4%), and chloramphenicol (12.0%) and expressed a MDR phenotype (62.4%). A total of 29.7% S. aureus isolates harbored the classical SEs genes (sea, seb, sec, and sed). The sea and seb genes were the most frequent SEs genes detected. Of note, 22% of the SEs genes positive S. aureus harbored two or three SEs genes, and 16 isolates were confirmed with the capacity to simultaneously produce two or three enterotoxin types. Moreover, nearly 50% of the MRSA isolates were positive for at least one SE gene in this study. Therefore, it is important to monitor the antimicrobial susceptibility and enterotoxigenicity of MDR S. aureus and MRSA in the food chain and to use these data to develop food safety measures, designed to reduce the contamination and transmission of this bacterium. PMID:29209290

METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA ...

African Journals Online (AJOL)

Nosocomial infections caused by methicillin-resistant strains of Staphylococcus aureus often pose therapeutic dilemma to the clinicians because of the multi resistant nature of these strains of Staphylococcus aureus. Outbreaks of both nosocomial and community acquired infections are also frequent and difficult to control.

Persistence of livestock-associated antibiotic-resistant Staphylococcus aureus among industrial hog operation workers in North Carolina over 14 days

Science.gov (United States)

Nadimpalli, Maya; Rinsky, Jessica L; Wing, Steve; Hall, Devon; Stewart, Jill; Larsen, Jesper; Nachman, Keeve E; Love, Dave C; Pierce, Elizabeth; Pisanic, Nora; Strelitz, Jean; Harduar-Morano, Laurel; Heaney, Christopher D

2015-01-01

Objectives This study aimed to evaluate the persistence of nasal carriage of Staphylococcus aureus, methicillin-resistant S. aureus and multidrug-resistant S. aureus over 14 days of follow-up among industrial hog operation workers in North Carolina. Methods Workers anticipating at least 24 h away from work were enrolled June–August 2012. Participants self-collected a nasal swab and completed a study journal on the evening of day 1, and each morning and evening on days 2–7 and 14 of the study. S. aureus isolated from nasal swabs were assessed for antibiotic susceptibility, spa type and absence of the scn gene. Livestock association was defined by absence of scn. Results Twenty-two workers provided 327 samples. S. aureus carriage end points did not change with time away from work (mean 49 h; range >0–96 h). Ten workers were persistent and six were intermittent carriers of livestock-associated S. aureus. Six workers were persistent and three intermittent carriers of livestock-associated multidrug-resistant S. aureus. One worker persistently carried livestock-associated methicillin-resistant S. aureus. Six workers were non-carriers of livestock-associated S. aureus. Eighty-two per cent of livestock-associated S. aureus demonstrated resistance to tetracycline. A majority of livestock-associated S. aureus isolates (n=169) were CC398 (68%) while 31% were CC9. No CC398 and one CC9 isolate was detected among scn-positive isolates. Conclusions Nasal carriage of livestock-associated S. aureus, multidrug-resistant S. aureus and methicillin-resistant S. aureus can persist among industrial hog operation workers over a 14-day period, which included up to 96 h away from work. PMID:25200855

Reverse Transcription Polymerase Chain Reaction-based System for Simultaneous Detection of Multiple Lily-infecting Viruses

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Ji Yeon Kwon

2013-09-01

Full Text Available A detection system based on a multiplex reverse transcription (RT polymerase chain reaction (PCR was developed to simultaneously identify multiple viruses in the lily plant. The most common viruses infecting lily plants are the cucumber mosaic virus (CMV, lily mottle virus (LMoV, lily symptomless virus (LSV. Leaf samples were collected at lily-cultivation facilities located in the Kangwon province of Korea and used to evaluate the detection system. Simplex and multiplex RT-PCR were performed using virus-specific primers to detect single-or mixed viral infections in lily plants. Our results demonstrate the selective detection of 3 different viruses (CMV, LMoV and LSV by using specific primers as well as the potential of simultaneously detecting 2 or 3 different viruses in lily plants with mixed infections. Three sets of primers for each target virus, and one set of internal control primers were used to evaluate the detection system for efficiency, reliability, and reproducibility.

PARTIAL CHARACTERIZATION OF A LYTIC METHICILLIN RESISTANT-STAPHYLOCOCCUS AUREUS BACTERIOPHAGE

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Sulaiman Al-Yousef

2014-12-01

Full Text Available A marked increase in the infection incidence caused by methicillin-resistant Staphylococcus aureus (MRSA strains has been noted in medical practice in recent years. This study was conducted to study the biological and characterize of MRSA-phage. Methicillin resistance of Staphylococcus aureus was detected and confirmed by determining of the MIC of oxacillin by the standard agar dilution method. Phage was biologically purified using single plaque technique, then phage characterization were studied using host range, adsorption time, particle morphology and its structural protein. MRSA phage showing lytic nature was purified by repeated plating after picking of single isolated plaques. This phage is active against all 11 isolates either of S. aureus or MRSA tested as hosts. Phage produced clear plaques indicating their lytic nature. This phage was concentrated employing polyethylene glycol (PEG-NaCl precipitation method. Morphologically, MRSA Phage has a hexagonal head having a long non-contractile tail, indicating his icosahedral nature. Adsorption studies showed 100% adsorption of MRSA-Phage after 35 minutes of exposure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE experimentation indicated that the phage particles contain one major structural protein (about 30 Kda.

Nasal carriage of Methicillin-resistant Staphylococcus aureus among healthy population of Kashmir, India

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B A Fomda

2014-01-01

Full Text Available Background: Nasal colonisation with community acquired methicillin resistant Staphylococcus aureus (CA-MRSA is being increasingly reported, especially in places where people are in close contact and where hygiene is compromised. The aim of this study was to find out prevalence of methicillin resistant S.aureus (MRSA colonising anterior nares of healthy subjects. Materials and Methods: Nasal swabs of healthy subjects were collected aseptically and cultured using standard microbiological protocols. Antibiotic susceptibility was done by Kirby-Bauer disc diffusion method according to CLSI guidelines. Methicillin resistance was detected by cefoxitin disc diffusion method and confirmed by minimum inhibitory concentration (MIC and amplification of mecA gene by PCR. Strain typing of MRSA strains was done by PFGE. Results: Out of 820 samples, S.aureus was isolated from 229 (27.92% subjects. Of the 229 isolates, 15 were methicillin resistant. All S. aureus isolates were susceptible to vancomycin. Nasal carriage of MRSA was found to be 1.83% among healthy population. The isolates were found to be polyclonal by PFGE analysis. Conclusion: High prevalence of MRSA is a cause of concern and strategies to interrupt transmission should be implemented.

Complete Genome Sequence of the Quality Control Strain Staphylococcus aureus subsp. aureus ATCC 25923.

Science.gov (United States)

Treangen, Todd J; Maybank, Rosslyn A; Enke, Sana; Friss, Mary Beth; Diviak, Lynn F; Karaolis, David K R; Koren, Sergey; Ondov, Brian; Phillippy, Adam M; Bergman, Nicholas H; Rosovitz, M J

2014-11-06

Staphylococcus aureus subsp. aureus ATCC 25923 is commonly used as a control strain for susceptibility testing to antibiotics and as a quality control strain for commercial products. We present the completed genome sequence for the strain, consisting of the chromosome and a 27.5-kb plasmid. Copyright © 2014 Treangen et al.

Prevalence and Characterization of Staphylococcus aureus Cultured From Raw Milk Taken From Dairy Cows With Mastitis in Beijing, China

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Wei Wang

2018-06-01

Full Text Available The colonization of dairy herds and subsequent contamination of raw milk by Staphylococcus aureus (S. aureus, especially those expressing a multi-drug resistance (MDR, biofilm and toxins producing ability, remains an important issue for both the dairy producer and public health. In this study, we investigated the prevalence, antimicrobial resistance, virulence, and genetic diversity of S. aureus in raw milk taken from 2 dairy farms in Beijing, China. Ninety (46.2%, 90/195 samples were positive for S. aureus. Resistant to penicillin (PEN (31.3%, ciprofloxacin (18.8% and enrofloxacin (15.6% were the most often observed. Isolates cultured from farm B showed significantly higher resistance to penicillin (73.9%, ciprofloxacin (34.8%, enrofloxacin (34.8%, tilmicosin (17.4%, and erythromycin (17.4% than those from farm A (p < 0.05. Totally, 94.8% S. aureus harbored at least one virulence gene and the pvl (93.8%, sec (65.6%, and sea (60.4% genes were the most frequently detected. The pvl and sec genes were more often detected in isolates from farm A (97.3% and 84.9% respectively than those from farm B (p < 0.05. Of all 77 staphylococcus enterotoxin (SE-positive isolates, more than 90% could produce enterotoxins and 70.1% could produce two types. Biofilm related genes (icaA/D, clf/B, can, and fnbA were detected in all96 isolates. All 96 isolates could produce biofilm with 8.3, 70.8, and 18.8% of the isolates demonstrating weak, moderate and strong biofilm formation, respectively. A total of 5 STs, 7 spa types (1 novel spa type t17182, 3agr types (no agrII, and 14 SmaI-pulso-types were found in this study. PFGE cluster II-CC1-ST1-t127-agr III was the most prevalent clone (56.3%. Isolates of agr III (PFGE Cluster I/II-CC1-ST1-t127/2279 had higher detection of virulence genes than those of agr I and agr IV. TheMSSA-ST398-t1456-agr I clone expressed the greatest MDRbut with no virulence genes and weakly biofilm formation. Our finding indicated a relatively

Real-time quantitative PCR of Staphylococcus aureus and application in restaurant meals.

Science.gov (United States)

Berrada, H; Soriano, J M; Mañes, J; Picó, Y

2006-01-01

Staphylococcus aureus is considered the second most common pathogen to cause outbreaks of food poisoning, exceeded only by Campylobacter. Consumption of foods containing this microorganism is often identified as the cause of illness. In this study, a rapid, reliable, and sensitive real-time quantitative PCR was developed and compared with conventional culture methods. Real-time quantitative PCR was carried out by purifying DNA extracts of S. aureus with a Staphylococcus sample preparation kit and quantifying it in the LightCycler system with hybridization probes. The assay was linear from a range of 10 to 10(6) S. aureus cells (r2 > 0.997). The PCR reaction presented an efficiency of >85%. Accuracy of the PCR-based assay, expressed as percent bias, was around 13%, and the precision, expressed as a percentage of the coefficient of variation, was 7 to 10%. Intraday and interday variability were studied at 10(2) CFU/g and was 12 and 14%, respectively. The proposed method was applied to the analysis of 77 samples of restaurant meals in Valencia (Spain). In 11.6% of samples S. aureus was detected by real-time quantitative PCR, as well as by the conventional microbiological method. An excellent correspondence between real-time quantitative PCR and microbiological numbers (CFU/g) was observed with deviations of < 28%.

Molecular characterization of a prevalent ribocluster of methicillin-sensitiveStaphylococcus aureus from orthopedic implant infections. Correspondencewith MLST CC30

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Lucio eMontanaro

2016-02-01

Full Text Available ABSTRACTStaphylococcus aureus is the leading etiologic agent of orthopedic implant infections. Here a ribocluster of 27 S. aureus strains underwent further molecular characterization and subtyping by multilocus sequence typing (MLST and spa-typing. This cluster had been detected by automated ribotyping (with EcoRI as restriction enzyme of 200 S. aureus isolates from periprosthetic infections come for revision at the Rizzoli Orthopaedic Institute. The ribocluster, consisting of agr type III isolates, with a 74% co-presence of bone sialoprotein-binding (bbp and collagen-binding (cna genes, turned out devoid of mecA and IS256 and exhibited a high prevalence of toxic shock syndrome toxin gene (tst, 85%. Sequences achieved by spa typing and MLST were analyzed by BURP and goeBURST. Two predominant spa types, t012 (32% and t021 (36%, and one predominant sequence type, ST30 (18/27, 67%, a Staphylococcus aureus lineage spread worldwide and regarded as the ancestor of MLST CC30, were identified. Two new sequence types (ST2954, ST2960 and one new spa type (t13129 were detected for the first time. BURP clustered the isolates into two spa clonal complexes, CC021/012 (22/27, 81% and CC166 (4/27, 15%, plus one singleton, while goeBURST recognized solely MLST CC30. Interestingly, the 27-strains cluster detected by ribotyping corresponded exactly to CC30.

Antimicrobial resistant coagulase positive Staphylococcus aureus ...

African Journals Online (AJOL)

ADEYEYE

S. aureus is associated with many clinical syndromes including tenosynovitis, omphalitis, femoral head necrosis, .... Markey, 2008) where occurrence of multidrug ... Staphylococcus aureus isolates from bovine mastitis in. Denmark. Veterinary.

Meta-Analysis of Transcriptional Responses to Mastitis-Causing Escherichia coli.

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Sidra Younis

Full Text Available Bovine mastitis is a widespread disease in dairy cows, and is often caused by bacterial mammary gland infection. Mastitis causes reduced milk production and leads to excessive use of antibiotics. We present meta-analysis of transcriptional profiles of bovine mastitis from 10 studies and 307 microarrays, allowing identification of much larger sets of affected genes than any individual study. Combining multiple studies provides insight into the molecular effects of Escherichia coli infection in vivo and uncovers differences between the consequences of E. coli vs. Staphylococcus aureus infection of primary mammary epithelial cells (PMECs. In udders, live E. coli elicits inflammatory and immune defenses through numerous cytokines and chemokines. Importantly, E. coli infection causes downregulation of genes encoding lipid biosynthesis enzymes that are involved in milk production. Additionally, host metabolism is generally suppressed. Finally, defensins and bacteria-recognition genes are upregulated, while the expression of the extracellular matrix protein transcripts is silenced. In PMECs, heat-inactivated E. coli elicits expression of ribosomal, cytoskeletal and angiogenic signaling genes, and causes suppression of the cell cycle and energy production genes. We hypothesize that heat-inactivated E. coli may have prophylactic effects against mastitis. Heat-inactivated S. aureus promotes stronger inflammatory and immune defenses than E. coli. Lipopolysaccharide by itself induces MHC antigen presentation components, an effect not seen in response to E. coli bacteria. These results provide the basis for strategies to prevent and treat mastitis and may lead to the reduction in the use of antibiotics.

Bivariate Genomic Footprinting Detects Changes in Transcription Factor Activity

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Songjoon Baek

2017-05-01

Full Text Available In response to activating signals, transcription factors (TFs bind DNA and regulate gene expression. TF binding can be measured by protection of the bound sequence from DNase digestion (i.e., footprint. Here, we report that 80% of TF binding motifs do not show a measurable footprint, partly because of a variable cleavage pattern within the motif sequence. To more faithfully portray the effect of TFs on chromatin, we developed an algorithm that captures two TF-dependent effects on chromatin accessibility: footprinting and motif-flanking accessibility. The algorithm, termed bivariate genomic footprinting (BaGFoot, efficiently detects TF activity. BaGFoot is robust to different accessibility assays (DNase-seq, ATAC-seq, all examined peak-calling programs, and a variety of cut bias correction approaches. BaGFoot reliably predicts TF binding and provides valuable information regarding the TFs affecting chromatin accessibility in various biological systems and following various biological events, including in cases where an absolute footprint cannot be determined.

Transfer of Antibiotic Resistance in Staphylococcus aureus

DEFF Research Database (Denmark)

Haaber, Jakob; Penadés, José R; Ingmer, Hanne

2017-01-01

Staphylococcus aureus is a serious human pathogen with remarkable adaptive powers. Antibiotic-resistant clones rapidly emerge mainly by acquisition of antibiotic-resistance genes from other S. aureus strains or even from other genera. Transfer is mediated by a diverse complement of mobile genetic...... of plasmids that can be transferred by conjugation and the efficiency with which transduction occurs. Here, we review the main routes of antibiotic resistance gene transfer in S. aureus in the context of its biology as a human commensal and a life-threatening pathogen. Staphylococcus aureus cells...... are effective in exchanging mobile genetic elements, including antibiotic-resistance genes.During colonization or infection of host organisms, the exchange appears to be particularly effective.Bacteriophage-mediated transfer involves both transduction and autotransduction, which may enable lysogenic S. aureus...

Methicillin-Susceptible, Vancomycin-Resistant Staphylococcus aureus, Brazil

OpenAIRE

Panesso , Diana; Planet , Paul J.; Diaz , Lorena; Hugonnet , Jean-Emannuel; Tran , Truc T.; Narechania , Apurva; Munita , José M.; Rincon , Sandra; Carvajal , Lina P.; Reyes , Jinnethe; Londono , Alejandra; Smith , Hannah; Sebra , Robert; Deikus , Gintaras; Weinstock , George M

2015-01-01

International audience; We report characterization of a methicillin-susceptible, vancomycin-resistant bloodstream isolate of Staphylococcus aureus recovered from a patient in Brazil. Emergence of vancomycin resistance in methicillin-susceptible S. aureus would indicate that this resistance trait might be poised to disseminate more rapidly among S. aureus and represents a major public health threat.

Detection of diverse genotypes of Methicillin-resistant Staphylococcus aureus from hospital personnel and the environment in Armenia

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Hermine V. Mkrtchyan

2017-02-01

Full Text Available Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA is a public health concern internationally. Studies examining a range of cohorts have been reported from various regions of the world, but little is known about the molecular epidemiology of MRSA in Armenia. Methods Between May and September 2013, twenty isolates of methicillin-resistant Staphylococcus aureus (MRSA; mecA positive were recovered from hospital personnel (n = 10; 9 females, 1 male and environmental sites (n = 10 in the maternity ward of one of the teaching hospitals in Armenia. Results Multi-locus sequence type clonal complex (MLST-CC assignments inferred from spa typing data revealed the majority belonged to 3 pandemic lineages of MRSA including: t008-CC8-SCCmecV (n = 10; 7 from personnel; t021-CC30-SCCmecIV (n = 5; all environmental; and t1523-CC45 (n = 2; 1 from personnel, one harboured SCCmecV the other was SCCmec non-typable. The remainder identified as belonging to genotype t364-CC182, both of which harboured a novel SCCmec cassette with kdp, rif5, ccrB2 and ccrC detected by PCR (both from personnel; and t325-CC88-SCCmecIV (n = 1; environmental. All MRSA were negative for the Panton-Valentine Leukocidin (PVL locus and three CC8 strains were positive for the arginine catabolic element (ACME. Conclusions In this small study, we report for the first time of the occurrence of diverse MRSA genotypes belonging to both pandemic and more sporadic international clones in Armenia harbouring the smaller SCCmec types and/or ACME, both of which have been associated with strain fitness. Further surveillance is warranted to better understand the prevalence, clinical and molecular epidemiology of MRSA throughout Armenia.

Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Simultaneous Detection of mecA, nuc, and femB by Loop-Mediated Isothermal Amplification (LAMP).

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Chen, Changguo; Zhao, Qiangyuan; Guo, Jianwei; Li, Yanjun; Chen, Qiuyuan

2017-08-01

The aim of this study was to develop a rapid detection assay to identify methicillin-resistant Staphylococcus aureus by simultaneous testing for the mecA, nuc, and femB genes using the loop-mediated isothermal amplification (LAMP) method. LAMP primers were designed using online bio-software ( http://primerexplorer.jp/e/ ), and amplification reactions were performed in an isothermal temperature bath. The products were then examined using 2% agarose gel electrophoresis. MecA, nuc, and femB were confirmed by triplex TaqMan real-time PCR. For better naked-eye inspection of the reaction result, hydroxy naphthol blue (HNB) was added to the amplification system. Within 60 min, LAMP successfully amplified the genes of interest under isothermal conditions at 63 °C. The results of 2% gel electrophoresis indicated that when the Mg 2+ concentration in the reaction system was 6 μmol, the amplification of the mecA gene was relatively good, while the amplification of the nuc and femB genes was better at an Mg 2+ concentration of 8 μmol. Obvious color differences were observed by adding 1 μL (3.75 mM) of HNB into 25 μL reaction system. The LAMP assay was applied to 128 isolates cases of methicillin-resistant Staphylococcus aureus, which were separated from the daily specimens and identified by Vitek microbial identification instruments. The results were identical for both LAMP and PCR. LAMP offers an alternative detection assay for mecA, nuc, and femB and is faster than other methods.

VanA and VanB Positive Vancomycin-resistant Staphylococcus aureus Among Clinical Isolates in Shiraz, South of Iran

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Sareh Saadat

2014-09-01

Full Text Available Objective: The purpose of this study was to determine the prevalence of vancomycin-resistant Staphylococcus aureus isolated from clinical samples in Shiraz hospitals. Methods: From March to December 2012, 100 S. aureus isolates (mainly from wound and blood were collected from three hospitals in Shiraz, south of Iran. After identification of Staphylococcus aureus by biochemical, microbiological and molecular methods, antibiotic susceptibility testing was performed by Kirby-Bauer disc diffusion test for 13 different antibiotics. Vancomycin-resistant Staphylococcus aureus isolates were determined by vancomycin agar screening test and PCR for vancomycin resistant genes (vanA and vanB. Results: The lowest and highest resistance was seen for quinupristin-dalfopristin (n=1 and ampicillin (n=95, respectively. Vancomycin agar screening test showed that 37 isolates can grow on these media. Further study by PCR also detected vanA and/or vanB genes in all of these strains. Also, 19 isolates showed either vanA or vanB but were susceptible according to vancomycin agar screening test. In total, vanA and vanB resistant genes were detected in 34% and 37% of clinical isolates, respectively. Conclusion: The results showed that the frequency of vancomycin resistance genes (vanA, vanB is very high in Staphylococcus aureus strains isolated from patients in south of Iran. Thus, urgent interventions are needed to keep the emergence and transmission of these isolates to a minimum.

Occurrence of Staphylococcus aureus in raw milk produced in dairy farms in São Paulo state, Brazil

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Helena Fagundes

2010-06-01

Full Text Available The objective of the present study was to evaluate the occurrence of Staphylococcus aureus in milk produced in 37 farms located in the regions of Ribeirão Preto and São Carlos, state of São Paulo, Brazil. Two-hundred and eight samples of milk from individual cows showing subclinical mastitis, and 37 samples of bulk tank milk were analyzed. S. aureus strains were detected in 18 (7.3% milk samples: 14 (6.7% from samples of individual cows, and 4 (10.8% from bulk tank milk. Two individual milk samples (14.3% and two bulk milk samples contained enterotoxigenic S. aureus. PFGE analysis revealed the genetic heterogeneity of the strains isolated from raw milk, which presented to 13 S. aureus patterns. Results confirmed the potential transmission of staphylococcal food poisoning to consumers via milk of cows affected by subclinical mastitis, mainly when raw milk is ingested.

Genetic relatedness and virulence factors of bovine Staphylococcus aureus isolated from teat skin and milk.

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da Costa, L B; Rajala-Schultz, P J; Hoet, A; Seo, K S; Fogt, K; Moon, B S

2014-11-01

The objective of this study was to assess the role of teat skin colonization in Staphylococcus aureus intramammary infections (IMI) by evaluating genetic relatedness of Staph. aureus isolates from milk and teat skin of dairy cows using pulsed-field gel electrophoresis and characterizing the isolates based on the carriage of virulence genes. Cows in 4 known Staph. aureus-positive herds were sampled and Staph. aureus was detected in 43 quarters of 20 cows, with 10 quarters positive in both milk and skin (20 isolates), 18 positive only in milk, and 15 only on teat skin. Quarters with teat skin colonized with Staph. aureus were 4.5 times more likely to be diagnosed with Staph. aureus IMI than quarters not colonized on teat skin. Three main clusters were identified by pulsed-field gel electrophoresis using a cutoff of 80% similarity. All 3 clusters included both milk and skin isolates. The majority of isolates (72%) belonged to one predominant cluster (B), with 60% of isolates in the cluster originating from milk and 40% from teat skin. Genotypic variability was observed within 10 pairs (formed by isolates originating from milk and teat skin of the same quarter), where isolates in 5 out of the 10 pairs belonged to the same cluster. Forty-two virulence factors were screened using PCR. Some virulence factors were carried more frequently by teat skin isolates than by milk isolates or isolates from quarters with high somatic cell counts. Isolates in the predominant cluster B carried virulence factors clfA and clfB significantly more often than isolates in the minor clusters, which may have assisted them in becoming predominant in the herds. The present findings suggest that teat skin colonization with Staph. aureus can be an important factor involved in Staph. aureus IMI. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Quantitative Proteomic Analysis of Staphylococcus aureus Treated With Punicalagin, a Natural Antibiotic From Pomegranate That Disrupts Iron Homeostasis and Induces SOS.

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Cooper, Bret; Islam, Nazrul; Xu, Yunfeng; Beard, Hunter S; Garrett, Wesley M; Gu, Ganyu; Nou, Xiangwu

2018-05-01

Staphylococcus aureus, a bacterial, food-borne pathogen of humans, can contaminate raw fruits and vegetables. While physical and chemical methods are available to control S. aureus, scientists are searching for inhibitory phytochemicals from plants. One promising compound from pomegranate is punicalagin, a natural antibiotic. To get a broader understanding of the inhibitory effect of punicalagin on S. aureus growth, high-throughput mass spectrometry and quantitative isobaric labeling was used to investigate the proteome of S. aureus after exposure to a sublethal dose of punicalagin. Nearly half of the proteins encoded by the small genome were interrogated, and nearly half of those exhibited significant changes in accumulation. Punicalagin treatment altered the accumulation of proteins and enzymes needed for iron acquisition, and it altered amounts of enzymes for glycolysis, citric acid cycling, protein biosynthesis, and purine and pyrimidine biosynthesis. Punicalagin treatment also induced an SOS cellular response to damaged DNA. Transcriptional comparison of marker genes shows that the punicalagin-induced iron starvation and SOS responses resembles those produced by EDTA and ciprofloxacin. These results show that punicalagin adversely alters bacterial growth by disrupting iron homeostasis and that it induces SOS, possibly through DNA biosynthesis inhibition. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role for β-catenin and HOX transcription factors in Caenorhabditis elegans and mammalian host epithelial-pathogen interactions

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Irazoqui, Javier E.; Ng, Aylwin; Xavier, Ramnik J.; Ausubel, Frederick M.

2008-01-01

We used the model nematode Caenorhabditis elegans infected with the human pathogen Staphylococcus aureus to identify components of epithelial immunity. Transcriptional profiling and reverse genetic analysis revealed that mutation of the C. elegans β-catenin homolog bar-1 or the downstream homeobox gene egl-5 results in a defective response and hypersensitivity to S. aureus infection. Epistasis analysis showed that bar-1 and egl-5 function in parallel to previously described C. elegans immune-response pathways. Overexpression of human homologs of egl-5 modulated NF-κB-dependent TLR2 signaling in epithelial cells. These data suggest that β-catenin and homeobox genes play an important and conserved role in innate immune defense. PMID:18981407

Role for beta-catenin and HOX transcription factors in Caenorhabditis elegans and mammalian host epithelial-pathogen interactions.

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Irazoqui, Javier E; Ng, Aylwin; Xavier, Ramnik J; Ausubel, Frederick M

2008-11-11

We used the model nematode Caenorhabditis elegans infected with the human pathogen Staphylococcus aureus to identify components of epithelial immunity. Transcriptional profiling and reverse genetic analysis revealed that mutation of the C. elegans beta-catenin homolog bar-1 or the downstream homeobox gene egl-5 results in a defective response and hypersensitivity to S. aureus infection. Epistasis analysis showed that bar-1 and egl-5 function in parallel to previously described C. elegans immune-response pathways. Overexpression of human homologs of egl-5 modulated NF-kappaB-dependent TLR2 signaling in epithelial cells. These data suggest that beta-catenin and homeobox genes play an important and conserved role in innate immune defense.

[Carriage of Staphylococcus aureus among food service workers].

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Alarcón-Lavín, María Paula; Oyarzo, Carolina; Escudero, Carlos; Cerda-Leal, Fabiola; Valenzuela, Francisco J

2017-12-01

Background Staphylococcus aureus produces 11 serotypes of endotoxins that may cause food poisoning. Aim To determine the prevalence of type A enterotoxigenic Staphylococcus aureus carriage among food service workers in Chillan, Chile. Material and Methods Pharyngeal swabs were obtained from 100 food service workers and were cultured in Agar plates. After identifying the presence of Staphylococcus aureus, DNA was extracted to identify type A toxin by conventional PCR. Results Thirty eight percent of samples were colonized with Staphylococcus aureus. Among these, 26% were toxin A producers. Conclusions Half of the sampled workers carried Staphylococcus aureus and a quarter of these produced type A enterotoxin.

Genotypes and enterotoxicity of Staphylococcus aureus isolated from the hands and nasal cavities of flight-catering employees.

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Hatakka, M; Björkroth, K J; Asplund, K; Mäki-Petäys, N; Korkeala, H J

2000-11-01

Hand and nasal samples of flight-catering staff were collected from 1995 to 1997 to find employees carrying Staphylococcus aureus. Altogether 153 hand samples and 136 nose samples were taken. Nasal sampling showed a higher prevalence of S. aureus among food handlers (29%) than hand sampling (9%). A high proportion of the strains (46%) were enterotoxigenic, and a considerable amount of food handlers carried enterotoxigenic S. aureus, 6% and 12% according to hand and nasal sampling, respectively. Pulsed-field gel electrophoresis macrorestriction profiles revealed a total of 32 different types associated with the 35 employees carrying S. aureus. In most cases, the same type colonized both the hand and nose of a person. Despite the wide variety of types found, one strain colonized five persons and the second most common strain was associated with four food handlers. The predominant toxin produced was B, which was produced by the most common strain. The results showed that nasal sampling is a good way to detect S. aureus carriers, whereas hand sampling may fail to reveal carriers. The high proportion of enterotoxigenic strains show that a food handler harboring S. aureus must be considered a potential source of enterotoxigenic strains for airline meals.

Petrifilm rapid S. aureus Count Plate method for rapid enumeration of Staphylococcus aureus in selected foods: collaborative study.

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Silbernagel, K M; Lindberg, K G

2001-01-01

A rehydratable dry-film plating method for Staphylococcus aureus in foods, the 3M Petrifilm Rapid S. aureus Count Plate method, was compared with AOAC Official Method 975.55 (Staphylococcus aureus in Foods). Nine foods-instant nonfat dried milk, dry seasoned vegetable coating, frozen hash browns, frozen cooked chicken patty, frozen ground raw pork, shredded cheddar cheese, fresh green beans, pasta filled with beef and cheese, and egg custard-were analyzed for S. aureus by 13 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample and 3 levels of inoculated test sample, each in duplicate. The mean log counts for the methods were comparable for pasta filled with beef and cheese; frozen hash browns; cooked chicken patty; egg custard; frozen ground raw pork; and instant nonfat dried milk. The repeatability and reproducibility variances of the Petrifilm Rapid S. aureus Count Plate method were similar to those of the standard method.

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China.

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Yang, Xiaojuan; Yu, Shubo; Wu, Qingping; Zhang, Jumei; Wu, Shi; Rong, Dongli

2018-01-01

The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST) and polymerase chain reaction (PCR) analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE) genes ( sea , seb , sec , sed , see , seg , seh , sei , sej ), the exfoliative toxin genes ( eta and etb ), the toxic shock syndrome toxin-1 gene ( tst ), and the Panton-Valentine leucocidin-encoding gene ( pvl ). The isolates encompassed 26 different sequence types (STs), including four new STs (ST3482, ST3484, ST3485, ST3504), clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7%) detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%), sea (33.3%), and seg (33.3%). The classical SE genes ( sea - see ), which contribute significantly to staphylococcal food poisoning (SFP), were detected in 72.5% of the S. aureus isolates. In addition, pvl , eta , etb , and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies.

Multilocus Sequence Typing and Virulence-Associated Gene Profile Analysis of Staphylococcus aureus Isolates From Retail Ready-to-Eat Food in China

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Xiaojuan Yang

2018-03-01

Full Text Available The aim of this study was to characterize the subtypes and virulence profiles of 69 Staphylococcus aureus isolates obtained from retail ready-to-eat food in China. The isolates were analyzed using multilocus sequence typing (MLST and polymerase chain reaction (PCR analysis of important virulence factor genes, including the staphylococcal enterotoxin (SE genes (sea, seb, sec, sed, see, seg, seh, sei, sej, the exfoliative toxin genes (eta and etb, the toxic shock syndrome toxin-1 gene (tst, and the Panton-Valentine leucocidin-encoding gene (pvl. The isolates encompassed 26 different sequence types (STs, including four new STs (ST3482, ST3484, ST3485, ST3504, clustered in three clonal complexes and 17 singletons. The most prevalent STs were ST1, ST6, and ST15, constituting 34.8% of all isolates. Most STs (15/26, 57.7% detected have previously been associated with human infections. All 13 toxin genes examined were detected in the S. aureus isolates, with 84.1% of isolates containing toxin genes. The three most prevalent toxin genes were seb (36.2%, sea (33.3%, and seg (33.3%. The classical SE genes (sea–see, which contribute significantly to staphylococcal food poisoning (SFP, were detected in 72.5% of the S. aureus isolates. In addition, pvl, eta, etb, and tst were found in 11.6, 10.1, 10.1, and 7.2% of the S. aureus isolates, respectively. Strains ST6 carrying sea and ST1 harboring sec-seh enterotoxin profile, which are the two most common clones associated with SFP, were also frequently detected in the food samples in this study. This study indicates that these S. aureus isolates present in Chinese ready-to-eat food represents a potential public health risk. These data are valuable for epidemiological studies, risk management, and public health strategies.

Molecular Characterization of Endocarditis-Associated Staphylococcus aureus

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Nethercott, Cara; Mabbett, Amanda N.; Totsika, Makrina; Peters, Paul; Ortiz, Juan C.; Nimmo, Graeme R.; Coombs, Geoffrey W.

2013-01-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted. PMID:23616460

Molecular characterization of endocarditis-associated Staphylococcus aureus.

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Nethercott, Cara; Mabbett, Amanda N; Totsika, Makrina; Peters, Paul; Ortiz, Juan C; Nimmo, Graeme R; Coombs, Geoffrey W; Walker, Mark J; Schembri, Mark A

2013-07-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk.

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Meyrand, A; Boutrand-Loei, S; Ray-Gueniot, S; Mazuy, C; Gaspard, C E; Jaubert, G; Perrin, G; Lapeyre, C; Vernozy-Rozand, C

1998-09-01

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml-1. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10(3) cfu ml-1 at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3.2 ng g-1 of cheese made with an initial population of 10(3)-10(6) cfu ml-1. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.

The Activity of Cotinus coggygria Scop. Leaves on Staphylococcus aureus Strains in Planktonic and Biofilm Growth Forms

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Katarína Rendeková

2015-12-01

Full Text Available The purpose of this study was to detect the effectiveness of Cotinus coggygria Scop. leaves methanol extract against planktonic and biofilm growth forms of Staphylococcus aureus. The antimicrobial activity was determined by the broth microdilution test. Minimal inhibitory concentrations and minimal bactericidal concentrations were detected against two collection and ten clinical S. aureus strains. Anti-biofilm activity of the tested extract was detected using 24 h bacterial biofilm on the surface of microtiter plate wells. The biofilm inhibitory activity was evaluated visually after 24 h interaction of extract with biofilm, and the eradicating activity by a regrowth method. The tested extract showed bactericidal activity against all S. aureus strains (methicillin susceptible or methicillin resistant in concentrations ranging from 0.313 to 0.625 mg·mL−1. Biofilm inhibitory concentrations were 10-times higher and biofilm eradicating concentrations 100-times higher (8 and 32 mg·mL−1, respectively. The phytochemical analysis of C. coggygria leaves 60% methanol extract performed by LC-DAD-MS/MS revealed quercetin rhamnoside, methyl gallate, and methyl trigallate as main constituents. Results of our study indicate that C. coggygria, rich in tannins and flavonoids, seems to be a prospective topical antibacterial agent with anti-biofilm activity.

A new real-time PCR assay for rapid identification of the S. aureus/MRSA strains

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Ivan Manga

2013-01-01

Full Text Available The Methicillin-resistant Staphylococcus aureus (MRSA with the livestock-associated MRSA (LA-MRSA are of great interest to scientists and general public. The aim of our study was to present a new more rapid and reliable diagnostic method working on the RT-PCR platform applicable for monitoring of MRSA/S. aureus. The parallel testing of the S. aureus specific nuc gene sequence and the mecA gene sequence was utilised for this purpose. A collection of ten S. aureus/MRSA reference strains, fifteen genetically related non S. aureus reference strains and fifty-six environmental samples was employed for estimation of the assay performance and parameters. The environmental samples acquired in the Czech livestock farms were represented with the livestock and human nasal mucosae or skin swabs, the slaughter meat swabs and were chosen preferentially from individuals with previously confi rmed or suspected positive MRSA/S. aureus cases. The classic selective cultivation approach with the biochemical test and agar disk diffusion test was accepted as reference diagnostic method. As there were no culture positive samples that were negative using RT-PCR, our method featured with 100% sensitivity in comparison to reference method. The limit of detection allowed to identify from tens to hundreds copies of S. aureus/MRSA genome. Further, the RT-PCR assay featured with 100% inclusivity and 95% exclusivity at Cq value below 30. These parameters suggested on powerful and reliable diagnostic method with real potential of practical utilisation. We consider our method as ideal for testing of individual suspected colonies, when the results can be acquired in less than 1.5 hour.

Direct bacterial loop-mediated isothermal amplification detection on the pathogenic features of the nosocomial pathogen - Methicillin resistant Staphylococcus aureus strains with respiratory origins.

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Lin, Qun; Xu, Pusheng; Li, Jiaowu; Chen, Yin; Feng, Jieyi

2017-08-01

Loop-mediated isothermal amplification based detection assays using bacterial culture or colony for direct detection of methicillin resistant Staphylococcus aureus(MRSA) had been developed and evaluated, followed by its extensive application on a large scale of clinical MRSA isolated from respiratory origins, including nasal swabs and sputums. Six primers, including outer primers, inner primers and loop primers, were specifically designed for recognizing eight distinct sequences on four targets: 16SrRNA, femA, mecA and orfX. Twenty-seven reference strains were used to develop, evaluate and optimize this assay. Then, a total of 532 clinical MRSA isolates were employed for each detected targets. And the results were determined through both visual observation of the color change by naked eye and electrophoresis. The specific of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 40 min. The limit of detections (LOD) of bacteria culture LAMP assays were determined to be 10 4  CFU/ml for 16S rRNA, femA, as well as orfX and 10 5  CFU/ml for mecA, respectively. The established novel assays on MRSA detection may provide new strategies for rapid detection of foodborne pathogens. Copyright © 2017. Published by Elsevier Ltd.

spa typing and antimicrobial resistance of Staphylococcus aureus from healthy humans, pigs and dogs in Tanzania.

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Katakweba, Abdul Sekemani; Muhairwa, Amandus Pachificus; Espinosa-Gongora, Carmen; Guardabassi, Luca; Mtambo, Madundo M A; Olsen, John Elmerdahl

2016-02-28

Staphylococcus aureus is an opportunistic pathogen causing infections in humans and animals. Here we report for the first time the prevalence of nasal carriage, spa typing and antimicrobial resistance of S. aureus in a Tanzanian livestock community. Nasal swabs were taken from 100 humans, 100 pigs and 100 dogs in Morogoro Municipal. Each swab was enriched in Mueller Hinton broth with 6.5% NaCl and subcultured on chromogenic agar for S. aureus detection. Presumptive S. aureus colonies were confirmed to the species level by nuc PCR and analysed by spa typing. Antimicrobial susceptibility patterns were determined by disc diffusion method. S. aureus was isolated from 22% of humans, 4% of pigs and 11% of dogs. A total of 21 spa types were identified: 13, 7 and 1 in human, dogs, and pigs, respectively. Three spa types (t314, t223 and t084) were shared between humans and dogs. A novel spa type (t10779) was identified in an isolate recovered from a colonized human. Antimicrobials tested revealed resistance to ampicillin in all isolates, moderate resistances to other antimicrobials with tetracycline resistance being the most frequent. S. aureus carrier frequencies in dogs and humans were within the expected range and low in pigs. The S. aureus spa types circulating in the community were generally not shared by different hosts and majority of types belonged to known clones. Besides ampicillin resistance, moderate levels of antimicrobial resistance were observed irrespective of the host species from which the strains were isolated.

[Septicaemia in piglets associated with a positive finding of a methicillin-resistant S. aureus strain].

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Graage, R; Ganter, M; Verspohl, J; Strommenger, B; Waldmann, K-H; Baumgärtner, W; Hennig-Pauka, I

2014-01-01

Staphylococcus (S.) aureus has been associated with septicaemia, mastitis, vaginitis, metritis, osteomyelitis, and endocarditis. This case report demonstrates S. aureus-induced septicaemia in suckling pig- lets. Three days after birth, littermates displayed severe ecchymosis and cyanosis, with a 50% mortality rate. The surviving littermates were cross-fostered, but died 1 day later. Other piglets, which were allowed to suck at the sow, developed similar clinical signs. Haematological findings were anaemia, thrombocytopenia, and leukopenia; therefore, neither isohaemolytic anaemia nor septicaemia could be excluded as differential diagnoses. At necropsy, petechial bleeding on inner organ surfaces and free blood in body cavities were found. Bacteriological examination of the sow's milk and of the spleen of one piglet detected a methicillin-resistent S. aureus strain (MRSA CC398), which was in all likelihood the cause of the disease. Potential differential diagnoses are discussed.

Subinhibitory quinupristin/dalfopristin attenuates virulence of Staphylococcus aureus.

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Koszczol, Carmen; Bernardo, Katussevani; Krönke, Martin; Krut, Oleg

2006-09-01

The semi-synthetic streptogramin quinupristin/dalfopristin antibiotic exerts potent bactericidal activity against Staphylococcus aureus. We investigated whether, like other bactericidal antibiotics used at subinhibitory concentrations, quinupristin/dalfopristin enhances release of toxins by Gram-positive cocci. The activity of quinupristin/dalfopristin on exotoxin release by S. aureus was investigated by 2D SDS-PAGE combined with MALDI-TOF/MS analysis and by western blotting. We show that quinupristin/dalfopristin at subinhibitory concentrations reduces the release of S. aureus factors that induce tumour necrosis factor secretion in macrophages. Furthermore, quinupristin/dalfopristin but not linezolid attenuated S. aureus-mediated killing of infected host cells. When added to S. aureus cultures at different stages of bacterial growth, quinupristin/dalfopristin reduced in a dose-dependent manner the release of specific virulence factors (e.g. autolysin, protein A, alpha- and beta-haemolysins, lipases). In contrast, other presumably non-toxic exoproteins remained unchanged. The results of the present study suggest that subinhibitory quinupristin/dalfopristin inhibits virulence factor release by S. aureus, which might be especially helpful for the treatment of S. aureus infections, where both bactericidal as well as anti-toxin activity may be advantageous.

Detection of Staphylococcus Aureus Enterotoxin A and B Genes with PCR-EIA and a Hand-Held Electrochemical Sensor

National Research Council Canada - National Science Library

Aitichou, Mohamed; Henkins, Robert; Sultana, Afroz M; Ulrich, Robert G; Ibrahim, M. S

2004-01-01

... S. aureus DNA, and genomic DNA from Alcaligens, Bacillus, Bacteroides, Bordetella, Burkholderia, Clostridium, Comanonas, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella...

Neutrophil evasion strategies by Streptococcus pneumoniae and Staphylococcus aureus.

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Lewis, Megan L; Surewaard, Bas G J

2018-03-01

Humans are well equipped to defend themselves against bacteria. The innate immune system employs diverse mechanisms to recognize, control and initiate a response that can destroy millions of different microbes. Microbes that evade the sophisticated innate immune system are able to escape detection and could become pathogens. The pathogens Streptococcus pneumoniae and Staphylococcus aureus are particularly successful due to the development of a wide variety of virulence strategies for bacterial pathogenesis and they invest significant efforts towards mechanisms that allow for neutrophil evasion. Neutrophils are a primary cellular defense and can rapidly kill invading microbes, which is an indispensable function for maintaining host health. This review compares the key features of Streptococcus pneumoniae and Staphylococcus aureus in epidemiology, with a specific focus on virulence mechanisms utilized to evade neutrophils in bacterial pathogenesis. It is important to understand the complex interactions between pathogenic bacteria and neutrophils so that we can disrupt the ability of pathogens to cause disease.

Staphylococcus aureus and healthcare-associated infections

NARCIS (Netherlands)

Ekkelenkamp, M.B.

2011-01-01

Many medical procedures breach or suppress patients’ natural defences, leaving them vulnerable to infections which would not occur in healthy humans: “healthcare-associated infections”. Healthcare-associated infections caused by the bacterium Staphylococcus aureus (S. aureus) are probably the most

Genotypes and oxacillin resistance of Staphylococcus aureus from chicken and chicken meat in Poland.

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Krupa, P; Bystroń, J; Bania, J; Podkowik, M; Empel, J; Mroczkowska, A

2014-12-01

The genotypes and oxacillin resistance of 263 Staphylococcus aureus isolates cultured from chicken cloacae (n = 138) and chicken meat (n = 125) was analyzed. Fifteen spa types were determined in the studied S. aureus population. Among 5 staphylococcal protein A gene (spa) types detected in S. aureus from chicken, t002, t3478, and t13620 were the most frequent. Staphylococcus aureus isolates from meat were assigned to 14 spa types. Among them, the genotypes t002, t056, t091, t3478, and t13620 were dominant. Except for 4 chicken S. aureus isolates belonging to CC398, the remaining 134 isolates were clustered into multilocus sequence clonal complex (CC) 5. Most of meat-derived isolates were assigned to CC5, CC7, and CC15, and to the newly described spa-CC12954 complex belonging to CC1. Except for t011 (CC398), all other spa types found among chicken isolates were also present in isolates from meat. Four S. aureus isolated from chicken and one from meat were identified as methicillin-resistant S. aureus (MRSA) with oxacillin minimum inhibitory concentrations from 16 to 64 μg/mL. All MRSA were assigned to spa types belonging to ST398, and included 4 animal spa t011 SCCmecV isolates and 1 meat-derived spa t899, SCCmecIV isolate. Borderline oxacillin-resistant S. aureus (BORSA) isolates, shown to grow on plates containing 2 to 3 μg/mL of oxacillin, were found within S. aureus isolates from chicken (3 isolates) and from meat (19 isolates). The spa t091 and t084 dominated among BORSA from chicken meat, whereas t548 and t002 were found within animal BORSA. We report for the first time the presence of MRSA in chicken in Poland. We demonstrate that MRSA CC398 could be found in chicken meat indicating potential of introduction of animal-associated genotypes into the food chain. We also report for the first time the possibility of transmission of BORSA isolates from chicken to meat. ©2014 Poultry Science Association Inc.

Screening of nasal carriage of methicillin-resistant Staphylococcus aureus during admission of patients to Frantz Fanon Hospital, Blida, Algeria

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Mohamed Amine Ouidri

2018-05-01

Full Text Available A study was performed of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA strains isolated from nasal preoperative samples. Of 663 samples assessed, staphylococcus was detected in 143 (21.57%. The disc diffusion method (cefoxitin 30 μg, a screening test (oxacillin 6 μg/mL and a search for Protein Binding Additional Penicillin 2 (PLP2a allowed the detection and confirmation of resistance to methicillin for 36 strains, a rate of 5.43% of the total population studied. Eight MRSA carriers received care in the trauma service, 14 in cardiology, five in ear, nose and throat, four in neurosurgery and paediatrics, and one in SCI. Thirty-six methicillin-resistant of the nasal portage strains are in their great majority, 27 of 36, rather limited multi-R character (two to three families namely resistance: tetracyclines, fluoroquinolones, aminoglycosides, macrolides. One of the MRSA strains was found to have intermediate sensitivity to vancomycin. Keywords: Antibiotic resistance, Healthy volunteers, MRSA, Prevalence, Staphylococcus aureus

The sensitivity status of community-acquired Staphylococcus aureus ...

African Journals Online (AJOL)

Community acquired Staphylococcus aureus was isolated from various infectious sites in two private laboratories in Kano-city, Nigeria. A total of 247 (11%) Staphylococcu aureus isolates were recovered from all infectious sites except cerebro-spinal fluid. The least Staphylococcus aureus isolates were found in urine ...

Growth and adherence of Staphylococcus aureus were enhanced through the PGE2 produced by the activated COX-2/PGE2 pathway of infected oral epithelial cells.

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Yuxia Wang

Full Text Available Staphylococcus aureus is a major pathogen of varieties of oral mucous infection. Prostaglandin E2 (PGE2 is a pro-inflammatory factor and Cyclooxygenase 2 (COX-2 is a critical enzyme of PGE2 biosynthesis. The purpose of this study is to investigate whether Staphylococcus aureus can increase PGE2 production of oral epithelial cells and how PGE2 functions in the growth and adherence of Staphylococcus aureus. mRNA levels of COX-2, fnbpA and fnbpB were estimated by quantitative PCR. PGE2 production was measured by Enzyme Linked Immunosorbent Assay (ELISA. The binding biomass of Staphylococcus aureus to human fibronectin was investigated by crystal violet staining and confocal laser scanning microscopy and the adherent force was measured by atomic force microscope (AFM. The COX-2 mRNA level and PGE2 production were increased by Staphylococcus aureus. PGE2 promoted the growth and biofilm formation of Staphylococcus aureus, enhanced the attachment of Staphylococcus aureus to the human fibronectin as well as to the HOK cells. The transcription of fnbpB was up-regulated by PGE2 in both early and middle exponential phase but not fnbpA. These results suggest that the activation of COX-2/PGE2 pathway in oral epithelial cell by Staphylococcus aureus can in turn facilitate the growth and the ability to adhere of the pathogen. These findings uncover a new function of PGE2 and may lead to the potential of COX-2/PGE2 targeting in the therapy of inflammation and cancer in both which the COX-2/PGE2 pathway were observed activated.

Evaluation of constitutive and inducible resistance to clindamycin in clinical samples of Staphylococcus aureus from a tertiary hospital

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Angelita Bottega

2014-10-01

Full Text Available Introduction Infections caused by methicillin-resistant Staphylococcus aureus (MRSA have become common in hospitals and the community environment, and this wide resistance has limited patient treatment. Clindamycin (CL represents an important alternative therapy for infections caused by S. aureus. Antimicrobial susceptibility testing using standard methods may not detect inducible CL resistance. This study was performed to detect the phenotypes of resistance to macrolides-lincosamides-streptogramin B (MLSB antibiotics, including CL, in clinical samples of S. aureus from patients at a tertiary hospital in Santa Maria, State of Rio Grande do Sul, Brazil. Methods One hundred and forty clinical isolates were submitted to the disk diffusion induction test (D-test with an erythromycin (ER disk positioned at a distance of 20mm from a CL disk. The results were interpreted according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI. Results In this study, 29 (20.7% of the 140 S. aureus samples were resistant to methicillin (MRSA, and 111 (79.3% were susceptible to methicillin (MSSA. The constitutive resistance phenotype (cMLSB was observed in 20 (14.3% MRSA samples and in 5 (3.6% MSSA samples, whereas the inducible resistance phenotype (iMLSB was observed in 3 (2.1% MRSA samples and in 8 (5.8% MSSA samples. Conclusions The D-test is essential for detecting the iMLSB phenotype because the early identification of this phenotype allows clinicians to choose an appropriate treatment for patients. Furthermore, this test is simple, easy to perform and inexpensive.

Molecular Characterization of Endocarditis-Associated Staphylococcus aureus

OpenAIRE

Nethercott, Cara; Mabbett, Amanda N.; Totsika, Makrina; Peters, Paul; Ortiz, Juan C.; Nimmo, Graeme R.; Coombs, Geoffrey W.; Walker, Mark J.; Schembri, Mark A.

2013-01-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated ...

Staphylococcus aureus penetrate the interkeratinocyte spaces created by skin-infiltrating neutrophils in a mouse model of impetigo.

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Imanishi, Ichiro; Hattori, Shinpei; Hisatsune, Junzo; Ide, Kaori; Sugai, Motoyuki; Nishifuji, Koji

2017-02-01

Impetigo is a bacterial skin disease characterized by intraepidermal neutrophilic pustules. Previous studies have demonstrated that exfoliative toxin producing staphylococci are isolated in the cutaneous lesions of human and canine impetigo. However, the mechanisms of intraepidermal splitting in impetigo remain poorly understood. To determine how staphylococci penetrate the living epidermis and create intraepidermal pustules in vivo using a mouse model of impetigo. Three Staphylococcus aureus strains harbouring the etb gene and three et gene negative strains were epicutaneously inoculated onto tape-stripped mouse skin. The skin samples were subjected to time course histopathological and immunofluorescence analyses to detect intraepidermal neutrophils and infiltrating staphylococci. To determine the role of neutrophils on intraepidermal bacterial invasion, cyclophosphamide (CPA) was injected intraperitoneally into the mice to cause leucopenia before the inoculation of etb gene positive strains. In mice inoculated with etb gene positive S. aureus, intraepidermal pustules resembling impetigo were detected as early as 4 h post-inoculation (hpi). Neutrophils in the epidermis were detected from 4 hpi, whereas intraepidermal staphylococci was detected from 6 hpi. The dimensions of the intraepidermal clefts created in mice inoculated with etb gene positive strains at 6 hpi were significantly larger than those in mice inoculated with et gene negative strains. In CPA treated mice, staphylococci or neutrophils were not detected in the deep epidermis until 6 hpi. Our findings indicate that intraepidermal neutrophils play an important role in S. aureus invasion into the living epidermis in a mouse model of impetigo. © 2016 ESVD and ACVD.

Inducible clindamycin and methicillin resistant Staphylococcus aureus in a tertiary care hospital, Kathmandu, Nepal.

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Adhikari, R P; Shrestha, S; Barakoti, A; Amatya, R

2017-07-11

Staphylococcus aureus, an important nosocomial pathogen, is frequently associated with infections in human. The management of the infections by it especially methicillin resistant ones is often difficult because methicillin resistant S. aureus is usually resistant to multiple antibiotics. Macrolide-lincosamide streptogramin B family of antibiotics is commonly used to treat such infections as an alternative to vancomycin. This study was conducted over the period of one and half year from November 2013-April 2015 in Microbiology laboratory of Nepal Medical College and Teaching Hospital, Kathmandu, Nepal to find the incidence of different phenotypes of MLS B resistance among S. aureus from clinical samples and their association with methicillin resistance. Two hundred seventy isolates of S. aureus were included in the study. Methicillin resistance was detected by cefoxitin disc diffusion method and inducible clindamycin resistance by erythromycin and clindamycin disc approximation test (D-test). Of the 270 clinical isolates of S. aureus, 25.1% (68/270) were MRSA. Erythromycin and clindamycin resistance was seen in 54.4% (147/270) and 41.8% (113/270) isolates respectively. Resistance to erythromycin and clindamycin were higher in MRSA as compared to MSSA (erythromycin-resistance: 88.2% Vs 39.1% and clindamycin-resistance: 79.4% Vs 41.8%). The overall prevalence of i MLS B and c MLS B phenotype was 11.48% (31/270) and 29.25% (79/270) respectively. Both i MLS B and c MLS B phenotypes predominated in MRSA strains. Detection rate of MRSA in our study shows the necessity to improve in healthcare practices and to formulate new policy for the control of MRSA infections. Clindamycin resistance in the form of i MLS B and c MLS B especially among MRSA emphasizes the need of D-test to be performed routinely in our set up while using clindamycin as an alternative choice to anti-staphylococcal antibiotics like vancomycin and linezolid in the treatment of staphylococcal infections.

Staphylococcus aureus Central Nervous System Infections in Children.

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Vallejo, Jesus G; Cain, Alexandra N; Mason, Edward O; Kaplan, Sheldon L; Hultén, Kristina G

2017-10-01

Central nervous system (CNS) infections caused by Staphylococcus aureus are uncommon in pediatric patients. We review the epidemiology, clinical features and treatment in 68 patients with a S. aureus CNS infection evaluated at Texas Children's Hospital. Cases of CNS infection in children with positive cerebrospinal fluid cultures or spinal epidural abscess (SEA) for S. aureus at Texas Children's Hospital from 2001 to 2013 were reviewed. Seventy cases of S. aureus CNS infection occurred in 68 patients. Forty-nine cases (70%) were secondary to a CNS device, 5 (7.1%) were postoperative meningitis, 9 (12.8%) were hematogenous meningitis and 7 (10%) were SEAs. Forty-seven (67.2%) were caused by methicillin-sensitive S. aureus (MSSA) and 23 (32.8%) by methicillin-resistant S. aureus (MRSA). Community-acquired infections were more often caused by MRSA that was clone USA300/pvl. Most patients were treated with nafcillin (MSSA) or vancomycin (MRSA) with or without rifampin. Among patients with MRSA infection, 50% had a serum vancomycin trough obtained with the median level being 10.6 μg/mL (range: 5.4-15.7 μg/mL). Only 1 death was associated with S. aureus infection. The epidemiology of invasive of S. aureus infections continues to evolve with MSSA accounting for most of the infections in this series. The majority of cases were associated with neurosurgical procedures; however, hematogenous S. aureus meningitis and SEA occurred as community-acquired infections in patients without predisposing factors. Patients with MRSA CNS infections had a favorable response to vancomycin, but the beneficial effect of combination therapy or targeting vancomycin trough concentrations of 15-20 μg/mL remains unclear.

The human nasal microbiota and Staphylococcus aureus carriage.

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Daniel N Frank

Full Text Available BACKGROUND: Colonization of humans with Staphylococcus aureus is a critical prerequisite of subsequent clinical infection of the skin, blood, lung, heart and other deep tissues. S. aureus persistently or intermittently colonizes the nares of approximately 50% of healthy adults, whereas approximately 50% of the general population is rarely or never colonized by this pathogen. Because microbial consortia within the nasal cavity may be an important determinant of S. aureus colonization we determined the composition and dynamics of the nasal microbiota and correlated specific microorganisms with S. aureus colonization. METHODOLOGY/PRINCIPAL FINDINGS: Nasal specimens were collected longitudinally from five healthy adults and a cross-section of hospitalized patients (26 S. aureus carriers and 16 non-carriers. Culture-independent analysis of 16S rRNA sequences revealed that the nasal microbiota of healthy subjects consists primarily of members of the phylum Actinobacteria (e.g., Propionibacterium spp. and Corynebacterium spp., with proportionally less representation of other phyla, including Firmicutes (e.g., Staphylococcus spp. and Proteobacteria (e.g. Enterobacter spp. In contrast, inpatient nasal microbiotas were enriched in S. aureus or Staphylococcus epidermidis and diminished in several actinobacterial groups, most notably Propionibacterium acnes. Moreover, within the inpatient population S. aureus colonization was negatively correlated with the abundances of several microbial groups, including S. epidermidis (p = 0.004. CONCLUSIONS/SIGNIFICANCE: The nares environment is colonized by a temporally stable microbiota that is distinct from other regions of the integument. Negative association between S. aureus, S. epidermidis, and other groups suggests microbial competition during colonization of the nares, a finding that could be exploited to limit S. aureus colonization.

Nasal Carriage of Staphylococcus aureus among Children in the Ashanti Region of Ghana.

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Eibach, Daniel; Nagel, Michael; Hogan, Benedikt; Azuure, Clinton; Krumkamp, Ralf; Dekker, Denise; Gajdiss, Mike; Brunke, Melanie; Sarpong, Nimako; Owusu-Dabo, Ellis; May, Jürgen

2017-01-01

Nasal carriage with Staphylococcus aureus is a common risk factor for invasive infections, indicating the necessity to monitor prevalent strains, particularly in the vulnerable paediatric population. This surveillance study aims to identify carriage rates, subtypes, antimicrobial susceptibilities and virulence markers of nasal S. aureus isolates collected from children living in the Ashanti region of Ghana. Nasal swabs were obtained from children aureus isolates were characterized by their antimicrobial susceptibility, the presence of genes encoding for Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) and further differentiated by spa-typing and multi-locus-sequence-typing. Out of 544 children 120 (22.1%) were colonized with S. aureus, with highest carriage rates during the rainy seasons (27.2%; p = 0.007), in females aged 6-8 years (43.7%) and males aged 8-10 years (35.2%). The 123 isolates belonged to 35 different spa-types and 19 sequence types (ST) with the three most prevalent spa-types being t355 (n = 25), t84 (n = 18), t939 (n = 13), corresponding to ST152, ST15 and ST45. Two (2%) isolates were methicillin-resistant S. aureus (MRSA), classified as t1096 (ST152) and t4454 (ST45), and 16 (13%) were resistant to three or more different antimicrobial classes. PVL and TSST-1 were detected in 71 (58%) and 17 (14%) isolates respectively. S. aureus carriage among Ghanaian children seems to depend on age, sex and seasonality. While MRSA rates are low, the high prevalence of PVL is of serious concern as these strains might serve not only as a source for severe invasive infections but may also transfer genes, leading to highly virulent MRSA clones.

Nasal Carriage of Staphylococcus aureus among Children in the Ashanti Region of Ghana.

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Daniel Eibach

Full Text Available Nasal carriage with Staphylococcus aureus is a common risk factor for invasive infections, indicating the necessity to monitor prevalent strains, particularly in the vulnerable paediatric population. This surveillance study aims to identify carriage rates, subtypes, antimicrobial susceptibilities and virulence markers of nasal S. aureus isolates collected from children living in the Ashanti region of Ghana.Nasal swabs were obtained from children < 15 years of age on admission to the Agogo Presbyterian Hospital between April 2014 and January 2015. S. aureus isolates were characterized by their antimicrobial susceptibility, the presence of genes encoding for Panton-Valentine leukocidin (PVL and toxic shock syndrome toxin-1 (TSST-1 and further differentiated by spa-typing and multi-locus-sequence-typing.Out of 544 children 120 (22.1% were colonized with S. aureus, with highest carriage rates during the rainy seasons (27.2%; p = 0.007, in females aged 6-8 years (43.7% and males aged 8-10 years (35.2%. The 123 isolates belonged to 35 different spa-types and 19 sequence types (ST with the three most prevalent spa-types being t355 (n = 25, t84 (n = 18, t939 (n = 13, corresponding to ST152, ST15 and ST45. Two (2% isolates were methicillin-resistant S. aureus (MRSA, classified as t1096 (ST152 and t4454 (ST45, and 16 (13% were resistant to three or more different antimicrobial classes. PVL and TSST-1 were detected in 71 (58% and 17 (14% isolates respectively.S. aureus carriage among Ghanaian children seems to depend on age, sex and seasonality. While MRSA rates are low, the high prevalence of PVL is of serious concern as these strains might serve not only as a source for severe invasive infections but may also transfer genes, leading to highly virulent MRSA clones.

Characterization of Enterotoxigenic Bacillus cereus sensu lato and Staphylococcus aureus Isolates and Associated Enterotoxin Production Dynamics in Milk or Meat-Based Broth.

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Walker-York-Moore, Laura; Moore, Sean C; Fox, Edward M

2017-07-15

Bacillus cereus sensu lato species, as well as Staphylococcus aureus , are important pathogenic bacteria which can cause foodborne illness through the production of enterotoxins. This study characterised enterotoxin genes of these species and examined growth and enterotoxin production dynamics of isolates when grown in milk or meat-based broth. All B. cereus s. l. isolates harboured nheA , hblA and entFM toxin genes, with lower prevalence of bceT and hlyII . When grown at 16 °C, toxin production by individual B. cereus s. l. isolates varied depending on the food matrix; toxin was detected at cell densities below 5 log 10 (CFU/mL). At 16 °C no staphylococcal enterotoxin C (SEC) production was detected by S. aureus isolates, although low levels of SED production was noted. At 30 °C all S. aureus isolates produced detectable enterotoxin in the simulated meat matrix, whereas SEC production was significantly reduced in milk. Relative to B. cereus s. l. toxin production, S. aureus typically required reaching higher cell numbers to produce detectable levels of enterotoxin. Phylogenetic analysis of the sec and sel genes suggested population evolution which correlated with animal host adaptation, with subgroups of bovine isolates or caprine/ovine isolates noted, which were distinct from human isolates. Taken together, this study highlights the marked differences in the production of enterotoxins both associated with different growth matrices themselves, but also in the behaviour of individual strains when exposed to different food matrices.

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

KAUST Repository

Hill-Cawthorne, Grant A.

2014-06-27

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. © 2014 Hill-Cawthorne et al.

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus.

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Grant A Hill-Cawthorne

Full Text Available Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec. We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA, a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (∼4% of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates.

Recombinations in staphylococcal cassette chromosome mec elements compromise the molecular detection of methicillin resistance in Staphylococcus aureus

KAUST Repository

Hill-Cawthorne, Grant A.; Hudson, Lyndsey O.; Abd El Ghany, Moataz; Piepenburg, Olaf; Nair, Mridul; Dodgson, Andrew; Forrest, Matthew S.; Clark, Taane G.; Pain, Arnab

2014-01-01

Clinical laboratories are increasingly using molecular tests for methicillin-resistant Staphylococcus aureus (MRSA) screening. However, primers have to be targeted to a variable chromosomal region, the staphylococcal cassette chromosome mec (SCCmec). We initially screened 726 MRSA isolates from a single UK hospital trust by recombinase polymerase amplification (RPA), a novel, isothermal alternative to PCR. Undetected isolates were further characterised using multilocus sequence, spa typing and whole genome sequencing. 96% of our tested phenotypically MRSA isolates contained one of the six orfX-SCCmec junctions our RPA test and commercially available molecular tests target. However 30 isolates could not be detected. Sequencing of 24 of these isolates demonstrated recombinations within the SCCmec element with novel insertions that interfered with the RPA, preventing identification as MRSA. This result suggests that clinical laboratories cannot rely solely upon molecular assays to reliably detect all methicillin-resistance. The presence of significant recombinations in the SCCmec element, where the majority of assays target their primers, suggests that there will continue to be isolates that escape identification. We caution that dependence on amplification-based molecular assays will continue to result in failure to diagnose a small proportion (?4%) of MRSA isolates, unless the true level of SCCmec natural diversity is determined by whole genome sequencing of a large collection of MRSA isolates. © 2014 Hill-Cawthorne et al.

Detection of prostate cancer-specific transcripts in extracellular vesicles isolated from post-DRE urine.

Science.gov (United States)

Pellegrini, Kathryn L; Patil, Dattatraya; Douglas, Kristen J S; Lee, Grace; Wehrmeyer, Kathryn; Torlak, Mersiha; Clark, Jeremy; Cooper, Colin S; Moreno, Carlos S; Sanda, Martin G

2017-06-01

The measurement of gene expression in post-digital rectal examination (DRE) urine specimens provides a non-invasive method to determine a patient's risk of prostate cancer. Many currently available assays use whole urine or cell pellets for the analysis of prostate cancer-associated genes, although the use of extracellular vesicles (EVs) has also recently been of interest. We investigated the expression of prostate-, kidney-, and bladder-specific transcripts and known prostate cancer biomarkers in urine EVs. Cell pellets and EVs were recovered from post-DRE urine specimens, with the total RNA yield and quality determined by Bioanalyzer. The levels of prostate, kidney, and bladder-associated transcripts in EVs were assessed by TaqMan qPCR and targeted sequencing. RNA was more consistently recovered from the urine EV specimens, with over 80% of the patients demonstrating higher RNA yields in the EV fraction as compared to urine cell pellets. The median EV RNA yield of 36.4 ng was significantly higher than the median urine cell pellet RNA yield of 4.8 ng. Analysis of the post-DRE urine EVs indicated that prostate-specific transcripts were more abundant than kidney- or bladder-specific transcripts. Additionally, patients with prostate cancer had significantly higher levels of the prostate cancer-associated genes PCA3 and ERG. Post-DRE urine EVs are a viable source of prostate-derived RNAs for biomarker discovery and prostate cancer status can be distinguished from analysis of these specimens. Continued analysis of urine EVs offers the potential discovery of novel biomarkers for pre-biopsy prostate cancer detection. © 2017 Wiley Periodicals, Inc.

Acceleration of the direct identification of Staphylococcus aureus versus coagulase-negative staphylococci from blood culture material: a comparison of six bacterial DNA extraction methods.

Science.gov (United States)

Loonen, A J M; Jansz, A R; Kreeftenberg, H; Bruggeman, C A; Wolffs, P F G; van den Brule, A J C

2011-03-01

To accelerate differentiation between Staphylococcus aureus and coagulase-negative staphylococci (CNS), this study aimed to compare six different DNA extraction methods from two commonly used blood culture materials, i.e. BACTEC and BacT/ALERT. Furthermore, we analysed the effect of reduced blood culture incubation for the detection of staphylococci directly from blood culture material. A real-time polymerase chain reaction (PCR) duplex assay was used to compare the six different DNA isolation protocols on two different blood culture systems. Negative blood culture material was spiked with methicillin-resistant S. aureus (MRSA). Bacterial DNA was isolated with automated extractor easyMAG (three protocols), automated extractor MagNA Pure LC (LC Microbiology Kit M(Grade)), a manual kit MolYsis Plus and a combination of MolYsis Plus and the easyMAG. The most optimal isolation method was used to evaluate reduced bacterial incubation times. Bacterial DNA isolation with the MolYsis Plus kit in combination with the specific B protocol on the easyMAG resulted in the most sensitive detection of S. aureus, with a detection limit of 10 CFU/ml, in BacT/ALERT material, whereas using BACTEC resulted in a detection limit of 100 CFU/ml. An initial S. aureus or CNS load of 1 CFU/ml blood can be detected after 5 h of incubation in BacT/ALERT 3D by combining the sensitive isolation method and the tuf LightCycler assay.

Dermoscopy of lichen aureus Dermatoscopia do liquen aureus

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Poliana Santin Portela

2013-04-01

Full Text Available Lichen aureus (also called "lichen purpuricus" is an uncommon subtype of pigmented purpuric dermatosis. Clinically characterized by rust macules, papules or plaques, it is a chronic disease which more often affects young adults and is localized mainly on the lower extremities. The diagnosis is made on the basis of clinical and histopathological features. Dermoscopy findings are useful to confirm clinical diagnosis.O líquen aureus (também denominado "liquen purpuricus" é um subtipo pouco comum entre as dermatoses purpúricas pigmentadas. Clinicamente caracterizado por máculas, pápulas ou placas de coloração ferruginosa, é doença crônica, que acomete mais frequentemente adultos jovens e localiza-se principalmente nos membros inferiores. O diagnóstico pode ser feito a partir das características clínicas e histopatológicas, sendo os achados dermatoscópicos úteis para corroborar o diagnóstico clínico.

Improved detection limit in rapid detection of human enterovirus 71 and coxsackievirus A16 by a novel reverse transcription-isothermal multiple-self-matching-initiated amplification assay.

Science.gov (United States)

Ding, Xiong; Nie, Kai; Shi, Lei; Zhang, Yong; Guan, Li; Zhang, Dan; Qi, Shunxiang; Ma, Xuejun

2014-06-01

Rapid detection of human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) is important in the early phase of hand-foot-and-mouth disease (HFMD). In this study, we developed and evaluated a novel reverse transcription-isothermal multiple-self-matching-initiated amplification (RT-IMSA) assay for the rapid detection of EV71 and CVA16 by use of reverse transcriptase, together with a strand displacement DNA polymerase. Real-time RT-IMSA assays using a turbidimeter and visual RT-IMSA assays to detect EV71 and CVA16 were established and completed in 1 h, and the reported corresponding real-time reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assays targeting the same regions of the VP1 gene were adopted as parallel tests. Through testing VP1 RNAs transcribed in vitro, the real-time RT-IMSA assays exhibited better linearity of quantification, with R(2) values of 0.952 (for EV71) and 0.967 (for CVA16), than the real-time RT-LAMP assays, which had R(2) values of 0.803 (for EV71) and 0.904 (for CVA16). Additionally, the detection limits of the real-time RT-IMSA assays (approximately 937 for EV71 and 67 for CVA16 copies/reaction) were higher than those of real-time RT-LAMP assays (approximately 3,266 for EV71 and 430 for CVA16 copies/reaction), and similar results were observed in the visual RT-IMSA assays. The new approaches also possess high specificities for the corresponding targets, with no cross-reactivity observed. In clinical assessment, compared to commercial reverse transcription-quantitative PCR (qRT-PCR) kits, the diagnostic sensitivities of the real-time RT-IMSA assays (96.4% for EV71 and 94.6% for CVA16) were higher than those of the real-time RT-LAMP assays (91.1% for EV71 and 90.8% for CVA16). The visual RT-IMSA assays also exhibited the same results. In conclusion, this proof-of-concept study suggests that the novel RT-IMSA assay is superior to the RT-LAMP assay in terms of detection limit and has the potential to rapidly detect EV71

Population structure of Staphylococcus aureus in China

OpenAIRE

Yan, Xiaomei

2015-01-01

The present PhD research was aimed at analysing the population structure of Staphylococcus aureus in China. Between 2000 and 2005 we found that patients from a single Chinese hospital showed increasing trends in antimicrobial resistance. Among methicillin-resistant S. aureus (MRSA), resistance against rifampicin doubled to 68%. Staphylococcal food poisoning (SFP) is frequent in China. Two predominant S. aureus lineages, ST6 and ST943, were identified causing outbreaks of SFP in Southern China...

Influenza B pneumonia with Staphylococcus aureus superinfection associated with parvovirus B19 and concomitant agranulocytosis.

Science.gov (United States)

Krell, S; Adams, I; Arnold, U; Kalinski, T; Aumann, V; König, W; König, B

2003-10-01

An 11-year-old patient with anamnestic fever for 3 days and signs of upper respiratory tract infection underwent fulminant Staphylococcus aureus pneumonia with concomitant agranulocytosis. From autopsia influenza B virus and parvovirus B19 were detected by nucleic acid amplification technique (NAT). Specific IgG but no IgM points to preexisting parvovirus B19 infection. Whether in this case agranulocytosis can be interpreted as early manifestation of reactivated parvovirus B19 infection is under discussion. Therefore, parvovirus B19 could have provoked a foudroyant course of influenza B pneumonia which was superinfected with S. aureus.

Occurrence of vancomycin-resistant Staphylococcus aureus in the oral cavity of patients with dental caries.

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Vellappally, Sajith; Divakar, Darshan Devang; Al Kheraif, Abdulaziz Abdullah; Ramakrishnaiah, Ravikumar; Alqahtani, Amer; Dalati, M H N; Anil, Sukumaran; Khan, Aftab Ahmed; Harikrishna Varma, P R

2017-09-01

Oral streptococci are the major group of microbes isolated from oral microflora. They represent frequent pathogens of infective endocarditis (IE), and it is assumed that in most of the cases oral streptococci are acquired via mucosa layer of oral cavity. Staphylococcus aureus is also frequently isolated from IE as it accounts for 20%-30% of all cases. Vancomycin has been the most reliable therapeutic agent against infections caused by methicillin-resistant S. aureus (MRSA). The main objective of this study was to examine the occurrence of S. aureus species in dental caries specimens. Antimicrobial susceptibility testing of S. aureus to four antibiotics namely vancomycin, linezolid, teicoplanin, and daptomycin was performed. Detection of vancomycin resistance was conducted using polymerase chain reaction. Among the tested 150 strains, 98 were MRSA and of that 54 were vancomycin sensitive and 27 were resistant. All 98 MRSA strains were positive for mecA and 36 yielded pvl, whereas 13 carried vanA and only 2 were positive for vanB. Majority of the isolates showed sensitivity toward daptomycin and linezolid. Strains of S. aureus exhibiting decreased susceptibility to different antibiotics like vancomycin, daptomycin, and linezolid severely compromise the therapeutic alternatives and require a considerable amount of time, public awareness, and integrative health-care strategies to prevent the emergence of resistance to these compounds.

Implantation of Corynebacterium pseudodiphtheriticum for elimination of Staphylococcus aureus from the nasal cavity in volunteers

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Viacheslav, Ilyin; Kiryukhina, Nataliya

Nasal carriage of Staphylococcus aureus is a well-documented risk factor of infection and inflammation of the skin, soft tissues and bacteremia. It is also known that most often etiology of these disorders is associated with autoinfection. The present-day methods of opportunistic pathogens eradication from the nasal cavity are based principally on the use of antiseptic and antibacterial agents. For instance, a local antibiotic mupirocin in the form of nasal ointment is considered to be the gold standard for the treatment of S. aureus carriage. The literature describes investigations showing how mupirocin can strengthen antibiotic resistance in S. aureus strains, including those with methicillin resistance (MRSA). It is also common knowledge that recolonization of the nasal mucous membrane takes place within several months after mupirocin treatment. This circumstance dictates the necessity to look for alternative ways of preventing the S. aureus carriage and methods of elimination. One of the methods of nasal S. aureus elimination is implantation of nonpathogenic microorganisms which will extrude opportunistic pathogens without impinging the symbiotic microbiota. Effectiveness of saline suspension of Corynebacterium pseudodiphtheriticum containing spray was assessed in a several chamber experiments with simulation of some spaceflight factors (dry immersion, isolation). Various schemes of application of preparations were applied. In all cases of corynebacteria application the strong inhibiting effect against S. aureus was detected. This fact opens a prospect of using nonpathogenic corynebacteria as a nasal probiotic. Administration of the nasal corynebacteria spray possibly prevented cross-infection by MRSA and appearance of staphylococcal infection. Further pre-clinical and clinical study of this bacterial therapy method is under development.

Antimicrobial susceptibility of Staphylococcus aureus causing bovine mastitis in Argentine dairy herds Sensibilidad a los antimicrobianos de Staphylococcus aureus causantes de mastitis bovina en rodeos lecheros de Argentina

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N.B. Russi

2008-06-01

Full Text Available We assessed the in vitro activity of selected antimicrobial agents against 95 Staphylococcus aureus strains causing both clinical and subclinical bovine mastitis belonging to 61 dairy farms from the Central dairy area of Argentina. Minimal inhibitory concentrations (MICs of penicillin, oxacillin, gentamicin, erythromycin, enrofloxacin and florfenicol were estimated. In addition, the agar diffusion test was performed. MIC50 and MIC90 were as follows: penicillin, 0.05 and 4 µg/ml; oxacillin, 0.25 and 0.25 µg/ml; gentamicin, 0.25 and 0.5 µg/ml; erythromycin 0.125 and 0.25 µg/ml; enrofloxacin 0.25 and 0.5 µg/ml, and florfenicol 4 and 8 µg/ml. b-lactamase activity was detected in 89% of 46 penicillin- resistant strains. Apart from penicillin, antimicrobial resistance in S. aureus causing bovine mastitis remains rare in Argentine dairy farms.Se evaluó la actividad in vitro de un grupo seleccionado de antimicrobianos contra 95 aislamientos de Staphylococcus aureus obtenidos de casos de mastitis bovina clínica y subclínica, en 61 rodeos lecheros de la cuenca central de Argentina. Fueron estimadas las concentraciones inhibitorias mínimas (CIM de penicilina, oxacilina, gentamicina, eritromicina, enrofloxacina y florfenicol. Además se realizó la prueba de difusión en agar. Las CIM50 y CIM90 obtenidas fueron: penicilina 0,05 y 4 µg/ml; oxacilina 0,25 y 0,25 µg/ml; gentamicina 0,25 y 0,5 µg/ml; eritromicina 0,125 y 0,25 µg/ml; enrofloxacina 0,25 y 0,5 µg/ml y florfenicol 4 y 8 µg/ml. Se detectó actividad de b-lactamasa en el 89% de las cepas resistentes a la penicilina. A excepción de lo observado para penicilina, la resistencia a los antimicrobianos en S. aureus causantes de mastitis bovina en Argentina parece ser un fenómeno poco frecuente.

Sae regulator factor impairs the response to photodynamic inactivation mediated by Toluidine blue in Staphylococcus aureus.

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Gándara, Lautaro; Mamone, Leandro; Dotto, Cristian; Buzzola, Fernanda; Casas, Adriana

2016-12-01

Photodynamic inactivation (PDI) involves the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of inactivating bacteria. Since the emergence of multi-resistant bacterial strains is becoming an increasing public health concern, PDI becomes an attractive choice. The aim of this work was to study the differential susceptibility to Toluidine blue (TB) mediated PDI (TB-PDI) of S. aureus mutants (RN6390 and Newman backgrounds) for different key regulators of virulence factors related to some extent to oxidative stress. Complete bacteria eradication of planktonic cultures of RN6390 S. aureus photosensitized with 13μM TB was obtained upon illumination with a low light dose of 4.2J/cm 2 from a non-coherent light source. Similarly, complete cell death was achieved applying 1.3μM TB and 19J/cm 2 light dose, showing that higher light doses can lead to equal cell death employing low photosensitizer concentrations. Interestingly, RN6390 in planktonic culture responded significantly better to TB-PDI than the Newman strain. We showed that deficiencies in rsbU, mgrA (transcription factors related to stress response) or agr (quorum sensing system involved in copper resistance to oxidative stress) did not modify the response of planktonic S. aureus to PDI. On the other hand, the two component system sae impaired the response to TB-PDI through a mechanism not related to the Eap adhesin. More severe conditions were needed to inactivate S. aureus biofilms (0.5mM TB, 157J/cm 2 laser light). In mutant sae biofilms, strain dependant differential susceptibilities are not noticed. Copyright © 2016 Elsevier B.V. All rights reserved.

Prevalence and characteristics of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus nasal colonization among a community-based diabetes population in Foshan, China.

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Lin, Jialing; Xu, Ping; Peng, Yang; Lin, Dongxin; Ou, Qianting; Zhang, Ting; Bai, Chan; Ye, Xiaohua; Zhou, Junli; Yao, Zhenjiang

2017-05-01

Evidence suggests that diabetes might cause an increase in colonization of Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) in community settings. We carried out a cross-sectional study to determine the prevalence and influencing factors of S. aureus and MRSA nasal colonization among a community-based diabetes population, and to identify the characteristics of the isolated strains. A total of 956 participants from 11 community settings were included in the study. Of the 529 diabetes participants, 46 were colonized with S. aureus and 22 were colonized with MRSA. Of the 427 non-diabetes participants, 25 were colonized with S. aureus and 12 were colonized with MRSA. Men (odds ratio 0.45, 95% confidence interval 0.20-0.99, P = 0.047) were less likely to have S. aureus nasal colonization, and those with well-controlled blood glucose (odds ratio 2.04, 95% confidence interval 1.01-4.13, P = 0.047) among the diabetes population were more likely to have S. aureus nasal colonization. The proportion of multidrug-resistant S. aureus strains in the diabetes population (52.17%) was higher than that in the non-diabetes population (28.00%; χ 2 = 3.848, P = 0.050). The most common clonal complex type and Staphylococcal chromosome cassette mec type of MRSA in diabetes population was clonal complex 5 (40.91%) and type IV (27.27%), respectively. The proportion of Panton-Valentine leukocidin gene in MRSA strains was 17.65%. There was great sequence type diversity in MRSA strains. The prevalence of MRSA in the community-based diabetes population was moderate, and the high proportions of multidrug-resistant S. aureus strains and diverse molecular characteristics in the diabetes population should be noticed. © 2016 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.

Prevalence of and risk factors associated with the presence of Staphylococcus aureus in the chronic wounds of patients treated in primary health care settings in Brazil

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Eliane Patricia Lino Pereira-Franchi

Full Text Available Abstract INTRODUCTION: Wounds can be colonized by methicillin-resistant Staphylococcus aureus (MRSA. METHODS: We evaluated the prevalence of S. aureus and MRSA in the wounds of patients treated at Basic Health Units in Brazil and identified risk factors associated with their presence. RESULTS: The prevalence rates of S. aureus and MRSA were 51.5% and 8.7%, respectively. There was a correlation between the presence of S. aureus in wounds and nostrils (p<0.01. A positive association was detected between S. aureus infection and previous benzylpenicillin use (p=0.02. No associations were observed for MRSA. CONCLUSIONS: Multidrug-resistant pathogens are present in primary healthcare settings in Brazil.

Increased Age-Dependent Risk of Death Associated With lukF-PV-Positive Staphylococcus aureus Bacteremia

DEFF Research Database (Denmark)

Knudsen, Trine A; Skov, Robert; Petersen, Andreas

2016-01-01

BACKGROUND: Panton-Valentine leucocidin is a Staphylococcus aureus virulence factor encoded by lukF-PV and lukS-PV that is infrequent in S aureus bacteremia (SAB), and, therefore, little is known about risk factors and outcome of lukF-PV/lukS-PV-positive SAB. METHODS: This report is a register......-based nationwide observational cohort study. lukF-PV was detected by polymerase chain reaction. Factors associated with the presence of lukF-PV were assessed by logistic regression analysis. Adjusted 30-day hazard ratios of mortality associated with lukF-PV status were computed by Cox proportional hazards...... regression analysis. RESULTS: Of 9490 SAB cases, 129 were lukF-PV-positive (1.4%), representing 14 different clonal complexes. lukF-PV was associated with younger age, absence of comorbidity, and methicillin-resistant S aureus. In unadjusted analysis, mortality associated with lukF-PV-positive SAB...

Transcription-induced DNA supercoiling: New roles of intranucleosomal DNA loops in DNA repair and transcription.

Science.gov (United States)

Gerasimova, N S; Pestov, N A; Kulaeva, O I; Clark, D J; Studitsky, V M

2016-05-26

RNA polymerase II (Pol II) transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damage hidden in chromatin structure.

Carriage of Staphylococcus aureus among Portuguese nursing students: A longitudinal cohort study over four years of education.

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Teresa Conceição

Full Text Available Staphylococcus aureus is a major human pathogen that can colonize healthy people mainly in the anterior nares. The aim of the present study was to evaluate S. aureus nasal colonization over time among Portuguese nursing students, including methicillin-resistant S. aureus (MRSA.In this longitudinal cohort study, we collected 280 nasal swabs from nursing students at 14 time points over four years of schooling (2012-2016. The isolates were characterized by pulsed-field gel electrophoresis (PFGE, spa typing, multilocus sequence typing (MLST, and SCCmec typing for MRSA. Among 47 students, 20 (43% carried methicillin-susceptible S. aureus (MSSA at admission, but none was colonized with MRSA. A total of 19 students (40% became colonized after exposure during the nursing training, out of which five carried MRSA. Overall, 39 students (83% had S. aureus detected at least once during the study period. Among the 97 MSSA isolates, most (65% belonged to four clones: PFGE A-ST30 (21%, B-ST72 (20%, C-ST508 (13%, and D-ST398 (11%. Three of the five MRSA carriers were colonized with the predominant clone circulating in Portuguese hospitals (ST22-IVh and two with ST3162-II. Colonization of nursing students was highly dynamic with continuous appearance of strains with distinct PFGE types in the same individual.A considerable proportion of students became colonized by S. aureus, including MRSA, during the nursing education, evidencing this population represents an important reservoir of S. aureus. Therefore, education on infection control measures in nursing schools is of major importance.

Rapid and easy detection of low-level resistance to vancomycin in methicillin-resistant Staphylococcus aureus by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Science.gov (United States)

Asakura, Kota; Azechi, Takuya; Sasano, Hiroshi; Matsui, Hidehito; Hanaki, Hideaki; Miyazaki, Motoyasu; Takata, Tohru; Sekine, Miwa; Takaku, Tomoiku; Ochiai, Tomonori; Komatsu, Norio; Shibayama, Keigo; Katayama, Yuki; Yahara, Koji

2018-01-01

Vancomycin-intermediately resistant Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are associated with treatment failure. hVISA contains only a subpopulation of cells with increased minimal inhibitory concentrations, and its detection is problematic because it is classified as vancomycin-susceptible by standard susceptibility testing and the gold-standard method for its detection is impractical in clinical microbiology laboratories. Recently, a research group developed a machine-learning classifier to distinguish VISA and hVISA from vancomycin-susceptible S. aureus (VSSA) according to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data. Nonetheless, the sensitivity of hVISA classification was found to be 76%, and the program was not completely automated with a graphical user interface. Here, we developed a more accurate machine-learning classifier for discrimination of hVISA from VSSA and VISA among MRSA isolates in Japanese hospitals by means of MALDI-TOF MS data. The classifier showed 99% sensitivity of hVISA classification. Furthermore, we clarified the procedures for preparing samples and obtaining MALDI-TOF MS data and developed all-in-one software, hVISA Classifier, with a graphical user interface that automates the classification and is easy for medical workers to use; it is publicly available at https://github.com/bioprojects/hVISAclassifier. This system is useful and practical for screening MRSA isolates for the hVISA phenotype in clinical microbiology laboratories and thus should improve treatment of MRSA infections.

Colonization of nursing professionals by Staphylococcus aureus La colonización de los profesionales de enfermería por Staphylococcus aureus A colonização dos profissionais de enfermagem por Staphylococcus aureus

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Josely Pinto de Moura

2011-04-01

Full Text Available This cross-sectional study aimed to investigate the presence of Staphylococcus aureus in the saliva of the nursing team of a teaching hospital in the interior of São Paulo State. Three saliva samples were collected from 351 individuals with an interval of two months between each collection. All ethical aspects were considered. In 867 (82.3% cultures there was no identification of Staphylococcus aureus in the saliva, in 88 (17.7% cultures Staphylococcus aureus was isolated, 26 (2.5% of which were resistant to methicillin. The prevalence of professionals colonized by Staphylococcus aureus was 41.0% (144/351, of which 7.1% (25/351 were characterized as methicillin-resistant Staphylococcus aureus. Transient carriers represented 81.2% and persistent carriers 18.8%. Resistance to mupirocin was 73.1% of MRSA and 9.3% of MSSA. The results demonstrate that it is the nurse and nursing technician that are the professional categories most susceptible to MRSA. Broader discussion on the thematic and interventions are needed.Se trata de un estudio transversal que tuvo como objetivo investigar la presencia de Staphylococcus aureus en la saliva del equipo de enfermería de un hospital escuela del interior del estado de Sao Paulo. Fueron recolectadas tres muestras de saliva de 351 individuos con intervalo de dos meses. Todos los aspectos éticos fueron contemplados. En 867 (82,3% culturas no hubo identificación de Staphylococcus aureus en la saliva, en 88 (17,7% culturas fue aislado Staphylococcus aureus, siendo 26 (2,5% resistentes a la meticilina. La prevalencia de profesionales colonizados por Staphylococcus aureus fue de 41,0% (144/351, de los cuales 7,1% (25/351 fueron caracterizados como Staphylococcus aureus resistentes a la meticilina. Los portadores transitorios representaron 81,2% y los persistentes 18,8%. La resistencia a la mupirocina fue de 73,1% entre los resistentes a la meticilina y 9,3% en los sensibles a la meticilina. Los resultados

Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

Science.gov (United States)

Guionie, O; Toquin, D; Sellal, E; Bouley, S; Zwingelstein, F; Allée, C; Bougeard, S; Lemière, S; Eterradossi, N

2007-02-01

Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

Evolution of methicillin-resistant Staphylococcus aureus towards increasing resistance

DEFF Research Database (Denmark)

Strommenger, Birgit; Bartels, Mette Damkjær; Kurt, Kevin

2014-01-01

To elucidate the evolutionary history of Staphylococcus aureus clonal complex (CC) 8, which encompasses several globally distributed epidemic lineages, including hospital-associated methicillin-resistant S. aureus (MRSA) and the highly prevalent community-associated MRSA clone USA300.......To elucidate the evolutionary history of Staphylococcus aureus clonal complex (CC) 8, which encompasses several globally distributed epidemic lineages, including hospital-associated methicillin-resistant S. aureus (MRSA) and the highly prevalent community-associated MRSA clone USA300....

A multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in Korean ready-to-eat food.

Science.gov (United States)

Lee, Nari; Kwon, Kyung Yoon; Oh, Su Kyung; Chang, Hyun-Joo; Chun, Hyang Sook; Choi, Sung-Wook

2014-07-01

A multiplex polymerase chain reaction (PCR) assay was developed for simultaneous detection of Escherichia coli O157:H7, Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes, and Staphylococcus aureus in various Korean ready-to-eat foods. The six specific primer pairs for multiplex PCR were selected based on the O157 antigen (rfbE) gene of E. coli O157:H7, the DNA gyrase subunit B (gyrB) gene of B. cereus, the toxin regulatory protein (toxR) gene of V. parahaemolyticus, the invasion protein A (invA) gene of Salmonella spp., the hemolysin (hly) gene of L. monocytogenes, and the thermonuclease (nuc) gene of S. aureus. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity assays for multiplex primer pairs were investigated by testing different strains. When this multiplex PCR assay was applied to evaluate the validity of detecting six foodborne pathogens in artificially inoculated several ready-to-eat food samples, the assay was able to specifically simultaneously detect as few as 1 colony-forming unit/mL of each pathogen after enrichment for 12 h. Their presence in naturally contaminated samples also indicates that the developed multiplex PCR assay is an effective and informative supplement for practical use.

Nasal Staphylococcus aureus and methicillin-resistant Staphylococcus aureus carriage among college student athletes in northern Taiwan

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Hong-Kai Wang

2017-08-01

Full Text Available Of 259 college students in northern Taiwan surveyed, nasal carriage rate of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA was 22.4% and 1.54%, respectively and no significant difference was found between athlete students and non-athlete students. Three of four MRSA isolates belonged to sequence type 59, the endemic community clone.

Detection of Enterovirus 71 gene from clinical specimens by reverse-transcription loop-mediated isothermal amplification

OpenAIRE

D Wang; X Wang; Y Geng; C An

2014-01-01

Purpose : The objective of this study was to develop a sensitive, specific and rapid approach to diagnose hand foot and mouth disease (HFMD) for an early treatment by using loop-mediated isothermal amplification (LAMP) technique. Materials and Methods : A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for detecting EV71 virus was developed, the specificity and sensitivity of RT-LAMP was tested, and the clinical specimens was assayed by the RT-LAMP comparing with conven...

Silver nanoparticles for the inhibition of Staphylococcus aureus

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Miguel Ángel Ortiz-Gila

2015-01-01

Full Text Available Existe un gran ecosistema microbiano en la cavidad oral donde Staphylococcus aureus ( S. aureus se puede encontrar, causando patologías orales tales como quelitis angular, las paperas y la mucositis estafilocócica. Estas enfermedades producidas por S. aureus en la cavidad oral son consecuencia de los factores de virulencia, toxinas y multiresistencia a los antibióticos, lo que contribuye a la infección. La colonización en la cavidad oral por S. aureus en pacientes sanos es de 24% a 36%. Sin embargo, la incidencia aumenta a 48% en pacientes con prótesis debido a la formación de biofilms en la superficie de las dentaduras postizas. Actualmente, no existe ningún tratamiento para infecciones orales sin el uso de antibióticos. Investigaciones recientes indican que las nanopartículas de plata (AgNPs son un material o estrategia para eliminar S. aureus debido a su efecto antibacteriano. Sin embargo, el mecanismo del efecto inhibidor de los iones de Ag sobre S. aureus es sólo parcialmente conocida y muy poco se ha informado. Por lo tanto, el propósito de la presente revisión sistemática es determinar las estrategias y retos de la utilización de biomateriales antimicrobianos con AgNPs frente a las infecciones orales de S. aureus.

Presence of Staphylococcus aureus and Shiga Toxigenic Escherichia coli O157:H7 in Raw Meat in Ağrı, Turkey

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Naim Deniz Ayaz

2016-08-01

Full Text Available Background: Staphylococcus aureus and Shiga toxin producing Escherichia coli O157:H7 (EHEC are significant foodborne pathogens worldwide. While S. aureus can cause mild superficial skin infections or life-threatening bacteremia and endocarditis, as well as toxininduced cases such as toxic shock syndrome; E. coli O157:H7 can cause symptoms from mild diarrhea to severe hemorrhagic colitis (HC, hemolytic uremic syndrome (HUS, thrombotic thrombocytopenic purpura (TTP. Objectives: The objectives of this study were to find out the prevalence and seasonal distribution of S. aureus in 214 frozen raw meat (turkey, chicken and beef and the prevalence of E. coli O157:H7 in 70 raw beef with the characterization of the E. coli O157:H7 isolate by multiplex polymerase chain reaction (PCR. Materials and Methods: For the detection of S. aureus, a total of 214 frozen raw meat samples including 74 turkey meat, 70 chicken meat and 70 beef cuts (approximately 2 × 3 cm cubic parts; and for the detection of E. coli O157:H7, a total of 70 frozen raw beef samples that all were produced from national companies and consumed in Ağrı, Turkey were analyzed. Results: Out of 214 meat samples, 25.7 % (18/70 of the beef, 11.4 % (8/74 of the chicken meat, and 5.4 % (4/70 of the turkey meat samples were contaminated with S. aureus. Out of 70 frozen raw beef samples, only 1 (1.4% was identified as both Shiga toxin 1 and 2producing E. coli O157:H7 by the detection of stx1, stx2, eaeA, hly, and fliCh7 according to multiplex PCR analysis. Conclusion: Our findings demonstrate that occurrence frequency of S. aureus was higher in frozen raw beef than in raw chicken and turkey meat samples. Although the prevalence of E. coli O157:H7 was low in beef, the presence of virulence genes, especially toxin genesrema in a significant public health concern.

Communications of Staphylococcus aureus and non-aureus Staphylococcus species from bovine intramammary infections and teat apex colonization

DEFF Research Database (Denmark)

Mahmmod, Yasser S.; Klaas, Ilka Christine; Svennesen, Line

2018-01-01

The role of non-aureus staphylococci (NAS) in the risk of acquisition of intramammary infections with Staphylococcus aureus is vague and still under debate. The objectives of this study were to (1) investigate the distribution patterns of NAS species from milk and teat skin in dairy herds with au...

Sodium Octanoate Modulates the Innate Immune Response of Bovine Mammary Epithelial Cells through the TLR2/P38/JNK/ERK1/2 Pathway: Implications during Staphylococcus aureus Internalization.

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Alva-Murillo, Nayeli; Ochoa-Zarzosa, Alejandra; López-Meza, Joel E

2017-01-01

Bovine mammary epithelial cells (bMECs) contribute to mammary gland defense against invading pathogens, such as Staphylococcus aureus (intracellular facultative), which is recognized by TLR2. In a previous report, we showed that sodium octanoate [NaO, a medium chain fatty acid (C8)] induces (0.25 mM) or inhibits (1 mM) S. aureus internalization into bMECs and differentially regulates the innate immune response (IIR). However, the molecular mechanisms have not been described, which was the aim of this study. The results showed that α5β1 integrin membrane abundance (MA) was increased in 0.25 mM NaO-treated cells, but TLR2 or CD36 MA was not modified. When these receptors were blocked individually, 0.25 mM NaO-increased S. aureus internalization was notably reduced. Interestingly, in this condition, the IIR of the bMECs was impaired because MAPK (p38, JNK, and ERK1/2) phosphorylation and the activation of transcription factors related to these pathways were decreased. In addition, the 1 mM NaO treatment induced TLR2 MA, but neither the integrin nor CD36 MA was modified. The reduction in S. aureus internalization induced by 1 mM NaO was increased further when TLR2 was blocked. In addition, the phosphorylation levels of the MAPKs increased, and 13 transcriptional factors related to the IIR were slightly activated (CBF, CDP, c-Myb, AP-1, Ets-1/Pea-3, FAST-1, GAS/ISRE, AP-2, NFAT-1, OCT-1, RAR/DR-5, RXR/DR-1, and Stat-3). Moreover, the 1 mM NaO treatment up-regulated gene expression of IL-8 and RANTES and secretion of IL-1β. Notably, when 1 mM NaO-treated bMECs were challenged with S. aureus , the gene expression of IL-8 and IL-10 increased, while IL-1β secretion was reduced. In conclusion, our results showed that α5β1 integrin, TLR2 and CD36 are involved in 0.25 mM NaO-increased S. aureus internalization in bMECs. In addition, 1 mM NaO activates bMECs via the TLR2 signaling pathways (p38, JNK, and ERK1/2), which improves IIR before S. aureus invasion. Additionally