The Causes of Post-Operative Meningitis: The Comparison Of Gram-Negative and Gram-Positive Pathogens.

Science.gov (United States)

Kurtaran, Behice; Kuscu, Ferit; Ulu, Aslihan; Inal, Ayse Seza; Komur, Suheyla; Kibar, Filiz; Cetinalp, Nuri Eralp; Ozsoy, Kerem Mazhar; Arslan, Yusuf Kemal; Aksu, Hasan Salih; Tasova, Yesim

2017-06-20

In this study, we aim to determine the microbiological etiology in critically ill neurosurgical patients with nosocomial meningitis (NM) and show the impact of Gram-negative rods and differences of patient's characteristics, clinical and prognostic measures between Gram-negative and Gram-positive meningitis. In this prospective, one center study we reviewed all adult patients hospitalized during a 12-year period and identified pathogens isolated from post-neurosurgical cases of NM. Demographic, clinical, and treatment characteristics were noted from the medical records. Of the 134 bacterial NM patients, 78 were male and 56 were female, with a mean age of 46±15.9 and median age of 50 (18-80) years. 141 strains isolated; 82 (58.2%) were Gram negative, 59 (41.8%) were Gram positive. Most common isolated microorganism was Acinetobacter baumannii (%34.8). In comparison of mortality data shows that the patients who have meningitis with Gram-negative pathogens have higher mortality than with Gram positives (p=0.034). The duration between surgery and meningitis was shorter in Gram negative meningitis cases compared to others (p=0.045) but the duration between the diagnosis and death was shorter in Gram-positive meningitis cases compared to Gram negatives (p= 0.017). CSF protein and lactate level were higher and glucose level was lower in cases of NM with Gram negatives (p value were respectively, 0.022, 0.039 and 0.049). As conclusions; in NM, Gram-negative pathogens were seen more frequently; A.baumanni was the predominant pathogen; and NM caused by Gram negatives had worse clinical and laboratory characteristic and prognostic outcome than Gram positives.

Methods for Confirming the Gram Reaction of Gram-variable Bacillus Species Isolated from Tobacco

Directory of Open Access Journals (Sweden)

Morin A

2014-12-01

Full Text Available Bacillus is a predominant genus of bacteria isolated from tobacco. The Gram stain is the most commonly used and most important of all diagnostic staining techniques in microbiology. In order to help confirm the Gram positivity of Bacillus isolates from tobacco, three methods using the chemical differences of the cell wall and membrane of Gram-positive and Gram-negative bacteria were investigated: the KOH (potassium hydroxide, the LANA (L-alanine-4-nitroanilide, and the vancomycin susceptibility tests. When colonies of Gram-negative bacteria are treated with 3% KOH solution, a slimy suspension is produced, probably due to destruction of the cell wall and liberation of deoxyribonucleic acid (DNA. Gram-positive cell walls resist KOH treatment. The LANA test reveals the presence of a cell wall aminopeptidase that hydrolyzes the L-alanine-4-nitroanilide in Gram-negative bacteria. This enzyme is absent in Gram-positive bacteria. Vancomycin is a glycopeptide antibiotic inhibiting the cell wall peptido-glycan synthesis of Gram-positive microorganisms. Absence of lysis with KOH, absence of hydrolysis of LANA, and susceptibility to vancomycin were used with the Gram reaction to confirm the Gram positivity of various Bacillus species isolated from tobacco. B. laevolacticus excepted, all Bacillus species tested showed negative reactions to KOH and LANA tests, and all species were susceptible to vancomycin (5 and 30 µg.

Mechanistic antimicrobial approach of extracellularly synthesized silver nanoparticles against gram positive and gram negative bacteria

Energy Technology Data Exchange (ETDEWEB)

Tamboli, Dhawal P.; Lee, Dae Sung, E-mail: daesung@knu.ac.kr

2013-09-15

Highlights: • Bacterial extracelluar enzymes stabilized the silver nanoparticles (AgNPs). • AgNPs formation was characterized by analytical techniques such as UV–vis, TEM, and FTIR. • AgNPs showed obvious antimicrobial activity against both gram positive and gram negative microorganisms. • A mechanism of AgNPs’ antimicrobial activity was proposed. -- Abstract: The development of eco-friendly and reliable processes for the synthesis of nanoparticles has attracted considerable interest in nanotechnology. In this study, an extracellular enzyme system of a newly isolated microorganism, Exiguobacterium sp. KNU1, was used for the reduction of AgNO{sub 3} solutions to silver nanoparticles (AgNPs). The extracellularly biosynthesized AgNPs were characterized by UV–vis spectroscopy, Fourier transform infra-red spectroscopy and transmission electron microscopy. The AgNPs were approximately 30 nm (range 5–50 nm) in size, well-dispersed and spherical. The AgNPs were evaluated for their antimicrobial effects on different gram negative and gram positive bacteria using the minimum inhibitory concentration method. Reasonable antimicrobial activity against Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was observed. The morphological changes occurred in all the microorganisms tested. In particular, E. coli exhibited DNA fragmentation after being treated with the AgNPs. Finally, the mechanism for their bactericidal activity was proposed according to the results of scanning electron microscopy and single cell gel electrophoresis.

Mechanistic antimicrobial approach of extracellularly synthesized silver nanoparticles against gram positive and gram negative bacteria

International Nuclear Information System (INIS)

Tamboli, Dhawal P.; Lee, Dae Sung

2013-01-01

Highlights: • Bacterial extracelluar enzymes stabilized the silver nanoparticles (AgNPs). • AgNPs formation was characterized by analytical techniques such as UV–vis, TEM, and FTIR. • AgNPs showed obvious antimicrobial activity against both gram positive and gram negative microorganisms. • A mechanism of AgNPs’ antimicrobial activity was proposed. -- Abstract: The development of eco-friendly and reliable processes for the synthesis of nanoparticles has attracted considerable interest in nanotechnology. In this study, an extracellular enzyme system of a newly isolated microorganism, Exiguobacterium sp. KNU1, was used for the reduction of AgNO 3 solutions to silver nanoparticles (AgNPs). The extracellularly biosynthesized AgNPs were characterized by UV–vis spectroscopy, Fourier transform infra-red spectroscopy and transmission electron microscopy. The AgNPs were approximately 30 nm (range 5–50 nm) in size, well-dispersed and spherical. The AgNPs were evaluated for their antimicrobial effects on different gram negative and gram positive bacteria using the minimum inhibitory concentration method. Reasonable antimicrobial activity against Salmonella typhimurium, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus was observed. The morphological changes occurred in all the microorganisms tested. In particular, E. coli exhibited DNA fragmentation after being treated with the AgNPs. Finally, the mechanism for their bactericidal activity was proposed according to the results of scanning electron microscopy and single cell gel electrophoresis

Distribution of multi-resistant Gram-negative versus Gram-positive bacteria in the hospital inanimate environment.

Science.gov (United States)

Lemmen, S W; Häfner, H; Zolldann, D; Stanzel, S; Lütticken, R

2004-03-01

We prospectively studied the difference in detection rates of multi-resistant Gram-positive and multi-resistant Gram-negative bacteria in the inanimate environment of patients harbouring these organisms. Up to 20 different locations around 190 patients were surveyed. Fifty-four patients were infected or colonized with methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant enterococci (VRE) and 136 with multi-resistant Gram-negative bacteria. The environmental detection rate for MRSA or VRE was 24.7% (174/705 samples) compared with 4.9% (89/1827 samples) for multi-resistant Gram-negative bacteria (PGram-positive bacteria were isolated more frequently than Gram-negatives from the hands of patients (PGram-positive and Gram-negative isolates. Our results suggest that the inanimate environment serves as a secondary source for MRSA and VRE, but less so for Gram-negative bacteria. Thus, strict contact isolation in a single room with complete barrier precautions is recommended for MRSA or VRE; however, for multi-resistant Gram-negative bacteria, contact isolation with barrier precautions for close contact but without a single room seems sufficient. This benefits not only the patients, but also the hospital by removing some of the strain placed on already over-stretched resources.

Type I and Type II mechanisms of antimicrobial photodynamic therapy: an in vitro study on gram-negative and gram-positive bacteria.

Science.gov (United States)

Huang, Liyi; Xuan, Yi; Koide, Yuichiro; Zhiyentayev, Timur; Tanaka, Masamitsu; Hamblin, Michael R

2012-08-01

Antimicrobial photodynamic therapy (APDT) employs a non-toxic photosensitizer (PS) and visible light, which in the presence of oxygen produce reactive oxygen species (ROS), such as singlet oxygen ((1) O(2), produced via Type II mechanism) and hydroxyl radical (HO(.), produced via Type I mechanism). This study examined the relative contributions of (1) O(2) and HO(.) to APDT killing of Gram-positive and Gram-negative bacteria. Fluorescence probes, 3'-(p-hydroxyphenyl)-fluorescein (HPF) and singlet oxygen sensor green reagent (SOSG) were used to determine HO(.) and (1) O(2) produced by illumination of two PS: tris-cationic-buckminsterfullerene (BB6) and a conjugate between polyethylenimine and chlorin(e6) (PEI-ce6). Dimethylthiourea is a HO(.) scavenger, while sodium azide (NaN(3)) is a quencher of (1) O(2). Both APDT and killing by Fenton reaction (chemical generation of HO(.)) were carried out on Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). Conjugate PEI-ce6 mainly produced (1) O(2) (quenched by NaN(3)), while BB6 produced HO(.) in addition to (1) O(2) when NaN(3) potentiated probe activation. NaN(3) also potentiated HPF activation by Fenton reagent. All bacteria were killed by Fenton reagent but Gram-positive bacteria needed a higher concentration than Gram-negatives. NaN(3) potentiated Fenton-mediated killing of all bacteria. The ratio of APDT killing between Gram-positive and Gram-negative bacteria was 2 or 4:1 for BB6 and 25:1 for conjugate PEI-ce6. There was a NaN(3) dose-dependent inhibition of APDT killing using both PEI-ce6 and BB6 against Gram-negative bacteria while NaN(3) almost failed to inhibit killing of Gram-positive bacteria. Azidyl radicals may be formed from NaN(3) and HO(.). It may be that Gram-negative bacteria are more susceptible to HO(.) while Gram-positive bacteria are more susceptible to (1) O(2). The differences in Na

c-di-GMP Regulates Various Phenotypes and Insecticidal Activity of Gram-Positive Bacillus thuringiensis

Directory of Open Access Journals (Sweden)

Yang Fu

2018-02-01

Full Text Available C-di-GMP has been well investigated to play significant roles in the physiology of many Gram-negative bacteria. However, its effect on Gram-positive bacteria is less known. In order to more understand the c-di-GMP functions in Gram-positive bacteria, we have carried out a detailed study on the c-di-GMP-metabolizing enzymes and their physiological functions in Bacillus thuringiensis, a Gram-positive entomopathogenic bacterium that has been applied as an insecticide successfully. We performed a systematic study on the ten putative c-di-GMP-synthesizing enzyme diguanylate cyclases (DGCs and c-di-GMP-degrading enzyme phosphodiesterases (PDEs in B. thuringiensis BMB171, and artificially elevated the intracellular c-di-GMP level in BMB171 by deleting one or more pde genes. We found increasing level of intracellular c-di-GMP exhibits similar activities as those in Gram-negative bacteria, including altered activities in cell motility, biofilm formation, and cell-cell aggregation. Unexpectedly, we additionally found a novel function exhibited by the increasing level of c-di-GMP to promote the insecticidal activity of this bacterium against Helicoverpa armigera. Through whole-genome transcriptome profile analyses, we found that 4.3% of the B. thuringiensis genes were differentially transcribed when c-di-GMP level was increased, and 77.3% of such gene products are involved in some regulatory pathways not reported in other bacteria to date. In summary, our study represents the first comprehensive report on the c-di-GMP-metabolizing enzymes, their effects on phenotypes, and the transcriptome mediated by c-di-GMP in an important Gram-positive bacterium.

An optimized staining technique for the detection of Gram positive and Gram negative bacteria within tissue.

Science.gov (United States)

Becerra, Sandra C; Roy, Daniel C; Sanchez, Carlos J; Christy, Robert J; Burmeister, David M

2016-04-12

Bacterial infections are a common clinical problem in both acute and chronic wounds. With growing concerns over antibiotic resistance, treatment of bacterial infections should only occur after positive diagnosis. Currently, diagnosis is delayed due to lengthy culturing methods which may also fail to identify the presence of bacteria. While newer costly bacterial identification methods are being explored, a simple and inexpensive diagnostic tool would aid in immediate and accurate treatments for bacterial infections. Histologically, hematoxylin and eosin (H&E) and Gram stains have been employed, but are far from optimal when analyzing tissue samples due to non-specific staining. The goal of the current study was to develop a modification of the Gram stain that enhances the contrast between bacteria and host tissue. A modified Gram stain was developed and tested as an alternative to Gram stain that improves the contrast between Gram positive bacteria, Gram negative bacteria and host tissue. Initially, clinically relevant strains of Pseudomonas aeruginosa and Staphylococcus aureus were visualized in vitro and in biopsies of infected, porcine burns using routine Gram stain, and immunohistochemistry techniques involving bacterial strain-specific fluorescent antibodies as validation tools. H&E and Gram stain of serial biopsy sections were then compared to a modification of the Gram stain incorporating a counterstain that highlights collagen found in tissue. The modified Gram stain clearly identified both Gram positive and Gram negative bacteria, and when compared to H&E or Gram stain alone provided excellent contrast between bacteria and non-viable burn eschar. Moreover, when applied to surgical biopsies from patients that underwent burn debridement this technique was able to clearly detect bacterial morphology within host tissue. We describe a modification of the Gram stain that provides improved contrast of Gram positive and Gram negative microorganisms within host

Gram staining.

Science.gov (United States)

Coico, Richard

2005-10-01

Named after Hans Christian Gram who developed the method in 1884, the Gram stain allows one to distinguish between Gram-positive and Gram-negative bacteria on the basis of differential staining with a crystal violet-iodine complex and a safranin counterstain. The cell walls of Gram-positive organisms retain this complex after treatment with alcohol and appear purple, whereas gram-negative organisms decolorize following such treatment and appear pink. The method described here is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures.

Methods for targetted mutagenesis in gram-positive bacteria

Science.gov (United States)

Yang, Yunfeng

2014-05-27

The present invention provides a method of targeted mutagenesis in Gram-positive bacteria. In particular, the present invention provides a method that effectively integrates a suicide integrative vector into a target gene in the chromosome of a Gram-positive bacterium, resulting in inactivation of the target gene.

Glass bead transformation method for gram-positive bacteria

OpenAIRE

Rattanachaikunsopon, Pongsak; Phumkhachorn, Parichat

2009-01-01

A simple, inexpensive and reproducible transformation method was developed for Gram-positive bacteria. It was based on agitation of bacterial protoplasts with glass beads in the presence of DNA and polyethylene glycol. By using this method, introduction of pGK12 into protoplasts of several strains of Gram-positive bacteria was achieved.

Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR.

Science.gov (United States)

Kim, Sung Joon; Chang, James; Singh, Manmilan

2015-01-01

Peptidoglycan is an essential component of cell wall in Gram-positive bacteria with unknown architecture. In this review, we summarize solid-state NMR approaches to address some of the unknowns in the Gram-positive bacteria peptidoglycan architecture: 1) peptidoglycan backbone conformation, 2) PG-lattice structure, 3) variations in the peptidoglycan architecture and composition, 4) the effects of peptidoglycan bridge-length on the peptidoglycan architecture in Fem mutants, 5) the orientation of glycan strands with respect to the membrane, and 6) the relationship between the peptidoglycan structure and the glycopeptide antibiotic mode of action. Solid-state NMR analyses of Staphylococcus aureus cell wall show that peptidoglycan chains are surprisingly ordered and densely packed. The peptidoglycan disaccharide backbone adopts 4-fold screw helical symmetry with the disaccharide unit periodicity of 40Å. Peptidoglycan lattice in the S. aureus cell wall is formed by cross-linked PG stems that have parallel orientations. The structural characterization of Fem-mutants of S. aureus with varying lengths of bridge structures suggests that the PG-bridge length is an important determining factor for the PG architecture. Copyright © 2014 Elsevier B.V. All rights reserved.

Amplifiable DNA from Gram-negative and Gram-positive bacteria by a low strength pulsed electric field method

Science.gov (United States)

Vitzthum, Frank; Geiger, Georg; Bisswanger, Hans; Elkine, Bentsian; Brunner, Herwig; Bernhagen, Jürgen

2000-01-01

An efficient electric field-based procedure for cell disruption and DNA isolation is described. Isoosmotic suspensions of Gram-negative and Gram-positive bacteria were treated with pulsed electric fields of Pulses had an exponential decay waveform with a time constant of 3.4 µs. DNA yield was linearly dependent on time or pulse number, with several thousand pulses needed. Electrochemical side-effects and electrophoresis were minimal. The lysates contained non-fragmented DNA which was readily amplifiable by PCR. As the method was not limited to samples of high specific resistance, it should be applicable to physiological fluids and be useful for genomic and DNA diagnostic applications. PMID:10734214

Comparative activity of ceftobiprole against Gram-positive and Gram-negative isolates from Europe and the Middle East: the CLASS study.

Science.gov (United States)

Rossolini, Gian M; Dryden, Matthew S; Kozlov, Roman S; Quintana, Alvaro; Flamm, Robert K; Läuffer, Jörg M; Lee, Emma; Morrissey, Ian; CLASS Study Group

2011-01-01

to assess the in vitro activity of ceftobiprole and comparators against a recent collection of Gram-positive and Gram-negative pathogens, in order to detect potential changes in susceptibility patterns, and to evaluate the Etest assay for ceftobiprole susceptibility testing. contemporary Gram-positive and Gram-negative isolates (excluding extended-spectrum β-lactamase-producing isolates) from across Europe and the Middle East were collected, and their susceptibility to ceftobiprole, vancomycin, teicoplanin, linezolid, ceftazidime and cefepime was assessed using the Etest method. Quality testing [using Etest and broth microdilution (BMD)] was conducted at a central reference laboratory. some 5041 Gram-positive and 4026 Gram-negative isolates were included. Against Gram-positive isolates overall, ceftobiprole had the lowest MIC50 (0.5 mg/L), compared with 1 mg/L for its comparators (vancomycin, teicoplanin and linezolid). Against methicillin-resistant Staphylococcus aureus, all four agents had a similar MIC90 (2 mg/L), but ceftobiprole had a 4-fold better MIC90 (0.5 mg/L) against methicillin-susceptible strains. Only 38 Gram-positive isolates were confirmed as ceftobiprole resistant. Among Gram-negative strains, 86.9%, 91.7% and 95.2% were susceptible to ceftobiprole, ceftazidime and cefepime, respectively. Pseudomonas aeruginosa was less susceptible to all three antimicrobials than any other Gram-negative pathogen. There was generally good agreement between local Etest results and those obtained at the reference laboratory (for ceftobiprole: 86.8% with Gram-negatives; and 94.7% with Gram-positives), as well as between results obtained by BMD and Etest methods (for ceftobiprole: 98.2% with Gram-negatives; and 98.4% with Gram-positives). ceftobiprole exhibits in vitro activity against a wide range of Gram-positive and Gram-negative pathogens, including multidrug-resistant strains. No changes in its known susceptibility profile were identified.

Prognostic factors and monomicrobial necrotizing fasciitis: gram-positive versus gram-negative pathogens

Directory of Open Access Journals (Sweden)

Hsu Wei-Hsiu

2011-01-01

Full Text Available Abstract Background Monomicrobial necrotizing fasciitis is rapidly progressive and life-threatening. This study was undertaken to ascertain whether the clinical presentation and outcome for patients with this disease differ for those infected with a gram-positive as compared to gram-negative pathogen. Methods Forty-six patients with monomicrobial necrotizing fasciitis were examined retrospectively from November 2002 to January 2008. All patients received adequate broad-spectrum antibiotic therapy, aggressive resuscitation, prompt radical debridement and adjuvant hyperbaric oxygen therapy. Eleven patients were infected with a gram-positive pathogen (Group 1 and 35 patients with a gram-negative pathogen (Group 2. Results Group 2 was characterized by a higher incidence of hemorrhagic bullae and septic shock, higher APACHE II scores at 24 h post-admission, a higher rate of thrombocytopenia, and a higher prevalence of chronic liver dysfunction. Gouty arthritis was more prevalent in Group 1. For non-survivors, the incidences of chronic liver dysfunction, chronic renal failure and thrombocytopenia were higher in comparison with those for survivors. Lower level of serum albumin was also demonstrated in the non-survivors as compared to those in survivors. Conclusions Pre-existing chronic liver dysfunction, chronic renal failure, thrombocytopenia and hypoalbuminemia, and post-operative dependence on mechanical ventilation represent poor prognostic factors in monomicrobial necrotizing fasciitis. Patients with gram-negative monobacterial necrotizing fasciitis present with more fulminant sepsis.

Bio sorption of some Rare Earth Elements and Yttrium by Gram Positive and Gram Negative Bacteria

International Nuclear Information System (INIS)

Ibrahim, H.A.

2012-01-01

The separate bio sorption of the REEs La, Sm, Eu and Dy together with yttrium upon the Gram positive bacteria Bacillus subtilis (B.subtilis) and Bacillus Licheniformis (B. Licheniformis),the Gram negative bacterium Escherichia coli (E. coli ) and Saccharomyces cervisiae (Yeast) was studied. The revelant factors of ph 1-6, contact time (30-180 min), the initial rare earth concentration (50-200 mg/l) have been studied. The amount of the accumulated element was strongly affected by its concentration.In addition, bio sorptive fractionation of Y and the studied REEs from a solution containing a mixture of these elements was also studied. From the obtained data, it was found that Langmuir isotherm model for both B.licheniformis and E.coli gives a best fit for the studied elements over the working range of concentration (50-200 mg/I). Transmission electron microscopy exhibited accumulation throughout the bacterial cell with some granular deposits in both the cell periphery and cytoplasm

Defining a role for Hfq in Gram-positive bacteria

DEFF Research Database (Denmark)

Nielsen, Jesper Sejrup; Lei, Lisbeth Kristensen; Ebersbach, Tine

2010-01-01

Small trans-encoded RNAs (sRNAs) modulate the translation and decay of mRNAs in bacteria. In Gram-negative species, antisense regulation by trans-encoded sRNAs relies on the Sm-like protein Hfq. In contrast to this, Hfq is dispensable for sRNA-mediated riboregulation in the Gram-positive species......-dependent and -independent mechanisms, thus adding another layer of complexity to sRNA-mediated riboregulation in Gram-positive species....

Antiadhesion agents against Gram-positive pathogens.

Science.gov (United States)

Cascioferro, Stella; Cusimano, Maria Grazia; Schillaci, Domenico

2014-01-01

A fundamental step of Gram-positive pathogenesis is the bacterial adhesion to the host tissue involving interaction between bacterial surface molecules and host ligands. This review is focused on antivirulence compounds that target Gram-positive adhesins and on their potential development as therapeutic agents alternative or complementary to conventional antibiotics in the contrast of pathogens. In particular, compounds that target the sortase A, wall theicoic acid inhibitors, carbohydrates able to bind bacterial proteins and proteins capable of influencing the bacterial adhesion, were described. We further discuss the advantages and disadvantages of this strategy in the development of novel antimicrobials and the future perspective of this research field still at its first steps.

Gram-positive bacteria persisting in the food production environment

DEFF Research Database (Denmark)

Knøchel, Susanne; Harmsen, Morten; Knudsen, Bettina

2008-01-01

Many gram-positive bacteria are able to form aggregates or biofilms and resist external stress factors and some gram-positive pathogenic bacteria such as Listeria monocytogenes and Bacillus cereus may persist in the food production environment for extended periods. Most research has focussed...

Antimicrobial Activity of Carbon Nanoparticles Isolated from Natural Sources against Pathogenic Gram-Negative and Gram-Positive Bacteria

International Nuclear Information System (INIS)

Varghese, S.; Jose, S.; Varghese, S.; Kuriakose, S.; Jose, S.

2013-01-01

This paper describes the isolation of carbon nanoparticles (CNPs) from kitchen soot, characterization of the CNPs by UV/visible spectroscopy, SEM and XRD, and their antimicrobial action. The antibacterial activity of the isolated carbon nanoparticles was tested against various pathogenic bacterial strains such as Gram-negative Proteus refrigere and Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus and Streptococcus haemolyticus. The inhibition zones were measured, and it was found that the carbon nanoparticles isolated from natural sources are active against these Gram-negative and Gram-positive bacterial strains

Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria

Directory of Open Access Journals (Sweden)

Muscariello Lidia

2006-05-01

Full Text Available Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters. Conclusion We propose that the CscA, CscB, CscC and Csc

Differential staining of bacteria: gram stain.

Science.gov (United States)

Moyes, Rita B; Reynolds, Jackie; Breakwell, Donald P

2009-11-01

In 1884, Hans Christian Gram, a Danish doctor, developed a differential staining technique that is still the cornerstone of bacterial identification and taxonomic division. This multistep, sequential staining protocol separates bacteria into four groups based on cell morphology and cell wall structure: Gram-positive cocci, Gram-negative cocci, Gram-positive rods, and Gram-negative rods. The Gram stain is useful for assessing bacterial contamination of tissue culture samples or for examining the Gram stain status and morphological features of bacteria isolated from mixed or isolated bacterial cultures. (c) 2009 by John Wiley & Sons, Inc.

Gram-positive rods prevailing in teeth with apical periodontitis undergoing root canal treatment.

Science.gov (United States)

Chávez de Paz, L E; Molander, A; Dahlén, G

2004-09-01

To identify Gram-positive rods from root canals of teeth with apical periodontitis and to examine their associations with other species. Consecutive root canal samples (RCSs) from 139 teeth undergoing root canal treatment were analyzed prospectively for cultivable microbes. Gram-positive rods in the first RCS submitted after chemo-mechanical preparation were categorised to genus level by selective media and gas-liquid chromatography (GLC), and identified to species level by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Associations between organisms were measured by odds ratios (OR). In the first samples submitted a total of 158 Gram-positive rods, 115 Gram-positive cocci, 26 Gram-negative rods and 9 Gram-negative cocci, were identified. At genus levels Gram-positive rods were classified into: Lactobacillus spp. (38%), Olsenella spp. (18%), Propionibacterium spp. (13%), Actinomyces spp. (12%), Bifidobacterium spp. (13%) and Eubacterium spp. (6%). The most frequent species were Olsenella uli, Lactobacillus paracasei and Propionibacterium propionicum. In subsequent samples taken during treatment, Gram-positive rods were also identified, although the number of strains was considerably reduced. Positive associations were observed between members of the genus lactobacilli and Gram-positive cocci (OR>2). Olsenella uli and Lactobacillus spp. predominated over other Gram-positive rods. A possible association exists between Lactobacillus spp. and Gram-positive cocci in root canals of teeth with apical periodontitis receiving treatment.

Selenoproteins in Archaea and Gram-positive bacteria.

Science.gov (United States)

Stock, Tilmann; Rother, Michael

2009-11-01

Selenium is an essential trace element for many organisms by serving important catalytic roles in the form of the 21st co-translationally inserted amino acid selenocysteine. It is mostly found in redox-active proteins in members of all three domains of life and analysis of the ever-increasing number of genome sequences has facilitated identification of the encoded selenoproteins. Available data from biochemical, sequence, and structure analyses indicate that Gram-positive bacteria synthesize and incorporate selenocysteine via the same pathway as enterobacteria. However, recent in vivo studies indicate that selenocysteine-decoding is much less stringent in Gram-positive bacteria than in Escherichia coli. For years, knowledge about the pathway of selenocysteine synthesis in Archaea and Eukarya was only fragmentary, but genetic and biochemical studies guided by analysis of genome sequences of Sec-encoding archaea has not only led to the characterization of the pathways but has also shown that they are principally identical. This review summarizes current knowledge about the metabolic pathways of Archaea and Gram-positive bacteria where selenium is involved, about the known selenoproteins, and about the respective pathways employed in selenoprotein synthesis.

Selective bowel decontamination results in gram-positive translocation.

Science.gov (United States)

Jackson, R J; Smith, S D; Rowe, M I

1990-05-01

Colonization by enteric gram-negative bacteria with subsequent translocation is believed to be a major mechanism for infection in the critically ill patient. Selective bowel decontamination (SBD) has been used to control gram-negative infections by eliminating these potentially pathogenic bacteria while preserving anaerobic and other less pathogenic organisms. Infection with gram-positive organisms and anaerobes in two multivisceral transplant patients during SBD led us to investigate the effect of SBD on gut colonization and translocation. Twenty-four rats received enteral polymixin E, tobramycin, amphotericin B, and parenteral cefotaxime for 7 days (PTA + CEF); 23 received parenteral cefotaxime alone (CEF), 19 received the enteral antibiotics alone (PTA), 21 controls received no antibiotics. Cecal homogenates, mesenteric lymph node (MLN), liver, and spleen were cultured. Only 8% of the PTA + CEF group had gram-negative bacteria in cecal culture vs 52% CEF, 84% PTA, and 100% in controls. Log Enterococcal colony counts were higher in the PTA + CEF group (8.0 + 0.9) vs controls (5.4 + 0.4) P less than 0.01. Translocation of Enterococcus to the MLN was significantly increased in the PTA + CEF group (67%) vs controls (0%) P less than 0.01. SBD effectively eliminates gram-negative organisms from the gut in the rat model. Overgrowth and translocation of Enterococcus suggests that infection with gram-positive organisms may be a limitation of SBD.

Dustborne and airborne gram-positive and gram-negative bacteria in high versus low ERMI homes

Science.gov (United States)

The study aimed at investigating Gram-positive and Gram-negative bacteria in moldy and non-moldy homes, as defined by the home's Environmental Relative Moldiness Index (ERMI) value. The ERMI values were determined from floor dust samples in 2010 and 2011 and homes were classified...

Multi-location gram-positive and gram-negative bacterial protein subcellular localization using gene ontology and multi-label classifier ensemble.

Science.gov (United States)

Wang, Xiao; Zhang, Jun; Li, Guo-Zheng

2015-01-01

It has become a very important and full of challenge task to predict bacterial protein subcellular locations using computational methods. Although there exist a lot of prediction methods for bacterial proteins, the majority of these methods can only deal with single-location proteins. But unfortunately many multi-location proteins are located in the bacterial cells. Moreover, multi-location proteins have special biological functions capable of helping the development of new drugs. So it is necessary to develop new computational methods for accurately predicting subcellular locations of multi-location bacterial proteins. In this article, two efficient multi-label predictors, Gpos-ECC-mPLoc and Gneg-ECC-mPLoc, are developed to predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. The two multi-label predictors construct the GO vectors by using the GO terms of homologous proteins of query proteins and then adopt a powerful multi-label ensemble classifier to make the final multi-label prediction. The two multi-label predictors have the following advantages: (1) they improve the prediction performance of multi-label proteins by taking the correlations among different labels into account; (2) they ensemble multiple CC classifiers and further generate better prediction results by ensemble learning; and (3) they construct the GO vectors by using the frequency of occurrences of GO terms in the typical homologous set instead of using 0/1 values. Experimental results show that Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently predict the subcellular locations of multi-label gram-positive and gram-negative bacterial proteins respectively. Gpos-ECC-mPLoc and Gneg-ECC-mPLoc can efficiently improve prediction accuracy of subcellular localization of multi-location gram-positive and gram-negative bacterial proteins respectively. The online web servers for Gpos-ECC-mPLoc and Gneg-ECC-mPLoc predictors are freely accessible

Bioengineered nisin A derivatives with enhanced activity against both Gram positive and Gram negative pathogens.

Directory of Open Access Journals (Sweden)

Des Field

Full Text Available Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G, with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.

Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

Science.gov (United States)

Field, Des; Begley, Maire; O’Connor, Paula M.; Daly, Karen M.; Hugenholtz, Floor; Cotter, Paul D.; Hill, Colin; Ross, R. Paul

2012-01-01

Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria. PMID:23056510

Peptidoglycan Recycling in Gram-Positive Bacteria Is Crucial for Survival in Stationary Phase

Science.gov (United States)

Borisova, Marina; Gaupp, Rosmarie; Duckworth, Amanda; Schneider, Alexander; Dalügge, Désirée; Mühleck, Maraike; Deubel, Denise; Unsleber, Sandra; Yu, Wenqi; Muth, Günther; Bischoff, Markus; Götz, Friedrich

2016-01-01

ABSTRACT Peptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms, Staphylococcus aureus, Bacillus subtilis, and Streptomyces coelicolor, all recycle the sugar N-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ in E. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P to N-acetylglucosamine-6-phosphate and d-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQ cells of S. aureus and B. subtilis revealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQ cultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQ mutants of S. aureus and B. subtilis showed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival. PMID:27729505

Electrochemical reduction of oxygen catalyzed by a wide range of bacteria including Gram-positive

Energy Technology Data Exchange (ETDEWEB)

Cournet, Amandine [Universite de Toulouse, UPS, LU49, Adhesion Bacterienne et Formation de Biofilms, 35 chemin des Maraichers, 31 062 Toulouse cedex 09 (France); Laboratoire de Genie Chimique CNRS, Universite de Toulouse, 4 allee Emile Monso, BP 84234, 31432 Toulouse cedex 04 (France); Delia, Marie-Line; Bergel, Alain [Laboratoire de Genie Chimique CNRS, Universite de Toulouse, 4 allee Emile Monso, BP 84234, 31432 Toulouse cedex 04 (France); Roques, Christine; Berge, Mathieu [Universite de Toulouse, UPS, LU49, Adhesion Bacterienne et Formation de Biofilms, 35 chemin des Maraichers, 31 062 Toulouse cedex 09 (France)

2010-04-15

Most bacteria known to be electrochemically active have been harvested in the anodic compartments of microbial fuel cells (MFCs) and are able to use electrodes as electron acceptors. The reverse phenomenon, i.e. using solid electrodes as electron donors, is not so widely studied. To our knowledge, most of the electrochemically active bacteria are Gram-negative. The present study implements a transitory electrochemical technique (cyclic voltammetry) to study the microbial catalysis of the electrochemical reduction of oxygen. It is demonstrated that a wide range of aerobic and facultative anaerobic bacteria are able to catalyze oxygen reduction. Among these electroactive bacteria, several were Gram-positive. The transfer of electrons was direct since no activity was obtained with the filtrate. These findings, showing a widespread property among bacteria including Gram-positive ones, open new and interesting routes in the field of electroactive bacteria research. (author)

The potent antimicrobial properties of cell penetrating peptide-conjugated silver nanoparticles with excellent selectivity for gram-positive bacteria over erythrocytes.

Science.gov (United States)

Liu, Lihong; Yang, Jun; Xie, Jianping; Luo, Zhentao; Jiang, Jiang; Yang, Yi Yan; Liu, Shaomin

2013-05-07

Silver nanoparticles are of great interest for use as antimicrobial agents. Studies aimed at producing potent nano-silver biocides have focused on manipulation of particle size, shape, composition and surface charge. Here, we report the cell penetrating peptide catalyzed formation of antimicrobial silver nanoparticles in N,N-dimethylformamide. The novel nano-composite demonstrated a distinctly enhanced biocidal effect toward bacteria (gram-positive Bacillus subtilis, gram-negative Escherichia coli) and pathogenic yeast (Candida albicans), as compared to triangular and extremely small silver nanoparticles. In addition, a satisfactory biocompatibility was verified by a haemolysis test. Our results provide a paradigm in developing strategies that can maximize the silver nanoparticle application potentials while minimizing the toxic effects.

RNases and Helicases in Gram-Positive Bacteria.

Science.gov (United States)

Durand, Sylvain; Condon, Ciaran

2018-04-01

RNases are key enzymes involved in RNA maturation and degradation. Although they play a crucial role in all domains of life, bacteria, archaea, and eukaryotes have evolved with their own sets of RNases and proteins modulating their activities. In bacteria, these enzymes allow modulation of gene expression to adapt to rapidly changing environments. Today, >20 RNases have been identified in both Escherichia coli and Bacillus subtilis , the paradigms of the Gram-negative and Gram-positive bacteria, respectively. However, only a handful of these enzymes are common to these two organisms and some of them are essential to only one. Moreover, although sets of RNases can be very similar in closely related bacteria such as the Firmicutes Staphylococcus aureus and B. subtilis , the relative importance of individual enzymes in posttranscriptional regulation in these organisms varies. In this review, we detail the role of the main RNases involved in RNA maturation and degradation in Gram-positive bacteria, with an emphasis on the roles of RNase J1, RNase III, and RNase Y. We also discuss how other proteins such as helicases can modulate the RNA-degradation activities of these enzymes.

Amino acids in cell wall of Gram-positive bacterium Micrococcus sp. hsn08 with flocculation activity on Chlorella vulgaris biomass.

Science.gov (United States)

Li, Yi; Xu, Yanting; Zheng, Tianling; Wang, Hailei

2018-02-01

The aim of this work was to investigate the flocculation mechanism by Gram-positive bacterium, Micrococcus sp. hsn08 as a means for harvesting Chlorella vulgaris biomass. Bacterial cells of Micrococcus sp. hsn08 were added into algal culture to harvest algal cells through direct contacting with algae to form flocs. Viability dependence test confirmed that flocculation activity does not depend on live bacteria, but on part of the peptidoglycan. The further investigation has determined that amino acids in cell wall play an important role to flocculate algal cells. Positively charged calcium can combine bacterial and algal cells together, and form a bridge between them, thereby forming the flocs, suggesting that ions bridging is the main flocculation mechanism. These results suggest that bacterial cells of Micrococcus sp. hsn08 can be applied to harvest microalgae biomass with the help of amino acids in cell wall. Copyright © 2017 Elsevier Ltd. All rights reserved.

Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

Science.gov (United States)

Fröhling, Antje; Schlüter, Oliver

2015-01-01

Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfill the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results. The aim of this study was to compare the inactivation effects of peracetic acid (PAA), ozonated water (O3), and cold atmospheric pressure plasma (CAPP) on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s) with 0.25% PAA at 10°C, and after treatment (10 s) with 3.8 mg l−1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l−1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process parameters. PMID

Flow cytometric evaluation of physico-chemical impact on Gram-positive and Gram-negative bacteria

Directory of Open Access Journals (Sweden)

Antje eFröhling

2015-09-01

Full Text Available Since heat sensitivity of fruits and vegetables limits the application of thermal inactivation processes, new emerging inactivation technologies have to be established to fulfil the requirements of food safety without affecting the produce quality. The efficiency of inactivation treatments has to be ensured and monitored. Monitoring of inactivation effects is commonly performed using traditional cultivation methods which have the disadvantage of the time span needed to obtain results.The aim of this study was to compare the inactivation effects of peracetic acid (PAA, ozonated water (O3 and cold atmospheric pressure plasma (CAPP on Gram-positive and Gram-negative bacteria using flow cytometric methods. E. coli cells were completely depolarized after treatment (15 s with 0.25 % PAA at 10 °C, and after treatment (10 s with 3.8 mg l-1 O3 at 12°C. The membrane potential of CAPP treated cells remained almost constant at an operating power of 20 W over a time period of 3 min, and subsequently decreased within 30 s of further treatment. Complete membrane permeabilization was observed after 10 s O3 treatment, but treatment with PAA and CAPP did not completely permeabilize the cells within 2 min and 4 min, respectively. Similar results were obtained for esterase activity. O3 inactivates cellular esterase but esterase activity was detected after 4 min CAPP treatment and 2 min PAA treatment. L. innocua cells and P. carotovorum cells were also permeabilized instantaneously by O3 treatment at concentrations of 3.8 ± 1 mg l-1. However, higher membrane permeabilization of L. innocua and P. carotovorum than of E. coli was observed at CAPP treatment of 20 W. The degree of bacterial damage due to the inactivation processes is highly dependent on treatment parameters as well as on treated bacteria. Important information regarding the inactivation mechanisms can be obtained by flow cytometric measurements and this enables the definition of critical process

Nucleotide sequence alignment of hdcA from Gram-positive bacteria.

Science.gov (United States)

Diaz, Maria; Ladero, Victor; Redruello, Begoña; Sanchez-Llana, Esther; Del Rio, Beatriz; Fernandez, Maria; Martin, Maria Cruz; Alvarez, Miguel A

2016-03-01

The decarboxylation of histidine -carried out mainly by some gram-positive bacteria- yields the toxic dietary biogenic amine histamine (Ladero et al. 2010 〈10.2174/157340110791233256〉 [1], Linares et al. 2016 〈http://dx.doi.org/10.1016/j.foodchem.2015.11.013〉〉 [2]). The reaction is catalyzed by a pyruvoyl-dependent histidine decarboxylase (Linares et al. 2011 〈10.1080/10408398.2011.582813〉 [3]), which is encoded by the gene hdcA. In order to locate conserved regions in the hdcA gene of Gram-positive bacteria, this article provides a nucleotide sequence alignment of all the hdcA sequences from Gram-positive bacteria present in databases. For further utility and discussion, see 〈http://dx.doi.org/ 10.1016/j.foodcont.2015.11.035〉〉 [4].

Revised mechanism of d-alanine incorporation into cell wall polymers in Gram-positive bacteria

Science.gov (United States)

Reichmann, Nathalie T.; Cassona, Carolina Picarra

2013-01-01

Teichoic acids (TAs) are important for growth, biofilm formation, adhesion and virulence of Gram-positive bacterial pathogens. The chemical structures of the TAs vary between bacteria, though they typically consist of zwitterionic polymers that are anchored to either the peptidoglycan layer as in the case of wall teichoic acid (WTA) or the cell membrane and named lipoteichoic acid (LTA). The polymers are modified with d-alanines and a lack of this decoration leads to increased susceptibility to cationic antimicrobial peptides. Four proteins, DltA–D, are essential for the incorporation of d-alanines into cell wall polymers and it has been established that DltA transfers d-alanines in the cytoplasm of the cell onto the carrier protein DltC. However, two conflicting models have been proposed for the remainder of the mechanism. Using a cellular protein localization and membrane topology analysis, we show here that DltC does not traverse the membrane and that DltD is anchored to the outside of the cell. These data are in agreement with the originally proposed model for d-alanine incorporation through a process that has been proposed to proceed via a d-alanine undecaprenyl phosphate membrane intermediate. Furthermore, we found that WTA isolated from a Staphylococcus aureus strain lacking LTA contains only a small amount of d-alanine, indicating that LTA has a role, either direct or indirect, in the efficient d-alanine incorporation into WTA in living cells. PMID:23858088

Cystic neutrophilic granulomatous mastitis: an underappreciated pattern strongly associated with gram-positive bacilli.

Science.gov (United States)

Renshaw, Andrew A; Derhagopian, Robert P; Gould, Edwin W

2011-09-01

Although granulomatous lobular mastitis is associated with gram-positive bacilli such as Corynebacterium, this association is not well known. We report 3 cases of mastitis caused by gram-positive bacilli. All 3 abscesses were suppurative with distinct enlarged cystic spaces in which rare gram-positive bacilli were identified. Two cases were also granulomatous. Cultures in all 3 cases were negative. All 3 patients recovered after biopsy and tetracycline-based therapy. Infection in the breast by gram-positive bacilli is associated with a distinct histologic pattern, including cystic spaces in the setting of neutrophilic/granulomatous inflammation that can be recognized and should prompt careful search for the organism within enlarged vacuoles.

Pleural effusion adenosine deaminase: a candidate biomarker to discriminate between Gram-negative and Gram-positive bacterial infections of the pleural space

Directory of Open Access Journals (Sweden)

Ruolin Li

2016-05-01

Full Text Available OBJECTIVES: Delay in the treatment of pleural infection may contribute to its high mortality. In this retrospective study, we aimed to evaluate the diagnostic accuracy of pleural adenosine deaminase in discrimination between Gram-negative and Gram-positive bacterial infections of the pleural space prior to selecting antibiotics. METHODS: A total of 76 patients were enrolled and grouped into subgroups according to Gram staining: 1 patients with Gram-negative bacterial infections, aged 53.2±18.6 years old, of whom 44.7% had empyemas and 2 patients with Gram-positive bacterial infections, aged 53.5±21.5 years old, of whom 63.1% had empyemas. The pleural effusion was sampled by thoracocentesis and then sent for adenosine deaminase testing, biochemical testing and microbiological culture. The Mann-Whitney U test was used to examine the differences in adenosine deaminase levels between the groups. Correlations between adenosine deaminase and specified variables were also quantified using Spearman’s correlation coefficient. Moreover, receiver operator characteristic analysis was performed to evaluate the diagnostic accuracy of pleural effusion adenosine deaminase. RESULTS: Mean pleural adenosine deaminase levels differed significantly between Gram-negative and Gram-positive bacterial infections of the pleural space (191.8±32.1 U/L vs 81.0±16.9 U/L, p<0.01. The area under the receiver operator characteristic curve was 0.689 (95% confidence interval: 0.570, 0.792, p<0.01 at the cutoff value of 86 U/L. Additionally, pleural adenosine deaminase had a sensitivity of 63.2% (46.0-78.2%; a specificity of 73.7% (56.9-86.6%; positive and negative likelihood ratios of 2.18 and 0.50, respectively; and positive and negative predictive values of 70.6% and 66.7%, respectively. CONCLUSIONS: Pleural effusion adenosine deaminase is a helpful alternative biomarker for early and quick discrimination of Gram-negative from Gram-positive bacterial infections of the

Antimicrobial activity of metal oxide nanoparticles against Gram-positive and Gram-negative bacteria: a comparative study

Directory of Open Access Journals (Sweden)

Azam A

2012-12-01

Full Text Available Ameer Azam,1,2 Arham S Ahmed,2 Mohammad Oves,3 Mohammad S Khan,3 Sami S Habib,1 Adnan Memic11Centre of Nanotechnology, King Abdulaziz University, Jeddah, Saudi Arabia; 2Centre of Excellence in Materials Science (Nanomaterials, 3Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, IndiaBackground: Nanomaterials have unique properties compared to their bulk counterparts. For this reason, nanotechnology has attracted a great deal of attention from the scientific community. Metal oxide nanomaterials like ZnO and CuO have been used industrially for several purposes, including cosmetics, paints, plastics, and textiles. A common feature that these nanoparticles exhibit is their antimicrobial behavior against pathogenic bacteria. In this report, we demonstrate the antimicrobial activity of ZnO, CuO, and Fe2O3 nanoparticles against Gram-positive and Gram-negative bacteria.Methods and results: Nanosized particles of three metal oxides (ZnO, CuO, and Fe2O3 were synthesized by a sol–gel combustion route and characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, and transmission electron microscopy techniques. X-ray diffraction results confirmed the single-phase formation of all three nanomaterials. The particle sizes were observed to be 18, 22, and 28 nm for ZnO, CuO, and Fe2O3, respectively. We used these nanomaterials to evaluate their antibacterial activity against both Gram-negative (Escherichia coli and Pseudomonas aeruginosa and Gram-positive (Staphylococcus aureus and Bacillus subtilis bacteria.Conclusion: Among the three metal oxide nanomaterials, ZnO showed greatest antimicrobial activity against both Gram-positive and Gram-negative bacteria used in this study. It was observed that ZnO nanoparticles have excellent bactericidal potential, while Fe2O3 nanoparticles exhibited the least bactericidal activity. The order of antibacterial activity was demonstrated to be the following: ZnO > CuO > Fe2O3

Inactivation dynamics of 222 nm krypton-chlorine excilamp irradiation on Gram-positive and Gram-negative foodborne pathogenic bacteria.

Science.gov (United States)

Kang, Jun-Won; Kim, Sang-Soon; Kang, Dong-Hyun

2018-07-01

The object of this study was to elucidate the bactericidal mechanism of a 222 nm Krypton Chlorine (KrCl) excilamp compared with that of a 254 nm Low Pressure mercury (LP Hg) lamp. The KrCl excilamp had higher bactericidal capacity against Gram-positive pathogenic bacteria (Staphylococcus aureus and L. monocytogenes) and Gram-negative pathogenic bacteria (S. Typhimurium and E. coli O157:H7) than did the LP Hg lamp when cell suspensions in PBS were irradiated with each type of UV lamp. It was found out that the KrCl excilamp induced cell membrane damage as a form of depolarization. From the study of respiratory chain dehydrogenase activity and the lipid peroxidation assay, it was revealed that cell membrane damage was attributed to inactivation of enzymes related to generation of membrane potential and occurrence of lipid peroxidation. Direct absorption of UV radiation which led to photoreaction through formation of an excited state was one of the causes inducing cell damage. Additionally, generation of ROS and thus occurrence of secondary damage can be another cause. The LP Hg lamp only induced damage to DNA but not to other components such as lipids or proteins. This difference was derived from differences of UV radiation absorption by cellular materials. Copyright © 2018 Elsevier Ltd. All rights reserved.

Daptomycin treatment in Gram-positive vascular graft infections

Directory of Open Access Journals (Sweden)

Francisco Arnaiz de las Revillas

2018-03-01

Full Text Available Background: Daptomycin is a bactericidal antibiotic approved for the treatment of skin and soft tissue infections and right-side endocarditis. However, there is a lack of published data outlining its usefulness in vascular graft infections (VGI. The aim of this study was to describe the clinical experience of daptomycin use in the treatment of VGI caused by Gram-positive bacteria. Methods: This was a retrospective cohort study of patients diagnosed with VGI receiving daptomycin at a tertiary care hospital during the period January 2010 to December 2012. Results: Of a total 1066 consecutive patients who had undergone vascular grafts (VG, 25 were diagnosed with VGI. Fifteen of these patients (11 prosthetic VG, three autologous VG, one both types received daptomycin (median dose 6.7 mg/kg/day, range 4.1–7.1 mg/kg/day; median age 69 years, range 45–83 years; 80% male. The infected bypass was removed in 13 cases. The most common reason for selecting daptomycin was kidney failure (53%. The Gram-positive organisms isolated were coagulase-negative Staphylococcus (n = 10, Staphylococcus aureus (n = 3 (two methicillin-resistant S. aureus, Enterococcus faecium (n = 2, and Enterococcus faecalis (n = 1. The mean follow-up was 69 months (interquartile range 48–72 months. Ten patients (66.7% achieved complete healing of the VGI. A recurrence of the infection was observed in 100% of patients in whom the bypass was not removed. Among patients who did not achieve complete healing, one needed a supracondylar amputation and one died as a consequence of infection. Five patients received treatment with rifampicin in addition to daptomycin and they were all cured. Conclusions: The use of daptomycin and surgery for Gram-positive VGI was effective and well tolerated, and this may be a good alternative for the treatment of VGI in patients with peripheral arterial disease in whom renal insufficiency is common. Keywords: Daptomycin, Gram-positive, Vascular

Clinical update on linezolid in the treatment of Gram-positive bacterial infections

Science.gov (United States)

Ager, Sally; Gould, Kate

2012-01-01

Gram-positive pathogens are a significant cause of morbidity and mortality in both community and health care settings. Glycopeptides have traditionally been the antibiotics of choice for multiresistant Gram-positive pathogens but there are problems with their use, including the emergence of glycopeptide-resistant strains, tissue penetration, and achieving and monitoring adequate serum levels. Newer antibiotics such as linezolid, a synthetic oxazolidinone, are available for the treatment of resistant Gram-positive bacteria. Linezolid is active against a wide range of Gram-positive bacteria and has been generally available for the treatment of Gram-positive infections since 2000. There are potential problems with linezolid use, including its bacteriostatic action and the relatively high incidence of reported adverse effects, particularly with long-term use. Long-term use may also be complicated by the development of resistance. However, linezolid has been shown to be clinically useful in the treatment of several serious infections where traditionally bacteriocidal agents have been required and many of its adverse effects are reversible on cessation. It has also been shown to be a cost-effective treatment option in several studies, with its high oral bioavailability allowing an early change from intravenous to oral formulations with consequent earlier patient discharge and lower inpatient costs. PMID:22787406

DMPD: Cellular reprogramming by gram-positive bacterial components: a review. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 16885502 Cellular reprogramming by gram-positive bacterial components: a review. Bu...(.csml) Show Cellular reprogramming by gram-positive bacterial components: a review. PubmedID 16885502 Title... Cellular reprogramming by gram-positive bacterial components: a review. Authors

Polymers for binding of the gram-positive oral pathogen Streptococcus mutans

Science.gov (United States)

Magennis, Eugene P.; Francini, Nora; Mastrotto, Francesca; Catania, Rosa; Redhead, Martin; Fernandez-Trillo, Francisco; Bradshaw, David; Churchley, David; Winzer, Klaus; Alexander, Cameron

2017-01-01

Streptococcus mutans is the most significant pathogenic bacterium implicated in the formation of dental caries and, both directly and indirectly, has been associated with severe conditions such as multiple sclerosis, cerebrovascular and peripheral artery disease. Polymers able to selectively bind S. mutans and/or inhibit its adhesion to oral tissue in a non-lethal manner would offer possibilities for addressing pathogenicity without selecting for populations resistant against bactericidal agents. In the present work two libraries of 2-(dimethylamino)ethyl methacrylate (pDMAEMA)-based polymers were synthesized with various proportions of either N,N,N-trimethylethanaminium cationic- or sulfobetaine zwitterionic groups. These copolymers where initially tested as potential macromolecular ligands for S. mutans NCTC 10449, whilst Escherichia coli MG1655 was used as Gram-negative control bacteria. pDMAEMA-derived materials with high proportions of zwitterionic repeating units were found to be selective for S. mutans, in both isolated and S. mutans–E. coli mixed bacterial cultures. Fully sulfobetainized pDMAEMA was subsequently found to bind/cluster preferentially Gram-positive S. mutans and S. aureus compared to Gram negative E. coli and V. harveyi. A key initial stage of S. mutans pathogenesis involves a lectin-mediated adhesion to the tooth surface, thus the range of potential macromolecular ligands was further expanded by investigating two glycopolymers bearing α-mannopyranoside and β-galactopyranoside pendant units. Results with these polymers indicated that preferential binding to either S. mutans or E. coli can be obtained by modulating the glycosylation pattern of the chosen multivalent ligands without incurring unacceptable cytotoxicity in a model gastrointestinal cell line. Overall, our results allowed to identify a structure–property relationship for the potential antimicrobial polymers investigated, and suggest that preferential binding to Gram-positive S

The rapid isolation of mutants of some Gram-positive bacteria

International Nuclear Information System (INIS)

Dijkhuizen, L.; Keijer, L.

1981-01-01

In this communication the authors describe a method for isolating at high frequency independent mutants of a number of Gram-positive bacteria. The method was originally developed for use with an Arthrobacter sp. and appears to work best with this and other coryneform bacteria. All the bacteria used were from the culture collections maintained at the University of Warwick or the Centre for Applied Microbiological Research. For mutagenesis using UV light, cells were grown in complex media and used while still in the logarithmic phase of growth. Details of the irradiation procedure are given in the text. (Auth.)

Simultaneous fluorescent gram staining and activity assessment of activated sludge bacteria.

Science.gov (United States)

Forster, Scott; Snape, Jason R; Lappin-Scott, Hilary M; Porter, Jonathan

2002-10-01

Wastewater treatment is one of the most important commercial biotechnological processes, and yet the component bacterial populations and their associated metabolic activities are poorly understood. The novel fluorescent dye hexidium iodide allows assessment of Gram status by differential absorption through bacterial cell walls. Differentiation between gram-positive and gram-negative wastewater bacteria was achieved after flow cytometric analysis. This study shows that the relative proportions of gram-positive and gram-negative bacterial cells identified by traditional microscopy and hexidium iodide staining were not significantly different. Dual staining of cells for Gram status and activity proved effective in analyzing mixtures of cultured bacteria and wastewater populations. Levels of highly active organisms at two wastewater treatment plants, both gram positive and gram negative, ranged from 1.5% in activated sludge flocs to 16% in the activated sludge fluid. Gram-positive organisms comprised Gram status and activity within activated sludge samples over a 4-day period showed significant differences over time. This method provides a rapid, quantitative measure of Gram status linked with in situ activity within wastewater systems.

Extracellular Electron Transfer Mediated by Flavins in Gram-positive Bacillus sp. WS-XY1 and Yeast Pichia stipitis

International Nuclear Information System (INIS)

Wu, Song; Xiao, Yong; Wang, Lu; Zheng, Yue; Chang, Kenlin; Zheng, Zhiyong; Yang, Zhaohui; Varcoe, John R.; Zhao, Feng

2014-01-01

Extracellular electron transfer (EET) of microorganisms represents a communicative bridge between the interior and exterior of the cells. Most prior EET studies have focused on Gram-negative bacteria. However, fungi and Gram-positive bacteria, that contain dense cellular walls, have rarely been reported. Herein, two model dense cell wall microorganisms (Bacillus sp. WS-XY1 and the yeast Pichia stipitis) were identified to be electrochemically active. Further analysis indicated that the two microorganisms were able to secrete flavins to mediate their EET. The discovery, that dense cell wall containing microorganisms can undertake mediated EET, adds to the body of knowledge towards building a comprehensive understanding of biogeochemical and bioelectrical processes

Detection and Antibiotic Susceptibility Pattern of Biofilm Producing Gram Positive and Gram Negative Bacteria Isolated From a Tertiary Care Hospital of Pakistan

Directory of Open Access Journals (Sweden)

Iqbal, M.

2011-01-01

Full Text Available Microorganisms adhere to non-living material or living tissue, and form biofilms made up of extracellular polymers/slime. Biofilm-associated microorganisms behave differently from free-floating bacteria with respect to growth rates and ability to resist antimicrobial treatments and therefore pose a public health problem. The objective of this study is to detect the prevalence of biofilm producers among Gram positive and Gram negative bacteria isolated from clinical specimens, and to study their antimicrobial susceptibility pattern. The study was carried out from October 2009 to March 2010, at the Department of Microbiology, Army Medical College/ National University of Sciences and Technology (NUST, Rawalpindi, Pakistan. Clinical specimens were received from various wards of a tertiary care hospital. These were dealt by standard microbiological procedures. Gram positive and Gram negative bacteria isolated were subjected to biofilm detection by congo red agar method (CRA. Antimicrobial susceptibility testing of those isolates, which showed positive results (slime production, was done according to the Kirby-Bauer disc diffusion technique. A total of 150 isolates were tested for the production of biofilm/slime. Among them, 81 isolates showed positive results. From these 81, 51 were Gram positive and 30 were Gram negative. All the 81(54% slime producers showed reduced susceptibility to majority of antibiotics. Bacterial biofilms are an important virulence factor associated with chronic nosocomial infection. Detection of biofilm forming organisms can help in appropriate antibiotic choice.

A classification system for plasmids from Enterococci and other Gram-positive bacteria

DEFF Research Database (Denmark)

Jensen, Lars Bogø; Garcia-Migura, Lourdes; Valenzuela, Antonio Jesus Sanchez

2010-01-01

A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiating....... Furthermore, conjugation experiments were performed obtaining 30 transconjugants when selecting for antimicrobial resistance. Among them 19 gave no positive amplicons indicating presence of rep-families not tested for in this experimental setup....

Mechanism of action of recombinant acc-royalisin from royal jelly of Asian honeybee against gram-positive bacteria.

Directory of Open Access Journals (Sweden)

Lirong Shen

Full Text Available The antibacterial activity of royalisin, an antimicrobial peptide from the royal jelly produced by honeybees, has been addressed extensively. However, its mechanism of action remains unclear. In this study, a recombinant royalisin, RAcc-royalisin from the royal jelly of Asian honeybee Apis cerana cerana, was expressed by fusing with glutathione S-transferase (GST in Escherichia coli BL21, isolated and purified. The agar dilution assays with inhibition zone showed that RAcc-royalisin, similar to nisin, inhibits the growth of Gram-positive bacteria. The antibacterial activity of RAcc-royalisin was associated with its concentration, and was weakened by heat treatment ranging from 55°C to 85°C for 15 min. Both RAcc-royalisin and nisin exhibited the minimum inhibitory concentrations (MIC of 62.5 µg/ml, 125 µg/ml, and 250 µg/ml against Gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. However, RAcc-royalisin did not show antimicrobial activity against tested Gram-negative bacterial and fungal strains. The antibacterial activity of RAcc-royalisin agrees well with the decrease in bacterial cell hydrophobicity, the leakage of 260-nm absorbing materials, and the observation by transmission electron microscopy, all indicating that RAcc-royalisin induced the disruption and dysfunction of cell walls and membranes. This is the first report detailing the antibacterial mechanism of royalisin against Gram-positive bacteria, and provides insight into the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent.

Neither Single nor a Combination of Routine Laboratory Parameters can Discriminate between Gram-positive and Gram-negative Bacteremia

Science.gov (United States)

Ratzinger, Franz; Dedeyan, Michel; Rammerstorfer, Matthias; Perkmann, Thomas; Burgmann, Heinz; Makristathis, Athanasios; Dorffner, Georg; Loetsch, Felix; Blacky, Alexander; Ramharter, Michael

2015-01-01

Adequate early empiric antibiotic therapy is pivotal for the outcome of patients with bloodstream infections. In clinical practice the use of surrogate laboratory parameters is frequently proposed to predict underlying bacterial pathogens; however there is no clear evidence for this assumption. In this study, we investigated the discriminatory capacity of predictive models consisting of routinely available laboratory parameters to predict the presence of Gram-positive or Gram-negative bacteremia. Major machine learning algorithms were screened for their capacity to maximize the area under the receiver operating characteristic curve (ROC-AUC) for discriminating between Gram-positive and Gram-negative cases. Data from 23,765 patients with clinically suspected bacteremia were screened and 1,180 bacteremic patients were included in the study. A relative predominance of Gram-negative bacteremia (54.0%), which was more pronounced in females (59.1%), was observed. The final model achieved 0.675 ROC-AUC resulting in 44.57% sensitivity and 79.75% specificity. Various parameters presented a significant difference between both genders. In gender-specific models, the discriminatory potency was slightly improved. The results of this study do not support the use of surrogate laboratory parameters for predicting classes of causative pathogens. In this patient cohort, gender-specific differences in various laboratory parameters were observed, indicating differences in the host response between genders. PMID:26522966

Miniaturized bead-beating device to automate full DNA sample preparation processes for gram-positive bacteria.

Science.gov (United States)

Hwang, Kyu-Youn; Kwon, Sung Hong; Jung, Sun-Ok; Lim, Hee-Kyun; Jung, Won-Jong; Park, Chin-Sung; Kim, Joon-Ho; Suh, Kahp-Yang; Huh, Nam

2011-11-07

We have developed a miniaturized bead-beating device to automate nucleic acids extraction from Gram-positive bacteria for molecular diagnostics. The microfluidic device was fabricated by sandwiching a monolithic flexible polydimethylsiloxane (PDMS) membrane between two glass wafers (i.e., glass-PDMS-glass), which acted as an actuator for bead collision via its pneumatic vibration without additional lysis equipment. The Gram-positive bacteria, S. aureus and methicillin-resistant S. aureus, were captured on surface-modified glass beads from 1 mL of initial sample solution and in situ lyzed by bead-beating operation. Then, 10 μL or 20 μL of bacterial DNA solution was eluted and amplified successfully by real-time PCR. It was found that liquid volume fraction played a crucial role in determining the cell lysis efficiency in a confined chamber by facilitating membrane deflection and bead motion. The miniaturized bead-beating operation disrupted most of S. aureus within 3 min, which turned out to be as efficient as the conventional benchtop vortexing machine or the enzyme-based lysis technique. The effective cell concentration was significantly enhanced with the reduction of initial sample volume by 50 or 100 times. Combination of such analyte enrichment and in situ bead-beating lysis provided an excellent PCR detection sensitivity amounting to ca. 46 CFU even for the Gram-positive bacteria. The proposed bead-beating microdevice is potentially useful as a nucleic acid extraction method toward a PCR-based sample-to-answer system. This journal is © The Royal Society of Chemistry 2011

Diversity of pigmented Gram-positive bacteria associated with marine macroalgae from Antarctica.

Science.gov (United States)

Leiva, Sergio; Alvarado, Pamela; Huang, Ying; Wang, Jian; Garrido, Ignacio

2015-12-01

Little is known about the diversity and roles of Gram-positive and pigmented bacteria in Antarctic environments, especially those associated with marine macroorganisms. This work is the first study about the diversity and antimicrobial activity of culturable pigmented Gram-positive bacteria associated with marine Antarctic macroalgae. A total of 31 pigmented Gram-positive strains were isolated from the surface of six species of macroalgae collected in the King George Island, South Shetland Islands. On the basis of 16S rRNA gene sequence similarities ≥99%, 18 phylotypes were defined, which were clustered into 11 genera of Actinobacteria (Agrococcus, Arthrobacter, Brachybacterium, Citricoccus, Kocuria, Labedella, Microbacterium, Micrococcus, Rhodococcus, Salinibacterium and Sanguibacter) and one genus of the Firmicutes (Staphylococcus). It was found that five isolates displayed antimicrobial activity against a set of macroalgae-associated bacteria. The active isolates were phylogenetically related to Agrococcus baldri, Brachybacterium rhamnosum, Citricoccus zhacaiensis and Kocuria palustris. The results indicate that a diverse community of pigmented Gram-positive bacteria is associated with Antartic macroalgae and suggest its potential as a promising source of antimicrobial and pigmented natural compounds. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Isolation of highly active monoclonal antibodies against multiresistant gram-positive bacteria.

Directory of Open Access Journals (Sweden)

Friederike S Rossmann

Full Text Available Multiresistant nosocomial pathogens often cause life-threatening infections that are sometimes untreatable with currently available antibiotics. Staphylococci and enterococci are the predominant Gram-positive species associated with hospital-acquired infections. These infections often lead to extended hospital stay and excess mortality. In this study, a panel of fully human monoclonal antibodies was isolated from a healthy individual by selection of B-cells producing antibodies with high opsonic killing against E. faecalis 12030. Variable domains (VH and VL of these immunoglobulin genes were amplified by PCR and cloned into an eukaryotic expression vector containing the constant domains of a human IgG1 molecule and the human lambda constant domain. These constructs were transfected into CHO cells and culture supernatants were collected and tested by opsonophagocytic assay against E. faecalis and S. aureus strains (including MRSA. At concentrations of 600 pg/ml, opsonic killing was between 40% and 70% against all strains tested. Monoclonal antibodies were also evaluated in a mouse sepsis model (using S. aureus LAC and E. faecium, a mouse peritonitis model (using S. aureus Newman and LAC and a rat endocarditis model (using E. faecalis 12030 and were shown to provide protection in all models at a concentration of 4 μg/kg per animal. Here we present a method to produce fully human IgG1 monoclonal antibodies that are opsonic in vitro and protective in vivo against several multiresistant Gram-positive bacteria. The monoclonal antibodies presented in this study are significantly more effective compared to another monoclonal antibody currently in clinical trials.

Gram-Positive Uropathogens, Polymicrobial Urinary Tract Infection, and the Emerging Microbiota of the Urinary Tract.

Science.gov (United States)

Kline, Kimberly A; Lewis, Amanda L

2016-04-01

Gram-positive bacteria are a common cause of urinary-tract infection (UTI), particularly among individuals who are elderly, pregnant, or who have other risk factors for UTI. Here we review the epidemiology, virulence mechanisms, and host response to the most frequently isolated Gram-positive uropathogens: Staphylococcus saprophyticus, Enterococcus faecalis, and Streptococcus agalactiae. We also review several emerging, rare, misclassified, and otherwise underreported Gram-positive pathogens of the urinary tract including Aerococcus, Corynebacterium, Actinobaculum, and Gardnerella. The literature strongly suggests that urologic diseases involving Gram-positive bacteria may be easily overlooked due to limited culture-based assays typically utilized for urine in hospital microbiology laboratories. Some UTIs are polymicrobial in nature, often involving one or more Gram-positive bacteria. We herein review the risk factors and recent evidence for mechanisms of bacterial synergy in experimental models of polymicrobial UTI. Recent experimental data has demonstrated that, despite being cleared quickly from the bladder, some Gram-positive bacteria can impact pathogenic outcomes of co-infecting organisms. When taken together, the available evidence argues that Gram-positive bacteria are important uropathogens in their own right, but that some can be easily overlooked because they are missed by routine diagnostic methods. Finally, a growing body of evidence demonstrates that a surprising variety of fastidious Gram-positive bacteria may either reside in or be regularly exposed to the urinary tract and further suggests that their presence is widespread among women, as well as men. Experimental studies in this area are needed; however, there is a growing appreciation that the composition of bacteria found in the bladder could be a potentially important determinant in urologic disease, including susceptibility to UTI.

Gram-negative and -positive bacteria differentiation in blood culture samples by headspace volatile compound analysis.

Science.gov (United States)

Dolch, Michael E; Janitza, Silke; Boulesteix, Anne-Laure; Graßmann-Lichtenauer, Carola; Praun, Siegfried; Denzer, Wolfgang; Schelling, Gustav; Schubert, Sören

2016-12-01

Identification of microorganisms in positive blood cultures still relies on standard techniques such as Gram staining followed by culturing with definite microorganism identification. Alternatively, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or the analysis of headspace volatile compound (VC) composition produced by cultures can help to differentiate between microorganisms under experimental conditions. This study assessed the efficacy of volatile compound based microorganism differentiation into Gram-negatives and -positives in unselected positive blood culture samples from patients. Headspace gas samples of positive blood culture samples were transferred to sterilized, sealed, and evacuated 20 ml glass vials and stored at -30 °C until batch analysis. Headspace gas VC content analysis was carried out via an auto sampler connected to an ion-molecule reaction mass spectrometer (IMR-MS). Measurements covered a mass range from 16 to 135 u including CO2, H2, N2, and O2. Prediction rules for microorganism identification based on VC composition were derived using a training data set and evaluated using a validation data set within a random split validation procedure. One-hundred-fifty-two aerobic samples growing 27 Gram-negatives, 106 Gram-positives, and 19 fungi and 130 anaerobic samples growing 37 Gram-negatives, 91 Gram-positives, and two fungi were analysed. In anaerobic samples, ten discriminators were identified by the random forest method allowing for bacteria differentiation into Gram-negative and -positive (error rate: 16.7 % in validation data set). For aerobic samples the error rate was not better than random. In anaerobic blood culture samples of patients IMR-MS based headspace VC composition analysis facilitates bacteria differentiation into Gram-negative and -positive.

Physico-Chemical-Managed Killing of Penicillin-Resistant Static and Growing Gram-Positive and Gram-Negative Vegetative Bacteria

Science.gov (United States)

Richmond, Robert Chaffee (Inventor); Schramm, Jr., Harry F. (Inventor); Defalco, Francis G. (Inventor); Farris, III, Alex F. (Inventor)

2012-01-01

Systems and methods for the use of compounds from the Hofmeister series coupled with specific pH and temperature to provide rapid physico-chemical-managed killing of penicillin-resistant static and growing Gram-positive and Gram-negative vegetative bacteria. The systems and methods represent the more general physico-chemical enhancement of susceptibility for a wide range of pathological macromolecular targets to clinical management by establishing the reactivity of those targets to topically applied drugs or anti-toxins.

Antimicrobial Activities of Leaf Extracts of Guava (Psidium guajava L.) on Two Gram-Negative and Gram-Positive Bacteria

Science.gov (United States)

Biswas, Bipul; Rogers, Kimberly; McLaughlin, Fredrick; Yadav, Anand

2013-01-01

Aim. To determine the antimicrobial potential of guava (Psidium guajava) leaf extracts against two gram-negative bacteria (Escherichia coli and Salmonella enteritidis) and two gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) which are some of foodborne and spoilage bacteria. The guava leaves were extracted in four different solvents of increasing polarities (hexane, methanol, ethanol, and water). The efficacy of these extracts was tested against those bacteria through a well-diffusion method employing 50 μL leaf-extract solution per well. According to the findings of the antibacterial assay, the methanol and ethanol extracts of the guava leaves showed inhibitory activity against gram-positive bacteria, whereas the gram-negative bacteria were resistant to all the solvent extracts. The methanol extract had an antibacterial activity with mean zones of inhibition of 8.27 and 12.3 mm, and the ethanol extract had a mean zone of inhibition of 6.11 and 11.0 mm against B. cereus and S. aureus, respectively. On the basis of the present finding, guava leaf-extract might be a good candidate in the search for a natural antimicrobial agent. This study provides scientific understanding to further determine the antimicrobial values and investigate other pharmacological properties. PMID:24223039

Antimicrobial Activities of Leaf Extracts of Guava (Psidium guajava L. on Two Gram-Negative and Gram-Positive Bacteria

Directory of Open Access Journals (Sweden)

Bipul Biswas

2013-01-01

Full Text Available Aim. To determine the antimicrobial potential of guava (Psidium guajava leaf extracts against two gram-negative bacteria (Escherichia coli and Salmonella enteritidis and two gram-positive bacteria (Staphylococcus aureus and Bacillus cereus which are some of foodborne and spoilage bacteria. The guava leaves were extracted in four different solvents of increasing polarities (hexane, methanol, ethanol, and water. The efficacy of these extracts was tested against those bacteria through a well-diffusion method employing 50 μL leaf-extract solution per well. According to the findings of the antibacterial assay, the methanol and ethanol extracts of the guava leaves showed inhibitory activity against gram-positive bacteria, whereas the gram-negative bacteria were resistant to all the solvent extracts. The methanol extract had an antibacterial activity with mean zones of inhibition of 8.27 and 12.3 mm, and the ethanol extract had a mean zone of inhibition of 6.11 and 11.0 mm against B. cereus and S. aureus, respectively. On the basis of the present finding, guava leaf-extract might be a good candidate in the search for a natural antimicrobial agent. This study provides scientific understanding to further determine the antimicrobial values and investigate other pharmacological properties.

Gram-negative diabetic foot osteomyelitis: risk factors and clinical presentation.

Science.gov (United States)

Aragón-Sánchez, Javier; Lipsky, Benjamin A; Lázaro-Martínez, Jose L

2013-03-01

Osteomyelitis frequently complicates infections in the feet of patients with diabetes. Gram-positive cocci, especially Staphylococcus aureus, are the most commonly isolated pathogens, but gram-negative bacteria also cause some cases of diabetic foot osteomyelitis (DFO). These gram-negatives require different antibiotic regimens than those commonly directed at gram-positives. There are, however, few data on factors related to their presence and how they influence the clinical picture. We conducted a retrospective study to determine the variables associated with the isolation of gram-negative bacteria from bone samples in cases of DFO and the clinical presentation of these infections. Among 341 cases of DFO, 150 had a gram-negative isolate (alone or combined with a gram-positive isolate) comprising 44.0% of all patients and 50.8% of those with a positive bone culture. Compared with gram-positive infections, wounds with gram-negative organisms more often had a fetid odor, necrotic tissue, signs of soft tissue infection accompanying osteomyelitis, and clinically severe infection. By multivariate analysis, the predictive variables related to an increased likelihood of isolating gram-negatives from bone samples were glycated hemoglobin gram-negatives had a statistically significantly higher prevalence of leukocytosis and higher white blood cell counts than those without gram-negatives. In conclusion, gram-negative organisms were isolated in nearly half of our cases of DFO and were associated with more severe infections, higher white blood cell counts, lower glycated hemoglobin levels, and wounds of traumatic etiology.

Nicotinoprotein methanol dehydrogenase enzymes in Gram-positive methylotrophic bacteria

NARCIS (Netherlands)

Hektor, Harm J.; Kloosterman, Harm; Dijkhuizen, Lubbert

2000-01-01

A novel type of alcohol dehydrogenase enzyme has been characterized from Gram-positive methylotrophic (Bacillus methanolicus, the actinomycetes Amycolatopsis methanolica and Mycobacterium gastri) and non-methylotrophic bacteria (Rhodococcus strains). Its in vivo role is in oxidation of methanol and

Antimicrobial effects of zero-valent iron nanoparticles on gram-positive Bacillus strains and gram-negative Escherichia coli strains

Directory of Open Access Journals (Sweden)

Yi-Huang Hsueh

2017-11-01

Full Text Available Abstract Background Zero-valent iron nanoparticles (ZVI NPs have been used extensively for the remediation of contaminated soil and groundwater. Owing to their large active surface area, they serve as strong and effective reductants. However, the ecotoxicity and bioavailability of ZVI NPs in diverse ecological media have not been evaluated in detail and most studies have focused on non-nano ZVI or Fe0. In addition, the antimicrobial properties of ZVI NPs have rarely been investigated, and the underlying mechanism of their toxicity remains unknown. Results In the present study, we demonstrate that ZVI NPs exhibited significant toxicity at 1000 ppm against two distinct gram-positive bacterial strains (Bacillus subtilis 3610 and Bacillus thuringiensis 407 but not against two gram-negative strains (Escherichia coli K12 and ATCC11634. Specifically, ZVI NPs caused at least a 4-log and 1-log reductions in cell numbers, respectively, in the two Bacillus strains, whereas no change was detected in the two E. coli strains. X-ray photoelectron spectroscopy, X-ray absorption near-edge, and extended X-ray absorption fine structure spectra confirmed that Bacillus cells exposed to ZVI NPs contained mostly Fe2O3 with some detectable FeS. This finding indicated that Fe0 nanoparticles penetrated the bacterial cells, where they were subsequently oxidized to Fe2O3 and FeS. RedoxSensor analysis and propidium iodide (PI staining showed decreased reductase activity and increased PI in both Bacillus strains treated with a high (1000 ppm concentration of ZVI NPs. Conclusion Taken together, these data show that the toxicity of ZVI NPs was derived from their oxidative properties, which may increase the levels of reactive oxygen species and lead to cell death.

In vitro activity of daptomycin against clinical isolates of Gram-positive bacteria.

Science.gov (United States)

Piper, Kerryl E; Steckelberg, James M; Patel, Robin

2005-08-01

We determined the activity of daptomycin, a recently FDA-approved antimicrobial agent, against clinical isolates of Gram-positive bacteria, including viridans group streptococci (16 Streptococcus mitis species group, 12 S. mutans species group, 9 S. anginosus species group, 8 S. sanguinis species group, 5 S. salivarius species group) from patients with infective endocarditis, 32 methicillin-resistant Staphylococcus aureus, 32 high-level penicillin-resistant Streptococcus pneumoniae, 38 vancomycin-resistant enterococci (including 1 linezolid-resistant isolate), and the following unusual Gram-positive bacteria: 3 Listeria monocytogenes, 4 Erysipelothrix rhusiopathiae, 9 Corynebacterium species, 10 Abiotrophia/Granulicatella species, 2 Rothia (Stomatococcus) mucilaginosus, and 4 Gemella morbillorum. Daptomycin minimum inhibitory concentration (MIC)(90) values for the viridans group streptococci, methicillin-resistant S. aureus, penicillin-resistant S. pneumoniae, and Enterococcus species were 0.5, 0.5, endocarditis as well as against several types of unusual Gram-positive bacteria that can cause endocarditis.

Diesel degradation and biosurfactant production by Gram-positive ...

African Journals Online (AJOL)

The ability of Gram-positive bacteria to degrade diesel increased in a comparable trend as its biosurfactant production increased. The E24 index was highest at 87.6% for isolate D9. Isolates D2, D9 and D10, were identified as Paenibacillus sp. whilst isolate DJLB was found to belong to Stenotrophomonas sp. This study ...

Endocarditis : Effects of routine echocardiography during Gram-positive bacteraemia

NARCIS (Netherlands)

Vos, F J; Bleeker-Rovers, C P; Sturm, P D; Krabbe, P F M; van Dijk, A P J; Oyen, W J G; Kullberg, B J

2011-01-01

BACKGROUND: Despite firm recommendations to perform echocardiography in high-risk patients with Gram-positive bacteraemia, routine echocardiography is not embedded in daily practice in many settings. The aim of this study was to evaluate whether a regime including routine echocardiography results in

Linezolid: a promising option in the treatment of Gram-positives.

Science.gov (United States)

Zahedi Bialvaei, Abed; Rahbar, Mohammad; Yousefi, Mehdi; Asgharzadeh, Mohammad; Samadi Kafil, Hossein

2017-02-01

Linezolid, an oxazolidinone antimicrobial agent that acts by inhibiting protein synthesis in a unique fashion, is used in the treatment of community-acquired pneumonia, skin and soft-tissue infections and other infections caused by Gram-positive bacteria including VRE and methicillin-resistant staphylococci. Currently, linezolid resistance among these pathogens remains low, commonly linezolid susceptibility testing for this important agent and should be taken into account when considering its therapeutic use. Considering the importance of linezolid in the treatment of infections caused by Gram-positive bacteria, this review was undertaken to optimize the clinical use of this antibiotic. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Fighting infections due to multidrug-resistant Gram-positive pathogens.

Science.gov (United States)

Cornaglia, G

2009-03-01

Growing bacterial resistance in Gram-positive pathogens means that what were once effective and inexpensive treatments for infections caused by these bacteria are now being seriously questioned, including penicillin and macrolides for use against pneumococcal infections and-in hospitals-oxacillin for use against staphylococcal infections. As a whole, multidrug-resistant (MDR) Gram-positive pathogens are rapidly becoming an urgent and sometimes unmanageable clinical problem. Nevertheless, and despite decades of research into the effects of antibiotics, the actual risk posed to human health by antibiotic resistance has been poorly defined; the lack of reliable data concerning the outcomes resulting from antimicrobial resistance stems, in part, from problems with study designs and the methods used in resistence determination. Surprisingly little is known, too, about the actual effectiveness of the many types of intervention aimed at controlling antibiotic resistance. New antibiotics active against MDR Gram-positive pathogens have been recently introduced into clinical practice, and the antibiotic pipeline contains additional compounds at an advanced stage of development, including new glycopeptides, new anti-methicillin-resistant Staphylococcus aureus (MRSA) beta-lactams, and new diaminopyrimidines. Many novel antimicrobial agents are likely to be niche products, endowed with narrow antibacterial spectra and/or targeted at specific clinical problems. Therefore, an important educational goal will be to change the current, long-lasting attitudes of both physicians and customers towards broad-spectrum and multipurpose compounds. Scientific societies, such as the European Society of Clinical Microbiology and Infectious Diseases (ESCMID), must play a leading role in this process.

Gram-positive bacteria as an antigen topically applied into gingival sulcus of immunized rat accelerates periodontal destruction.

Science.gov (United States)

Nagano, F; Kaneko, T; Yoshinaga, Y; Ukai, T; Kuramoto, A; Nakatsu, S; Oshino, K; Ichimura, I; Hara, Y

2013-08-01

Periodontitis is generally accepted to relate to gram-negative bacteria, and the host defense system influences its onset and progression. However, little is known about the relation between gram-positive bacteria and periodontitis. In this study, we topically applied gram-positive and gram-negative bacterial suspensions to the gingival sulcus in rats after immunization, and then histopathologically examined their influence on periodontal destruction. Rats previously immunized with heat-treated and sonicated Staphylococcus aureus or Aggregatibacter actinomycetemcomitans were used as immunized groups. The non-immunized group received only sterile phosphate-buffered saline. In each animal, S. aureus or A. actinomycetemcomitans suspension was applied topically to the palatal gingival sulcus of first molars every 24 h for 10 d. Blood samples were collected and the serum level of anti-S. aureus or anti-A. actinomycetemcomitans immunoglobulin G (IgG) antibodies was determined by enzyme-linked immunosorbent assay. The first molar regions were resected and observed histopathologically. Osteoclasts were stained with tartrate-resistant acid phosphatase (TRAP). The formation of immune complexes was confirmed by immunohistological staining of C1qB. Serum levels of anti-S. aureus and anti-A. actinomycetemcomitans IgG antibodies in the immunized groups were significantly higher than those in the non-immunized groups were. The loss of attachment, increase in apical migration of the junctional epithelium, and decreases in alveolar bone level and number of TRAP-positive multinuclear cells in each immunized group were significantly greater than in each non-immunized group. The presence of C1qB was observed in the junctional epithelium and adjacent connective tissue in the immunized groups. Heat-treated and sonicated S. aureus and A. actinomycetemcomitans induced attachment loss in rats immunized with their suspensions. Our results suggest that not only gram-negative but also gram-positive

A pathway closely related to the (D)-tagatose pathway of gram-negative enterobacteria identified in the gram-positive bacterium Bacillus licheniformis.

Science.gov (United States)

Van der Heiden, Edwige; Delmarcelle, Michaël; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M; Galleni, Moreno; Joris, Bernard

2013-06-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus.

A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

OpenAIRE

Van der Heiden, Edwige; Delmarcelle, Michaël; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M.; Galleni, Moreno; Joris, Bernard

2013-01-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus.

A Pathway Closely Related to the d-Tagatose Pathway of Gram-Negative Enterobacteria Identified in the Gram-Positive Bacterium Bacillus licheniformis

Science.gov (United States)

Van der Heiden, Edwige; Lebrun, Sarah; Freichels, Régine; Brans, Alain; Vastenavond, Christian M.; Galleni, Moreno; Joris, Bernard

2013-01-01

We report the first identification of a gene cluster involved in d-tagatose catabolism in Bacillus licheniformis. The pathway is closely related to the d-tagatose pathway of the Gram-negative bacterium Klebsiella oxytoca, in contrast to the d-tagatose 6-phosphate pathway described in the Gram-positive bacterium Staphylococcus aureus. PMID:23524682

Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate.

Science.gov (United States)

Barnini, Simona; Ghelardi, Emilia; Brucculeri, Veronica; Morici, Paola; Lupetti, Antonella

2015-06-18

Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media. Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0). The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits.

A flow-cytometric gram-staining technique for milk-associated bacteria.

Science.gov (United States)

Holm, Claus; Jespersen, Lene

2003-05-01

A Gram-staining technique combining staining with two fluorescent stains, Oregon Green-conjugated wheat germ agglutinin (WGA) and hexidium iodide (HI) followed by flow-cytometric detection is described. WGA stains gram-positive bacteria while HI binds to the DNA of all bacteria after permeabilization by EDTA and incubation at 50 degrees C for 15 min. For WGA to bind to gram-positive bacteria, a 3 M potassium chloride solution was found to give the highest fluorescence intensity. A total of 12 strains representing some of the predominant bacterial species in bulk tank milk and mixtures of these were stained and analyzed by flow cytometry. Overall, the staining method showed a clear differentiation between gram-positive and gram-negative bacterial populations. For stationary-stage cultures of seven gram-positive bacteria and five gram-negative bacteria, an average of 99% of the cells were correctly interpreted. The method was only slightly influenced by the growth phase of the bacteria or conditions such as freezing at -18 degrees C for 24 h. For any of these conditions, an average of at least 95% of the cells were correctly interpreted. When stationary-stage cultures were stored at 5 degrees C for 14 days, an average of 86% of the cells were correctly interpreted. The Gram-staining technique was applied to the flow cytometry analysis of bulk tank milk inoculated with Staphylococcus aureus and Escherichia coli. These results demonstrate that the technique is suitable for analyzing milk samples without precultivation.

Thusin, a novel two-component lantibiotic with potent antimicrobial activity against several Gram-positive pathogens

Directory of Open Access Journals (Sweden)

Bingyue Xin

2016-07-01

Full Text Available Due to the rapidly increasing prevalence of multidrug-resistant bacterial strains, the need for new antimicrobial drugs to treat infections has become urgent. Bacteriocins, which are antimicrobial peptides of bacterial origin, are considered potential alternatives to conventional antibiotics and have attracted widespread attention in recent years. Among these bacteriocins, lantibiotics, especially two-component lantibiotics, exhibit potent antimicrobial activity against some clinically relevant Gram-positive pathogens and have potential applications in the pharmaceutical industry. In this study, we characterized a novel two-component lantibiotic termed thusin that consists of Thsα, Thsβ and Thsβ' (mutation of Thsβ, A14G and that was isolated from a B. thuringiensis strain BGSC 4BT1. Thsα and Thsβ (or Thsβ' exhibit optimal antimicrobial activity at a 1:1 ratio and act sequentially to affect target cells, and they are all highly thermostable (100°C for 30 min and pH tolerant (pH 2.0 to 9.0. Thusin shows remarkable efficacy against all tested Gram-positive bacteria and greater activities than two known lantibiotics thuricin 4A-4 and ticin A4, and one antibiotic vancomycin against various bacterial pathogens (Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus (MRSA, Staphylococcus sciuri, Enterococcus faecalis, and Streptococcus pneumoniae. Moreover, thusin is also able to inhibit the outgrowth of Bacillus cereus spores. The potent antimicrobial activity of thusin against some Gram-positive pathogens indicates that it has potential for the development of new drugs.

Cytokine profile in severe gram-positive and gram-negative abdominal sepsis

Science.gov (United States)

Surbatovic, Maja; Popovic, Nada; Vojvodic, Danilo; Milosevic, Ivan; Acimovic, Gordana; Stojicic, Milan; Veljovic, Milic; Jevdjic, Jasna; Djordjevic, Dragan; Radakovic, Sonja

2015-01-01

Sepsis is a principal cause of death in critical care units worldwide and consumes considerable healthcare resources. The aim of our study was to determine whether the early cytokine profile can discriminate between Gram-positive and Gram-negative bacteraemia (GPB and GNB, respectively) and to assess the prognostic value regarding outcome in critically ill patients with severe abdominal sepsis. The outcome measure was hospital mortality. Blood samples were obtained from 165 adult patients with confirmed severe abdominal sepsis. Levels of the proinflammatory mediators TNF-α, IL-8, IL-12 and IFN-γ and the anti-inflammatory mediators IL-1ra, IL-4, IL-10 and TGF-β1 were determined and correlated with the nature of the bacteria isolated from the blood culture and outcome. The cytokine profile in our study indicated that the TNF-α levels were 2-fold, IL-8 were 3.3-fold, IFN-γ were 13-fold, IL-1ra were 1.05-fold, IL-4 were 1.4-fold and IL-10 were 1.83-fold higher in the GNB group compared with the GPB group. The TNF-α levels were 4.7-fold, IL-8 were 4.6-fold, IL-1ra were 1.5-fold and IL-10 were 3.3-fold higher in the non-survivors compared with the survivors. PMID:26079127

Techniques for controlling variability in gram staining of obligate anaerobes.

Science.gov (United States)

Johnson, M J; Thatcher, E; Cox, M E

1995-01-01

Identification of anaerobes recovered from clinical samples is complicated by the fact that certain gram-positive anaerobes routinely stain gram negative; Peptostreptococcus asaccharolyticus, Eubacterium plautii, Clostridium ramosum, Clostridium symbiosum, and Clostridium clostridiiforme are among the nonconformists with regard to conventional Gram-staining procedures. Accurate Gram staining of American Type Culture Collection strains of these anaerobic bacteria is possible by implementing fixing and staining techniques within a gloveless anaerobic chamber. Under anaerobic conditions, gram-positive staining occurred in all test organisms with "quick" fixing techniques with both absolute methanol and formalin. The results support the hypothesis that, when anaerobic bacteria are exposed to oxygen, a breakdown of the physical integrity of the cell wall occurs, introducing Gram stain variability in gram-positive anaerobes. PMID:7538512

A Synthetic Dual Drug Sideromycin Induces Gram-Negative Bacteria To Commit Suicide with a Gram-Positive Antibiotic.

Science.gov (United States)

Liu, Rui; Miller, Patricia A; Vakulenko, Sergei B; Stewart, Nichole K; Boggess, William C; Miller, Marvin J

2018-05-10

Many antibiotics lack activity against Gram-negative bacteria because they cannot permeate the outer membrane or suffer from efflux and, in the case of β-lactams, are degraded by β-lactamases. Herein, we describe the synthesis and studies of a dual drug conjugate (1) of a siderophore linked to a cephalosporin with an attached oxazolidinone. The cephalosporin component of 1 is rapidly hydrolyzed by purified ADC-1 β-lactamase to release the oxazolidinone. Conjugate 1 is active against clinical isolates of Acinetobacter baumannii as well as strains producing large amounts of ADC-1 β-lactamase. Overall, the results are consistent with siderophore-mediated active uptake, inherent activity of the delivered dual drug, and in the presence of β-lactamases, intracellular release of the oxazolidinone upon cleavage of the cephalosporin to allow the freed oxazolidinone to inactivate its target. The ultimate result demonstrates that Gram-positive oxazolidinone antibiotics can be made to be effective against Gram-negative bacteria by β-lactamase triggered release.

Ultrastructure, inferred porosity, and gram-staining character of Methanospirillum hungatei filament termini describe a unique cell permeability for this archaeobacterium

International Nuclear Information System (INIS)

Beveridge, T.J.; Sprott, G.D.; Whippey, P.

1991-01-01

By light microscopy, Methanospirillum hungatei GP1 stains gram positive at the terminal ends of each multicellular filament and gram negative at all regions in between. This phenomenon was studied further by electron microscopy and energy-dispersive X-ray spectroscopy of Gram-stained cells, using a platinum compound to replace Gram's iodine. Crystal violet-platinum precipitates could be found only in the terminal cells of each filament, which suggested that the multilamellar plugs at the filament ends were involved with stain penetration. When sheaths were isolated by sodium dodecyl sulfate-dithiothreitol treatment, the end plugs could be ejected and their layers could be separated from one another by 0.1 M NaOH treatment. Each plug consisted of at least three individual layers; two were particulate and possessed 14.0-nm particles hexagonally arranged on their surfaces with a spacing of a = b = 18.0 nm, whereas the other was a netting of 12.5-nm holes with spacings and symmetry identical to those of the particulate layers. Optical diffraction and computer image reconstruction were used to clarify the structures of each layer in an intact plug and to provide a high-resolution image of their interdigitated structures. The holes through this composite were three to six times larger than those through the sheath. Accordingly, we propose that the terminal plugs of M. hungatei allow the access of larger solutes than does the sheath and that this is the reason why the end cells of each filament stain gram positive whereas more internal cells are gram negative. Intuitively, since the cell spacers which partition the cells from one another along the filament contain plugs identical in structure to terminal plugs, the diffusion of large solutes for these cells would be unidirectional along the filament-cell axis

In vivo metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal microorganisms and ruminants and its use as a marker of bacterial biomass

International Nuclear Information System (INIS)

Masson, H.A.; Denholm, A.M.; Ling, J.R.

1991-01-01

Cells of Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino [G- 3 H] pimelic acid ([ 3 H]DAP) as models of gram-positive and gram-negative bacteria, respectively. Two experiments were conducted to study the in vivo metabolism of 2,2'-diaminopimelic acid (DAP) in sheep. In experiment 1, cells of [ 3 H]DAP-labeled B. megaterium GW1 were infused into the rumen of one sheep and the radiolabel was traced within microbial samples, digesta, and the whole animal. Bacterially bound [ 3 H]DAP was extensively metabolized, primarily (up to 70% after 8 h) via decarboxylation to [ 3 H]lysine by both ruminal protozoa and ruminal bacteria. Recovery of infused radiolabel in urine and feces was low (42% after 96 h) and perhaps indicative of further metabolism by the host animal. In experiment 2, [ 3 H]DAP-labeled B. megaterium GW1 was infused into the rumens of three sheep and [ 3 H]DAP-labeled E. coli W7-W5 was infused into the rumen of another sheep. The radioactivity contents of these mutant bacteria were insufficient to use as tracers, but the metabolism of DAP was monitored in the total, free, and peptidyl forms. Free DAP, as a proportion of total DPA in duodenal digesta, varied from 0 to 9.5%, whereas peptidyl DAP accounted for 8.3 to 99.2%

Treatment of gram-positive deep sternal wound infections in cardiac surgery -experiences with daptomycin-

Directory of Open Access Journals (Sweden)

Coskun Kasim O

2011-09-01

Full Text Available Abstract The reported incidence of deep sternal wound infection (DSWI after cardiac surgery is 0.4-5% with Staphylococcus aureus being the most common pathogen isolated from infected wound sternotomies and bacteraemic blood cultures. This infection is associated with a higher morbidity and mortality than other known aetiologies. Little is reported about the optimal antibiotic management. The aim of the study is to quantify the application of daptomycin treatment of DSWI due to gram-positive organisms post cardiac surgery. We performed an observational analysis in 23 cases of post sternotomy DSWI with gram-positive organisms February 2009 and September 2010. When the wound appeared viable and the microbiological cultures were negative, the technique of chest closure was individualised to the patient. The incidence of DSWI was 1.46%. The mean dose of daptomycin application was 4.4 ± 0.9 mg/kg/d and the average duration of the daptomycin application was 14.47 ± 7.33 days. In 89% of the patients VAC therapy was used. The duration from daptomycin application to sternal closure was 18 ± 13.9 days. The parameters of infection including, fibrinogen (p = 0.03, white blood cell count (p = 0.001 and C-reactive protein (p = 0.0001 were significantly reduced after daptomycin application. We had no mortality and wound healing was successfully achieved in all patients. Treatment of DSWI due to gram-positive organisms with a daptomycin-containing antibiotic regimen is safe, effective and promotes immediate improvement of local wound conditions. Based on these observations, daptomycin may offer a new treatment option for expediting surgical management of DSWI after cardiac surgery.

Comparative activity of tigecycline and tetracycline on Gram-negative and Gram-positive bacteria revealed by a multicentre study in four North European countries

DEFF Research Database (Denmark)

Nilsson, Lennart E; Frimodt-Møller, Niels; Vaara, Martti

2011-01-01

This study involves a multicentre surveillance of tigecycline and tetracycline activity against Gram-negative and Gram-positive bacteria from primary care centres (PCCs), general hospital wards (GHWs) and intensive care units (ICUs) in Denmark (n = 9), Finland (n = 10), Norway (n = 7) and Sweden (n...

Trans-generational Immune Priming Protects the Eggs Only against Gram-Positive Bacteria in the Mealworm Beetle

Science.gov (United States)

Dubuffet, Aurore; Zanchi, Caroline; Boutet, Gwendoline; Moreau, Jérôme; Teixeira, Maria; Moret, Yannick

2015-01-01

In many vertebrates and invertebrates, offspring whose mothers have been exposed to pathogens can exhibit increased levels of immune activity and/or increased survival to infection. Such phenomena, called “Trans-generational immune priming” (TGIP) are expected to provide immune protection to the offspring. As the offspring and their mother may share the same environment, and consequently similar microbial threats, we expect the immune molecules present in the progeny to be specific to the microbes that immune challenged the mother. We provide evidence in the mealworm beetle Tenebrio molitor that the antimicrobial activity found in the eggs is only active against Gram-positive bacteria, even when females were exposed to Gram-negative bacteria or fungi. Fungi were weak inducers of TGIP while we obtained similar levels of anti-Gram-positive activity using different bacteria for the maternal challenge. Furthermore, we have identified an antibacterial peptide from the defensin family, the tenecin 1, which spectrum of activity is exclusively directed toward Gram-positive bacteria as potential contributor to this antimicrobial activity. We conclude that maternal transfer of antimicrobial activity in the eggs of T. molitor might have evolved from persistent Gram-positive bacterial pathogens between insect generations. PMID:26430786

Trans-generational Immune Priming Protects the Eggs Only against Gram-Positive Bacteria in the Mealworm Beetle.

Directory of Open Access Journals (Sweden)

Aurore Dubuffet

2015-10-01

Full Text Available In many vertebrates and invertebrates, offspring whose mothers have been exposed to pathogens can exhibit increased levels of immune activity and/or increased survival to infection. Such phenomena, called "Trans-generational immune priming" (TGIP are expected to provide immune protection to the offspring. As the offspring and their mother may share the same environment, and consequently similar microbial threats, we expect the immune molecules present in the progeny to be specific to the microbes that immune challenged the mother. We provide evidence in the mealworm beetle Tenebrio molitor that the antimicrobial activity found in the eggs is only active against Gram-positive bacteria, even when females were exposed to Gram-negative bacteria or fungi. Fungi were weak inducers of TGIP while we obtained similar levels of anti-Gram-positive activity using different bacteria for the maternal challenge. Furthermore, we have identified an antibacterial peptide from the defensin family, the tenecin 1, which spectrum of activity is exclusively directed toward Gram-positive bacteria as potential contributor to this antimicrobial activity. We conclude that maternal transfer of antimicrobial activity in the eggs of T. molitor might have evolved from persistent Gram-positive bacterial pathogens between insect generations.

Subcellular localization for Gram positive and Gram negative bacterial proteins using linear interpolation smoothing model.

Science.gov (United States)

Saini, Harsh; Raicar, Gaurav; Dehzangi, Abdollah; Lal, Sunil; Sharma, Alok

2015-12-07

Protein subcellular localization is an important topic in proteomics since it is related to a protein׳s overall function, helps in the understanding of metabolic pathways, and in drug design and discovery. In this paper, a basic approximation technique from natural language processing called the linear interpolation smoothing model is applied for predicting protein subcellular localizations. The proposed approach extracts features from syntactical information in protein sequences to build probabilistic profiles using dependency models, which are used in linear interpolation to determine how likely is a sequence to belong to a particular subcellular location. This technique builds a statistical model based on maximum likelihood. It is able to deal effectively with high dimensionality that hinders other traditional classifiers such as Support Vector Machines or k-Nearest Neighbours without sacrificing performance. This approach has been evaluated by predicting subcellular localizations of Gram positive and Gram negative bacterial proteins. Copyright © 2015 Elsevier Ltd. All rights reserved.

Comparison of TiO2 and ZnO nanoparticles for photocatalytic degradation of methylene blue and the correlated inactivation of gram-positive and gram-negative bacteria

International Nuclear Information System (INIS)

Barnes, Robert J.; Molina, Rodrigo; Xu Jianbin; Dobson, Peter J.; Thompson, Ian P.

2013-01-01

Titanium dioxide (TiO 2 ) and zinc oxide (ZnO) nanoparticles are important photocatalysts and as such have been extensively studied for the removal of organic compounds from contaminated air and water and for microbial disinfection. Despite much research on the effect of TiO 2 and ZnO nanoparticles on different bacterial species, uncertainties remain about which bacteria are more sensitive to these compounds. Very few studies have directly compared the toxicity of ZnO to TiO 2 under both light and dark conditions. In addition, authors investigating the photocatalytic inactivation of TiO 2 and ZnO nanoparticles on bacteria have failed to investigate the reactive oxygen species (ROS) generation of the nanoparticles, making it difficult to correlate killing action with the generation of ROS. In this study, three types of metal nanoparticle (ZnO 2 ) have been characterised and ROS production assessed through the degradation of methylene blue (MB). The photocatalytic killing potential of three nanoparticle concentrations (0.01, 0.1 and 1 g/L) was then assessed on four representative bacteria: two gram-positive (S. aureus and B. subtilis) and two gram-negative (E. coli and P. aeruginosa). Results showed that out of the three nanoparticles tested, the TiO 2 nanoparticles generated more ROS than the ZnO nanoparticles, corresponding to a greater photocatalytic inactivation of three of the four species of bacteria examined. The MB decomposition results correlated well with the bacterial inactivation results with higher TiO 2 nanoparticle concentrations leading to greater ROS production and increased loss of cell viability. Although producing less ROS than the TiO 2 nanoparticles under ultraviolet light, the ZnO nanoparticles were toxic to two of the bacterial species even under dark conditions. In this study, no correlation between cell wall type and bacterial inactivation was observed for any of the nanoparticles tested although both gram-positive bacteria were sensitive to

Daptomycin: a novel lipopeptide antibiotic against Gram-positive pathogens

Directory of Open Access Journals (Sweden)

Andres Beiras-Fernandez

2010-08-01

Full Text Available Andres Beiras-Fernandez1,*, Ferdinand Vogt1,*, Ralf Sodian1, Florian Weis21Department of Cardiac Surgery, University Hospital Großhadern, Ludwig-Maximilian-University, Munich, Germany; 2Department of Anesthesiology, University Hospital Großhadern, Ludwig-Maximilian-University, Munich, Germany *Andres Beiras-Fernandez and Ferdinand Vogt contributed equally to this paperAbstract: The aim of this review is to summarize the historical background of drug resistance of Gram-positive pathogens as well as to describe in detail the novel lipopeptide antibiotic daptomycin. Pharmacological and pharmacokinetic aspects are reviewed and the current clinical use of daptomycin is presented. Daptomycin seems to be a reliable drug in the treatment of complicated skin and skin structure infections, infective right-sided endocarditis, and bacteremia caused by Gram-positive agents. Its unique mechanism of action and its low resistance profile, together with its rapid bactericidal action make it a favorable alternative to vancomycin in multi-drug resistant cocci. The role of daptomycin in the treatment of prosthetic material infections, osteomyelitis, and urogenital infections needs to be evaluated in randomized clinical trials.Keywords: daptomycin, multi-drug resistance, methicillin-resistant Staphylococcus aureus (MRSA, pneumonia, urinary tract infection, left-sided endocarditis

Tribolium castaneum defensins are primarily active against Gram-positive bacteria

Czech Academy of Sciences Publication Activity Database

Tonk, M.; Knorr, E.; Cabezas-Cruz, A.; Valdés, James J.; Kollewe, C.; Vilcinskas, A.

2015-01-01

Roč. 132, NOV 2015 (2015), s. 208-215 ISSN 0022-2011 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : Antimicrobial peptides * Defensin * Innate immunity * Insects * Tribolium castaneum * Gram-positive bacteria Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.198, year: 2015

European surveillance study on antimicrobial susceptibility of Gram-positive anaerobic cocci

DEFF Research Database (Denmark)

Brazier, J; Chmelar, D; Dubreuil, L

2008-01-01

Gram-positive anaerobic cocci (GPAC) are a heterogeneous group of microorganisms frequently isolated from local and systemic infections. In this study, the antimicrobial susceptibilities of clinical strains isolated in 10 European countries were investigated. After identification of 299 GPAC...

Discovery of novel cell wall-active compounds using P ywaC, a sensitive reporter of cell wall stress, in the model gram-positive bacterium Bacillus subtilis.

Science.gov (United States)

Czarny, T L; Perri, A L; French, S; Brown, E D

2014-06-01

The emergence of antibiotic resistance in recent years has radically reduced the clinical efficacy of many antibacterial treatments and now poses a significant threat to public health. One of the earliest studied well-validated targets for antimicrobial discovery is the bacterial cell wall. The essential nature of this pathway, its conservation among bacterial pathogens, and its absence in human biology have made cell wall synthesis an attractive pathway for new antibiotic drug discovery. Herein, we describe a highly sensitive screening methodology for identifying chemical agents that perturb cell wall synthesis, using the model of the Gram-positive bacterium Bacillus subtilis. We report on a cell-based pilot screen of 26,000 small molecules to look for cell wall-active chemicals in real time using an autonomous luminescence gene cluster driven by the promoter of ywaC, which encodes a guanosine tetra(penta)phosphate synthetase that is expressed under cell wall stress. The promoter-reporter system was generally much more sensitive than growth inhibition testing and responded almost exclusively to cell wall-active antibiotics. Follow-up testing of the compounds from the pilot screen with secondary assays to verify the mechanism of action led to the discovery of 9 novel cell wall-active compounds. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Outcomes of single organism peritonitis in peritoneal dialysis: gram negatives versus gram positives in the Network 9 Peritonitis Study.

Science.gov (United States)

Bunke, C M; Brier, M E; Golper, T A

1997-08-01

The use of the "peritonitis rate" in the management of patients undergoing peritoneal dialysis is assuming importance in comparing the prowess of facilities, care givers and new innovations. For this to be a meaningful outcome measure, the type of infection (causative pathogen) must have less clinical significance than the number of infections during a time interval. The natural history of Staphylococcus aureus, pseudomonas, and fungal peritonitis would not support that the outcome of an episode of peritonitis is independent of the causative pathogen. Could this concern be extended to other more frequently occurring pathogens? To address this, the Network 9 Peritonitis Study identified 530 episodes of single organism peritonitis caused by a gram positive organism and 136 episodes caused by a single non-pseudomonal gram negative (NPGN) pathogen. Coincidental soft tissue infections (exit site or tunnel) occurred equally in both groups. Outcomes of peritonitis were analyzed by organism classification and by presence or absence of a soft tissue infection. NPGN peritonitis was associated with significantly more frequent catheter loss, hospitalization, and technique failure and was less likely to resolve regardless of the presence or absence of a soft tissue infection. Hospitalization and death tended to occur more frequently with enterococcal peritonitis than with other gram positive peritonitis. The outcomes in the NPGN peritonitis group were significantly worse (resolution, catheter loss, hospitalization, technique failure) compared to coagulase negative staphylococcal or S. aureus peritonitis, regardless of the presence or absence of a coincidental soft tissue infection. Furthermore, for the first time, the poor outcomes of gram negative peritonitis are shown to be independent of pseudomonas or polymicrobial involvement or soft tissue infections. The gram negative organism appears to be the important factor. In addition, the outcome of peritonitis caused by S. aureus

A new technique for Gram staining paraffin-embedded tissue.

Science.gov (United States)

Engbaek, K; Johansen, K S; Jensen, M E

1979-01-01

Five techniques for Gram staining bacteria in paraffin sections were compared on serial sections of pulmonary tissues from eight bacteriological necropsies. Brown and Hopp's method was the most satisfactory for distinguishing Gram-positive and Gram-negative bacteria. However, this method cannot be recommended as the preparations were frequently overstained, and the Gram-negative bacteria were stained indistinctly. A modification of Brown and Hopps' method was developed which stains larger numbers of Gram-negative bacteria and differentiates well between different cell types and connective tissue, and there is no risk of overstaining. PMID:86548

Optimization of linezolid treatment regimens for Gram-positive bacterial infections based on pharmacokinetic/pharmacodynamic analysis.

Science.gov (United States)

Yang, Minjie; Zhang, Jing; Chen, Yuancheng; Liang, Xiaoyu; Guo, Yan; Yu, Jicheng; Zhu, Demei; Zhang, Yingyuan

2017-01-01

To optimize linezolid treatment regimens for Gram-positive bacterial infections based on pharmacokinetic/pharmacodynamic analysis. The minimum inhibitory concentration (MIC) distribution of 572 Gram-positive strains from patients with clinically confirmed infections was analyzed. Using the Monte Carlo simulation method, the cumulative fraction of response and probability of target attainment were determined for linezolid regimens of 600 mg q.12h and q.8h Results: Linezolid dosage of 600 mg q.12h yielded >90% cumulative fraction of response and probability of target attainment for staphylococcal infections with an MIC of ≤1 mg/l, enterococcal infections with higher MIC values required 600 mg q.8h. Linezolid 600 mg q.12h is still the clinically recommended empirical dosage for Gram-positive bacterial infections. However, as bacterial MICs increase, 600 mg q.8h may be required to achieve better efficacy.

High-level fluorescence labeling of gram-positive pathogens.

Directory of Open Access Journals (Sweden)

Simone Aymanns

Full Text Available Fluorescence labeling of bacterial pathogens has a broad range of interesting applications including the observation of living bacteria within host cells. We constructed a novel vector based on the E. coli streptococcal shuttle plasmid pAT28 that can propagate in numerous bacterial species from different genera. The plasmid harbors a promoterless copy of the green fluorescent variant gene egfp under the control of the CAMP-factor gene (cfb promoter of Streptococcus agalactiae and was designated pBSU101. Upon transfer of the plasmid into streptococci, the bacteria show a distinct and easily detectable fluorescence using a standard fluorescence microscope and quantification by FACS-analysis demonstrated values that were 10-50 times increased over the respective controls. To assess the suitability of the construct for high efficiency fluorescence labeling in different gram-positive pathogens, numerous species were transformed. We successfully labeled Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. equisimilis, Enterococcus faecalis, Enterococcus faecium, Streptococcus mutans, Streptococcus anginosus and Staphylococcus aureus strains utilizing the EGFP reporter plasmid pBSU101. In all of these species the presence of the cfb promoter construct resulted in high-level EGFP expression that could be further increased by growing the streptococcal and enterococcal cultures under high oxygen conditions through continuous aeration.

Higher order structure in the 3'-minor domain of small subunit ribosomal RNAs from a gram negative bacterium, a gram positive bacterium and a eukaryote

DEFF Research Database (Denmark)

Douthwaite, S; Christensen, A; Garrett, R A

1983-01-01

of additional higher order structure in the renatured free RNA. It can be concluded that a high level of conservation of higher order structure has occurred during the evolution of the gram negative and gram positive eubacteria and the eukaryote in both the double helical regions and the "unstructured" regions...

Recovery of vancomycin-resistant gram-positive cocci from children.

OpenAIRE

Green, M; Wadowsky, R M; Barbadora, K

1990-01-01

A cross-sectional survey of vancomycin-resistant gram-positive cocci (VRGPC) in the feces of children was initiated after several bacteremic infections with these organisms occurred at our hospital. A selective medium consisting of colistin-nalidixic acid agar, 5% sheep blood, vancomycin (5 mg/liter), and amphotericin B (8 mg/liter) was developed to isolate VRGPC. A single stool specimen submitted to the clinical microbiology laboratory from each of 48 patients was inoculated onto the medium....

The complete genome sequence of the Gram-positive bacterium Bacillus subtilis

NARCIS (Netherlands)

Kunst, F; Ogasawara, N; Moszer, [No Value; Albertini, AM; Alloni, G; Azevedo, [No Value; Bertero, MG; Bessieres, P; Bolotin, A; Borchert, S; Borriss, R; Boursier, L; Brans, A; Brignell, SC; Bron, S; Brouillet, S; Bruschi, CV; Caldwell, B; Capuano, [No Value; Carter, NM; Choi, SK; Codani, JJ; Connerton, IF; Cummings, NJ; Daniel, RA; Denizot, F; Devine, KM; Dusterhoft, A; Ehrlich, SD; Emmerson, PT; Entian, KD; Errington, J; Fabret, C; Ferrari, E; Foulger, D; Fujita, M; Fujita, Y; Fuma, S; Galizzi, A; Galleron, N; Ghim, SY; Glaser, P; Goffeau, A; Golightly, EJ; Grandi, G; Guiseppi, G; Guy, BJ; Haga, K; Haiech, J; Harwood, CR; Henaut, A; Hilbert, H; Holsappel, S; Hosono, S; Hullo, MF; Itaya, M; Jones, L; Joris, B; Karamata, D; Kasahara, Y; KlaerrBlanchard, M; Klein, C; Kobayashi, Y; Koetter, P; Koningstein, G; Krogh, S; Kumano, M; Kurita, K; Lapidus, A; Lardinois, S; Lauber, J; Lazarevic, [No Value; Lee, SM; Levine, A; Liu, H; Masuda, S; Mauel, C; Medigue, C; Medina, N; Mellado, RP; Mizuno, M; Moestl, D; Nakai, S; Noback, M; Noone, D; OReilly, M; Ogawa, K; Ogiwara, A; Oudega, B; Park, SH; Parro, [No Value; Pohl, TM; Portetelle, D; Porwollik, S; Prescott, AM; Presecan, E; Pujic, P; Purnelle, B; Rapoport, G; Rey, M; Reynolds, S; Rieger, M; Rivolta, C; Rocha, E; Roche, B; Rose, M; Sadaie, Y; Sato, T; Scanlan, E; Schleich, S; Schroeter, R; Scoffone, F; Sekiguchi, J; Sekowska, A; Seror, SJ; Serror, P; Shin, BS; Soldo, B; Sorokin, A; Tacconi, E; Takagi, T; Takahashi, H; Takemaru, K; Takeuchi, M; Tamakoshi, A; Tanaka, T; Terpstra, P; Tognoni, A; Tosato, [No Value; Uchiyama, S; Vandenbol, M; Vannier, F; Vassarotti, A; Viari, A; Wambutt, R; Wedler, E; Wedler, H; Weitzenegger, T; Winters, P; Wipat, A; Yamamoto, H; Yamane, K; Yasumoto, K; Yata, K; Yoshida, K; Yoshikawa, HF; Zumstein, E; Yoshikawa, H; Danchin, A

1997-01-01

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been

Metabolic versatility of Gram-positive microbial isolates from contaminated river sediments

International Nuclear Information System (INIS)

Narancic, Tanja; Djokic, Lidija; Kenny, Shane T.; O’Connor, Kevin E.; Radulovic, Vanja; Nikodinovic-Runic, Jasmina; Vasiljevic, Branka

2012-01-01

Highlights: ► Thirty-four isolated Gram-positive bacteria could degrade wide range of aromatic pollutants. ► Nine isolates could grow in the presence of extremely high levels of heavy metals. ► Twelve isolates accumulated polyphosphate, 3 polyhydroxybutyrate, 4 exopolysaccharides. ► The incidence of multiple antibiotic resistance markers among isolates was low. - Abstract: Gram-positive bacteria from river sediments affected by the proximity of a petrochemical industrial site were isolated and characterized with respect to their ability to degrade a wide range of aromatic compounds. In this study we identified metabolically diverse Gram-positive bacteria capable of growth on wide range aromatic compounds in the presence of heavy metals and with the ability to accumulate biopolymers. Thirty-four isolates that were able to use 9 or more common aromatic pollutants, such as benzene, biphenyl, naphthalene etc. as a sole source of carbon and energy included members of Bacillus, Arthrobacter, Rhodococcus, Gordonia, Streptomyces, and Staphylococcus genus. Rhodococcus sp. TN105, Gordonia sp. TN103 and Arthrobacter sp. TN221 were identified as novel strains. Nine isolates were able to grow in the presence of one or more metals (mercury, cadmium, nickel) at high concentration (100 mM). Seven isolates could degrade 15 different aromatic compounds and could grow in the presence of one or more heavy metals. Two of these isolates were resistant to multiple antibiotics including erythromycin and nalidixic acid. One third of isolates could accumulate at least one biopolymer. Twelve isolates (mainly Bacillus sp. and Arthrobacter sp.) accumulated polyphosphate, 3 Bacillus sp. accumulated polyhydroxybutyrate, while 4 isolates could accumulate exopolysaccharides.

Facile synthesis of gold nanoparticles on propylamine functionalized SBA-15 and effect of surface functionality of its enhanced bactericidal activity against gram positive bacteria

International Nuclear Information System (INIS)

Bhuyan, Diganta; Saikia, Mrinal; Saikia, Lakshi; Gogoi, Animesh; Saikia, Ratul

2015-01-01

The facile synthesis of an SBA-15-pr- + NH 3 .Au 0 nano-hybrid material by spontaneous autoreduction of aqueous chloroaurate anions on propylamine functionalized SBA-15 was successfully demonstrated. The as-synthesized SBA-15-pr- + NH 3 .Au 0 nano-hybrid material was well characterized using low and wide angle x-ray diffraction (XRD), N 2 adsorption–desorption isotherms, Fourier transform infrared (FTIR), transmission electron microscopy (TEM), scanning electron microscopy-energy dispersive x-ray spectroscopy (SEM-EDX), x-ray photoelectron spectroscopy (XPS), UV-Visible spectroscopy and atomic absorption spectroscopy (AAS). The activity of the nano-hybrid material as a potent bactericidal agent was successfully tested against Gram positive/negative bacteria viz. Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The colony killing percentage of Gram positive bacteria was found to be higher than Gram negative bacteria due to the stronger electrostatic interaction between the positively-charged amine functionality of SBA-15 and the negatively charged functionality of the bacterial cell wall. (paper)

Enhanced antiadhesive properties of chitosan/hyaluronic acid polyelectrolyte multilayers driven by thermal annealing: Low adherence for mammalian cells and selective decrease in adhesion for Gram-positive bacteria.

Science.gov (United States)

Muzzio, Nicolás E; Pasquale, Miguel A; Diamanti, Eleftheria; Gregurec, Danijela; Moro, Marta Martinez; Azzaroni, Omar; Moya, Sergio E

2017-11-01

The development of antifouling coatings with restricted cell and bacteria adherence is fundamental for many biomedical applications. A strategy for the fabrication of antifouling coatings based on the layer-by-layer assembly and thermal annealing is presented. Polyelectrolyte multilayers (PEMs) assembled from chitosan and hyaluronic acid were thermally annealed in an oven at 37°C for 72h. The effect of annealing on the PEM properties and topography was studied by atomic force microscopy, ζ-potential, circular dichroism and contact angle measurements. Cell adherence on PEMs before and after annealing was evaluated by measuring the cell spreading area and aspect ratio for the A549 epithelial, BHK kidney fibroblast, C2C12 myoblast and MC-3T3-E1 osteoblast cell lines. Chitosan/hyaluronic acid PEMs show a low cell adherence that decreases with the thermal annealing, as observed from the reduction in the average cell spreading area and more rounded cell morphology. The adhesion of S. aureus (Gram-positive) and E. coli (Gram-negative) bacteria strains was quantified by optical microscopy, counting the number of colony-forming units and measuring the light scattering of bacteria suspension after detachment from the PEM surface. A 20% decrease in bacteria adhesion was selectively observed in the S. aureus strain after annealing. The changes in mammalian cell and bacteria adhesion correlate with the changes in topography of the chitosan/hyaluronic PEMs from a rough fibrillar 3D structure to a smoother and planar surface after thermal annealing. Copyright © 2017. Published by Elsevier B.V.

Silver-doped manganese dioxide and trioxide nanoparticles inhibit both gram positive and gram negative pathogenic bacteria.

Science.gov (United States)

Kunkalekar, R K; Prabhu, M S; Naik, M M; Salker, A V

2014-01-01

Palladium, ruthenium and silver-doped MnO2 and silver doped Mn2O3 nanoparticles were synthesized by simple co-precipitation technique. SEM-TEM analysis revealed the nano-size of these synthesized samples. XPS data illustrates that Mn is present in 4+ and 3+ oxidation states in MnO2 and Mn2O3 respectively. Thermal analysis gave significant evidence for the phase changes with increasing temperature. Antibacterial activity of these synthesized nanoparticles on three Gram positive bacterial cultures (Staphylococcus aureus ATCC 6538, Streptococcus epidermis ATCC 12228, Bacillus subtilis ATCC 6633) and three Gram negative cultures (Escherichia coli ATCC 8739, Salmonella abony NCTC 6017 and Klebsiella pneumoniae ATCC 1003) was investigated using a disc diffusion method and live/dead assay. Only Ag-doped MnO2 and Ag-doped Mn2O3 nanoparticles showed antibacterial property against all six-test bacteria but Ag-doped MnO2 was found to be more effective than Ag-doped Mn2O3. Copyright © 2013 Elsevier B.V. All rights reserved.

Nanoparticle targeting of Gram-positive and Gram-negative bacteria for magnetic-based separations of bacterial pathogens

Science.gov (United States)

Lu, Hoang D.; Yang, Shirley S.; Wilson, Brian K.; McManus, Simon A.; Chen, Christopher V. H.-H.; Prud'homme, Robert K.

2017-04-01

Antimicrobial resistance is a healthcare problem of increasing significance, and there is increasing interest in developing new tools to address bacterial infections. Bacteria-targeting nanoparticles hold promise to improve drug efficacy, compliance, and safety. In addition, nanoparticles can also be used for novel applications, such as bacterial imaging or bioseperations. We here present the use of a scalable block-copolymer-directed self-assembly process, Flash NanoPrecipitation, to form zinc(II)-bis(dipicolylamine) modified nanoparticles that bind to both Gram-positive and Gram-negative bacteria with specificity. Particles have tunable surface ligand densities that change particle avidity and binding efficacy. A variety of materials can be encapsulated into the core of the particles, such as optical dyes or iron oxide colloids, to produce imageable and magnetically active bacterial targeting constructs. As a proof-of-concept, these particles are used to bind and separate bacteria from solution in a magnetic column. Magnetic manipulation and separation would translate to a platform for pathogen identification or removal. These magnetic and targeted nanoparticles enable new methods to address bacterial infections.

Identification of aerobic Gram-positive bacilli by use of Vitek MS.

Science.gov (United States)

Navas, Maria; Pincus, David H; Wilkey, Kathy; Sercia, Linda; LaSalvia, Margaret; Wilson, Deborah; Procop, Gary W; Richter, Sandra S

2014-04-01

The accuracy of Vitek MS mass spectrometric identifications was assessed for 206 clinically significant isolates of aerobic Gram-positive bacilli representing 20 genera and 38 species. The Vitek MS identifications were correct for 85% of the isolates (56.3% to the species level, 28.6% limited to the genus level), with misidentifications occurring for 7.3% of the isolates.

In vitro activities of dalbavancin and nine comparator agents against anaerobic gram-positive species and corynebacteria.

Science.gov (United States)

Goldstein, Ellie J C; Citron, Diane M; Merriam, C Vreni; Warren, Yumi; Tyrrell, Kerin; Fernandez, Helen T

2003-06-01

Dalbavancin is a novel semisynthetic glycopeptide with enhanced activity against gram-positive species. Its comparative in vitro activities and those of nine comparator agents, including daptomycin, vancomycin, linezolid, and quinupristin-dalfopristin, against 290 recent gram-positive clinical isolates strains, as determined by the NCCLS agar dilution method, were studied. The MICs of dalbavancin at which 90% of various isolates tested were inhibited were as follows: Actinomyces spp., 0.5 microg/ml; Clostridium clostridioforme, 8 microg/ml; C. difficile, 0.25 microg/ml; C. innocuum, 0.25 microg/ml; C. perfringens, 0.125 microg/ml; C. ramosum, 1 microg/ml; Eubacterium spp., 1 microg/ml; Lactobacillus spp., >32 microg/ml, Propionibacterium spp., 0.5 microg/ml; and Peptostreptococcus spp., 0.25 microg/ml. Dalbavancin was 1 to 3 dilutions more active than vancomycin against most strains. Dalbavancin exhibited excellent activity against gram-positive strains tested and warrants clinical evaluation.

The Toll pathway underlies host sexual dimorphism in resistance to both Gram-negative and Gram-positive bacteria in mated Drosophila.

Science.gov (United States)

Duneau, David F; Kondolf, Hannah C; Im, Joo Hyun; Ortiz, Gerardo A; Chow, Christopher; Fox, Michael A; Eugénio, Ana T; Revah, J; Buchon, Nicolas; Lazzaro, Brian P

2017-12-21

Host sexual dimorphism is being increasingly recognized to generate strong differences in the outcome of infectious disease, but the mechanisms underlying immunological differences between males and females remain poorly characterized. Here, we used Drosophila melanogaster to assess and dissect sexual dimorphism in the innate response to systemic bacterial infection. We demonstrated sexual dimorphism in susceptibility to infection by a broad spectrum of Gram-positive and Gram-negative bacteria. We found that both virgin and mated females are more susceptible than mated males to most, but not all, infections. We investigated in more detail the lower resistance of females to infection with Providencia rettgeri, a Gram-negative bacterium that naturally infects D. melanogaster. We found that females have a higher number of phagocytes than males and that ablation of hemocytes does not eliminate the dimorphism in resistance to P. rettgeri, so the observed dimorphism does not stem from differences in the cellular response. The Imd pathway is critical for the production of antimicrobial peptides in response to Gram-negative bacteria, but mutants for Imd signaling continued to exhibit dimorphism even though both sexes showed strongly reduced resistance. Instead, we found that the Toll pathway is responsible for the dimorphism in resistance. The Toll pathway is dimorphic in genome-wide constitutive gene expression and in induced response to infection. Toll signaling is dimorphic in both constitutive signaling and in induced activation in response to P. rettgeri infection. The dimorphism in pathway activation can be specifically attributed to Persephone-mediated immune stimulation, by which the Toll pathway is triggered in response to pathogen-derived virulence factors. We additionally found that, in absence of Toll signaling, males become more susceptible than females to the Gram-positive Enterococcus faecalis. This reversal in susceptibility between male and female Toll

Dose-Dependent Antimicrobial Activity of Silver Nanoparticles on Polycaprolactone Fibers against Gram-Positive and Gram-Negative Bacteria

Directory of Open Access Journals (Sweden)

Erick Pazos-Ortiz

2017-01-01

Full Text Available The adhesion ability and adaptability of bacteria, coupled with constant use of the same bactericides, have made the increase in the diversity of treatments against infections necessary. Nanotechnology has played an important role in the search for new ways to prevent and treat infections, including the use of metallic nanoparticles with antibacterial properties. In this study, we worked on the design of a composite of silver nanoparticles (AgNPS embedded in poly-epsilon-caprolactone nanofibers and evaluated its antimicrobial properties against various Gram-positive and Gram-negative microorganisms associated with drug-resistant infections. Polycaprolactone-silver composites (PCL-AgNPs were prepared in two steps. The first step consisted in the reduction in situ of Ag+ ions using N,N-dimethylformamide (DMF in tetrahydrofuran (THF solution, and the second step involved the simple addition of polycaprolactone before electrospinning process. Antibacterial activity of PCL-AgNPs nanofibers against E. coli, S. mutans, K. pneumoniae, S. aureus, P. aeruginosa, and B. subtilis was evaluated. Results showed sensibility of E. coli, K. pneumoniae, S. aureus, and P. aeruginosa, but not for B. subtilis and S. mutans. This antimicrobial activity of PCL-AgNPs showed significant positive correlations associated with the dose-dependent effect. The antibacterial property of the PCL/Ag nanofibers might have high potential medical applications in drug-resistant infections.

Blue green alga mediated synthesis of gold nanoparticles and its antibacterial efficacy against Gram positive organisms

Energy Technology Data Exchange (ETDEWEB)

Uma Suganya, K.S. [Centre for Ocean Research, Sathyabama University, Chennai 600 119 (India); Govindaraju, K., E-mail: govindtu@gmail.com [Centre for Ocean Research, Sathyabama University, Chennai 600 119 (India); Ganesh Kumar, V.; Stalin Dhas, T.; Karthick, V. [Centre for Ocean Research, Sathyabama University, Chennai 600 119 (India); Singaravelu, G. [Nanoscience Division, Department of Zoology, Thiruvalluvar University, Vellore 632115 (India); Elanchezhiyan, M. [Department of Microbiology, Dr ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai 600113 (India)

2015-02-01

Biofunctionalized gold nanoparticles (AuNPs) play an important role in design and development of nanomedicine. Synthesis of AuNPs from biogenic materials is environmentally benign and possesses high bacterial inhibition and bactericidal properties. In the present study, blue green alga Spirulina platensis protein mediated synthesis of AuNPs and its antibacterial activity against Gram positive bacteria is discussed. AuNPs were characterized using Ultraviolet–visible (UV–vis) spectroscopy, Fluorescence spectroscopy, Fourier Transform-Infrared (FTIR) spectroscopy, Raman spectroscopy, High Resolution-Transmission Electron Microscopy (HR-TEM) and Energy Dispersive X-ray analysis (EDAX). Stable, well defined AuNPs of smaller and uniform shape with an average size of ∼ 5 nm were obtained. The antibacterial efficacy of protein functionalized AuNPs were tested against Gram positive organisms Bacillus subtilis and Staphylococcus aureus. - Highlights: • Size controlled synthesis of gold nanoparticles from blue green alga Spirulina platensis • Stability of gold nanoparticles at different temperatures • Potent antibacterial efficacy against Gram positive organisms.

Inhibition of various gram-positive and gram-negative bacteria growth on selenium nanoparticle coated paper towels.

Science.gov (United States)

Wang, Qi; Larese-Casanova, Philip; Webster, Thomas J

2015-01-01

There are wide spread bacterial contamination issues on various paper products, such as paper towels hanging in sink splash zones or those used to clean surfaces, filter papers used in water and air purifying systems, and wrappings used in the food industry; such contamination may lead to the potential spread of bacteria and consequent severe health concerns. In this study, selenium nanoparticles were coated on normal paper towel surfaces through a quick precipitation method, introducing antibacterial properties to the paper towels in a healthy way. Their effectiveness at preventing biofilm formation was tested in bacterial assays involving Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus epidermidis. The results showed significant and continuous bacteria inhibition with about a 90% reduction from 24 to 72 hours for gram-positive bacteria including S. aureus and S. epidermidis. The selenium coated paper towels also showed significant inhibition of gram-negative bacteria like P. aeruginosa and E. coli growth at about 57% and 84%, respectively, after 72 hours of treatment. Therefore, this study established a promising selenium-based antibacterial strategy to prevent bacterial growth on paper products, which may lead to the avoidance of bacteria spreading and consequent severe health concerns.

Cardiolipin, a major phospholipid of gram-positive bacteria that is not readily extractable

NARCIS (Netherlands)

Filgueiras, M.H.; Kamp, J.A.F. op den

1980-01-01

Extraction of phospholipids from stationary phase grown cells of the Gram+ bacteria, Bacillus megaterium, Bacillus subtilis, Bacillus cereus and Micrococcus lysodeikticus was found to be incomplete with various commonly used extraction procedures. Phosphatidylglycerol and phosphatidyl-ethanolamine

In vitro activity of tigecycline and comparator agents against a global collection of Gram-negative and Gram-positive organisms: tigecycline Evaluation and Surveillance Trial 2004 to 2007.

Science.gov (United States)

Garrison, Mark W; Mutters, Reinier; Dowzicky, Michael J

2009-11-01

The Tigecycline Evaluation and Surveillance Trial began in 2004 to monitor the in vitro activity of tigecycline and comparator agents against a global collection of Gram-negative and Gram-positive pathogens. Against Gram negatives (n = 63 699), tigecycline MIC(90)'s ranged from 0.25 to 2 mg/L for Escherichia coli, Haemophilus influenzae, Acinetobacter baumannii, Klebsiella oxytoca, Enterobacter cloacae, Klebsiella pneumoniae, and Serratia marcescens (but was > or =32 for Pseudomonas aeruginosa). Against Gram-positive organisms (n = 32 218), tigecycline MIC(90)'s were between 0.06 and 0.25 mg/L for Streptococcus pneumoniae, Enterococcus faecium, Streptococcus agalactiae, Staphylococcus aureus, and Enterococcus faecalis. The in vitro activity of tigecycline was maintained against resistant phenotypes, including multidrug-resistant A. baumannii (9.2% of isolates), extended-spectrum beta-lactamase-producing E. coli (7.0%) and K. pneumoniae (14.0%), beta-lactamase-producing H. influenzae (22.2%), methicillin-resistant S. aureus (44.5%), vancomycin-resistant E. faecium (45.9%) and E. faecalis (2.8%), and penicillin-resistant S. pneumoniae (13.8%). Tigecycline represents a welcome addition to the armamentarium against difficult to treat organisms.

Gram staining for the treatment of peritonsillar abscess.

Science.gov (United States)

Takenaka, Yukinori; Takeda, Kazuya; Yoshii, Tadashi; Hashimoto, Michiko; Inohara, Hidenori

2012-01-01

Objective. To examine whether Gram staining can influence the choice of antibiotic for the treatment of peritonsillar abscess. Methods. Between 2005 and 2009, a total of 57 cases of peritonsillar abscess were analyzed with regard to cultured bacteria and Gram staining. Results. Only aerobes were cultured in 16% of cases, and only anaerobes were cultured in 51% of cases. Mixed growth of aerobes and anaerobes was observed in 21% of cases. The cultured bacteria were mainly aerobic Streptococcus, anaerobic Gram-positive cocci, and anaerobic Gram-negative rods. Phagocytosis of bacteria on Gram staining was observed in 9 cases. The bacteria cultured from these cases were aerobic Streptococcus, anaerobic Gram-positive cocci, and anaerobic Gram-negative rods. The sensitivity of Gram staining for the Gram-positive cocci and Gram-negative rods was 90% and 64%, respectively. The specificity of Gram staining for the Gram-positive cocci and Gram-negative rods was 62% and 76%, respectively. Most of the Gram-positive cocci were sensitive to penicillin, but some of anaerobic Gram-negative rods were resistant to penicillin. Conclusion. When Gram staining shows only Gram-positive cocci, penicillin is the treatment of choice. In other cases, antibiotics effective for the penicillin-resistant organisms should be used.

Transformation and Stability of Cloned Polysaccharidase Genes in Gram-Positive Bacteria

OpenAIRE

EKİNCİ, Mehmet Sait

2014-01-01

Polysaccharidase genes from rumen bacteria were transferred to and expressed in ruminal and non-ruminal Gram-positive bacteria. The transformation efficiency and genetic stability of polysaccharidase genes in bacteria from different habitats were investigated. PCR amplification of cloned polysaccharidase genes from Escherichia coli, Lactococcus lactis, Enterococcus faecalis, Streptococcus sanguis and S. bovis strain 26 showed that rearrangement of plasmid and the gene fragment did not occur. ...

Rational approaches to the therapy of nosocomial infections caused by gram-positive microorganisms in cancer p

Directory of Open Access Journals (Sweden)

V. V. Aginova

2017-01-01

Full Text Available Nosocomial infections caused by gram-positive organisms, including Staphylococcus aureus and enterococci (Enterococcus faecium and Enterococcus faecalis are steadily increasing in almost all clinics around the world. Cancer patients have a higher risk of hospital-acquired infections than non-cancer patients. Cancer patients are immunosuppressed due to increased use of broad-spectrum antibiotics and chemotherapy drugs, radiation therapy, surgery and use of steroids. This paper presents an analysis of resistance of gram-positive bacterial pathogens to antimicrobial agents to determine treatment strategy for cancer patients.

Antibacterial activity of sphingoid bases and fatty acids against Gram-positive and Gram-negative bacteria.

Science.gov (United States)

Fischer, Carol L; Drake, David R; Dawson, Deborah V; Blanchette, Derek R; Brogden, Kim A; Wertz, Philip W

2012-03-01

There is growing evidence that the role of lipids in innate immunity is more important than previously realized. How lipids interact with bacteria to achieve a level of protection, however, is still poorly understood. To begin to address the mechanisms of antibacterial activity, we determined MICs and minimum bactericidal concentrations (MBCs) of lipids common to the skin and oral cavity--the sphingoid bases D-sphingosine, phytosphingosine, and dihydrosphingosine and the fatty acids sapienic acid and lauric acid--against four Gram-negative bacteria and seven Gram-positive bacteria. Exact Kruskal-Wallis tests of these values showed differences among lipid treatments (P 500 μg/ml). Sapienic acid (MBC range, 31.3 to 375.0 μg/ml) was active against Streptococcus sanguinis, Streptococcus mitis, and Fusobacterium nucleatum but not active against Escherichia coli, Staphylococcus aureus, S. marcescens, P. aeruginosa, Corynebacterium bovis, Corynebacterium striatum, and Corynebacterium jeikeium (MBC > 500 μg/ml). Lauric acid (MBC range, 6.8 to 375.0 μg/ml) was active against all bacteria except E. coli, S. marcescens, and P. aeruginosa (MBC > 500 μg/ml). Complete killing was achieved as early as 0.5 h for some lipids but took as long as 24 h for others. Hence, sphingoid bases and fatty acids have different antibacterial activities and may have potential for prophylactic or therapeutic intervention in infection.

A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria.

Science.gov (United States)

Ferro, Roberto; Rennig, Maja; Hernández-Rollán, Cristina; Daley, Daniel O; Nørholm, Morten H H

2018-03-08

The market for recombinant proteins is on the rise, and Gram-positive strains are widely exploited for this purpose. Bacillus subtilis is a profitable host for protein production thanks to its ability to secrete large amounts of proteins, and Lactococcus lactis is an attractive production organism with a long history in food fermentation. We have developed a synbio approach for increasing gene expression in two Gram-positive bacteria. First of all, the gene of interest was coupled to an antibiotic resistance gene to create a growth-based selection system. We then randomised the translation initiation region (TIR) preceding the gene of interest and selected clones that produced high protein titres, as judged by their ability to survive on high concentrations of antibiotic. Using this approach, we were able to significantly increase production of two industrially relevant proteins; sialidase in B. subtilis and tyrosine ammonia lyase in L. lactis. Gram-positive bacteria are widely used to produce industrial enzymes. High titres are necessary to make the production economically feasible. The synbio approach presented here is a simple and inexpensive way to increase protein titres, which can be carried out in any laboratory within a few days. It could also be implemented as a tool for applications beyond TIR libraries, such as screening of synthetic, homologous or domain-shuffled genes.

Novel Antimicrobial Peptides That Inhibit Gram Positive Bacterial Exotoxin Synthesis

Science.gov (United States)

Merriman, Joseph A.; Nemeth, Kimberly A.; Schlievert, Patrick M.

2014-01-01

Gram-positive bacteria, such as Staphylococcus aureus, cause serious human illnesses through combinations of surface virulence factors and secretion of exotoxins. Our prior studies using the protein synthesis inhibitor clindamycin and signal transduction inhibitors glycerol monolaurate and α-globin and β-globin chains of hemoglobin indicate that their abilities to inhibit exotoxin production by S. aureus are separable from abilities to inhibit growth of the organism. Additionally, our previous studies suggest that inhibition of exotoxin production, in absence of ability to kill S. aureus and normal flora lactobacilli, will prevent colonization by pathogenic S. aureus, while not interfering with lactobacilli colonization. These disparate activities may be important in development of novel anti-infective agents that do not alter normal flora. We initiated studies to explore the exotoxin-synthesis-inhibition activity of hemoglobin peptides further to develop potential agents to prevent S. aureus infections. We tested synthesized α-globin chain peptides, synthetic variants of α-globin chain peptides, and two human defensins for ability to inhibit exotoxin production without significantly inhibiting S. aureus growth. All of these peptides were weakly or not inhibitory to bacterial growth. However, the peptides were inhibitory to exotoxin production with increasing activity dependent on increasing numbers of positively-charged amino acids. Additionally, the peptides could be immobilized on agarose beads or have amino acid sequences scrambled and still retain exotoxin-synthesis-inhibition. The peptides are not toxic to human vaginal epithelial cells and do not inhibit growth of normal flora L. crispatus. These peptides may interfere with plasma membrane signal transduction in S. aureus due to their positive charges. PMID:24748386

Flavins mediate extracellular electron transfer in Gram-positive Bacillus megaterium strain LLD-1

DEFF Research Database (Denmark)

You, Le-Xing; Liu, Li-Dan; Xiao, Yong

2018-01-01

The extracellular electron transfer (EET) mechanism of an isolated Gram-positive Bacillus megaterium strain (LLD-1), identified by 16S rRNA gene sequencing and physiological analysis, was investigated in the present study. The electrochemical activity of strain LLD-1 was confirmed by electrochemi...

The Effect of Charge at the Surface of Silver Nanoparticles on Antimicrobial Activity against Gram-Positive and Gram-Negative Bacteria: A Preliminary Study

International Nuclear Information System (INIS)

Abbaszadegan, A.; Ghahramani, Y.; Nabavizadeh, M.; Gholami, A.; Hemmateenejad, I.; Dorostkar, S.; Sharghi, H.

2014-01-01

The bactericidal efficiency of various positively and negatively charged silver nanoparticles has been extensively evaluated in literature, but there is no report on efficacy of neutrally charged silver nanoparticles. The goal of this study is to evaluate the role of electrical charge at the surface of silver nanoparticles on antibacterial activity against a panel of microorganisms. Three different silver nanoparticles were synthesized by different methods, providing three different electrical surface charges (positive, neutral, and negative). The antibacterial activity of these nanoparticles was tested against gram-positive (i.e., Staphylococcus aureus, Streptococcus mutans, and Streptococcus pyogenes) and gram-negative (i.e., Escherichia coli and Proteus vulgaris) bacteria. Well diffusion and micro-dilution tests were used to evaluate the bactericidal activity of the nanoparticles. According to the obtained results, the positively-charged silver nanoparticles showed the highest bactericidal activity against all microorganisms tested. The negatively charged silver nanoparticles had the least and the neutral nanoparticles had intermediate antibacterial activity. The most resistant bacteria were Proteus vulgaris. We found that the surface charge of the silver nanoparticles was a significant factor affecting bactericidal activity on these surfaces. Although the positively charged nanoparticles showed the highest level of effectiveness against the organisms tested, the neutrally charged particles were also potent against most bacterial species.

Características morfo-tintoriales en el ciclo celular de Acinetobacter baumannii por los métodos de Gram y 4’, 6-diamidino-2’-fenilindol, dihidrocloruro | Morphological and staining characteristics in cell cycle of Acinetobacter baumannii by the methods of Gram and 4´, 6-diamidino-2´-phenylindole, dihydrochloride

Directory of Open Access Journals (Sweden)

Evelin Flores Hernández

2017-11-01

Full Text Available Several studies have reported variations in bacterial staining by Gram, which are associated with the content of nucleic acids in the bacterial cell cycle. In order to give continuity to the microscopical studies pertaining to the bacterial cell cycle, the morphological and staining characteristics were evaluated by Gram and 4', 6-diamidino-2'-phenylindole, dihydrochloride (DAPI in the cell cycle of Acinetobacter baumannii. From the synchronic cultures of 4 strains of A. baumannii, the cell cycle was studied incubating them in aerobiosis at 30ºC, taking aliquots every 5 min for 2 h. In turn, the respective extensions were made and stained with Gram and DAPI for their posterior microscopical study. In the statistical analysis Fischer's test was used to associate morphological and the staining characteristics by Gram and DAPI. In 74% of cycle times, the cocoid form predominated and 67% in the negative Gram staining. When determining the mean and standard deviation of bacterial morphological values, it was observed that between 15 and 55 min the bacterium initiates a new cell cycle, decreasing its diameter and length. There was a statistically significant association (p < 0.05 between the lax and compact appearance of the material with the light and intense fluorescent, respectively; likewise, between the strong Gram positive character and the intense fluorescence. It is concluded that in the Gram positive reaction, the DNA is possibly involved and that the purple material of compact and lax aspect observed by Gram staining in the bacterial cytoplasm correspond to the nucleoid and the morphological changes obey to the cellular growth process. These findings contribute to explain the mechanism of Gram staining, as well as its variability and the pleomorphism observed in this bacterial genus.

Effects of rhodomyrtone on Gram-positive bacterial tubulin homologue FtsZ

Directory of Open Access Journals (Sweden)

Dennapa Saeloh

2017-02-01

Full Text Available Rhodomyrtone, a natural antimicrobial compound, displays potent activity against many Gram-positive pathogenic bacteria, comparable to last-defence antibiotics including vancomycin and daptomycin. Our previous studies pointed towards effects of rhodomyrtone on the bacterial membrane and cell wall. In addition, a recent molecular docking study suggested that the compound could competitively bind to the main bacterial cell division protein FtsZ. In this study, we applied a computational approach (in silico, in vitro, and in vivo experiments to investigate molecular interactions of rhodomyrtone with FtsZ. Using molecular simulation, FtsZ conformational changes were observed in both (S- and (R-rhodomyrtone binding states, compared with the three natural states of FtsZ (ligand-free, GDP-, and GTP-binding states. Calculations of free binding energy showed a higher affinity of FtsZ to (S-rhodomyrtone (−35.92 ± 0.36 kcal mol−1 than the GDP substrate (−23.47 ± 0.25 kcal mol−1 while less affinity was observed in the case of (R-rhodomyrtone (−18.11 ± 0.11 kcal mol−1. In vitro experiments further revealed that rhodomyrtone reduced FtsZ polymerization by 36% and inhibited GTPase activity by up to 45%. However, the compound had no effect on FtsZ localization in Bacillus subtilis at inhibitory concentrations and cells also did not elongate after treatment. Higher concentrations of rhodomyrtone did affect localization of FtsZ and also affected localization of its membrane anchor proteins FtsA and SepF, showing that the compound did not specifically inhibit FtsZ but rather impaired multiple divisome proteins. Furthermore, a number of cells adopted a bean-like shape suggesting that rhodomyrtone possibly possesses further targets involved in cell envelope synthesis and/or maintenance.

Endocarditis associated with cardiac catheterization due to a Gram-positive coccus designated Micrococcus mucilaginosus incertae sedis.

Science.gov (United States)

Rubin, S J; Lyons, R W; Murcia, A J

1978-01-01

A gram-positive coccus, presently named Micrococcus mucilaginosus incertae sedis, was isolated from 14 blood cultures from a patient with endocarditis. The first positive blood culture was drawn 5 days after the patient underwent cardiac catheterization. PMID:670378

A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria

DEFF Research Database (Denmark)

Ferro, Roberto; Rennig, Maja; Hernández Rollán, Cristina

2018-01-01

with a long history in food fermentation. We have developed a synbio approach for increasing gene expression in two Gram-positive bacteria. First of all, the gene of interest was coupled to an antibiotic resistance gene to create a growth-based selection system. We then randomised the translation initiation...... region (TIR) preceding the gene of interest and selected clones that produced high protein titres, as judged by their ability to survive on high concentrations of antibiotic. Using this approach, we were able to significantly increase production of two industrially relevant proteins; sialidase in B....... subtilis and tyrosine ammonia lyase in L. lactis. Gram-positive bacteria are widely used to produce industrial enzymes. High titres are necessary to make the production economically feasible. The synbio approach presented here is a simple and inexpensive way to increase protein titres, which can be carried...

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections.

Science.gov (United States)

Kobayashi, Naomi; Bauer, Thomas W; Tuohy, Marion J; Lieberman, Isador H; Krebs, Viktor; Togawa, Daisuke; Fujishiro, Takaaki; Procop, Gary W

2006-08-01

We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of "molecular Gram stain" with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results.

Gram staining in the diagnosis of acute septic arthritis.

Science.gov (United States)

Faraj, A A; Omonbude, O D; Godwin, P

2002-10-01

This study aimed at determining the sensitivity and specificity of Gram staining of synovial fluid as a diagnostic tool in acute septic arthritis. A retrospective study was made of 22 patients who had arthroscopic lavage following a provisional diagnosis of acute septic arthritis of the knee joint. Gram stains and cultures of the knee aspirates were compared with the clinical and laboratory parameters, to evaluate their usefulness in diagnosing acute arthritis. All patients who had septic arthritis had pain, swelling and limitation of movement. CRP was elevated in 90% of patients. The incidence of elevated white blood cell count was higher in the group of patients with a positive Gram stain study (60%) as compared to patients with a negative Gram stain study (33%). Gram staining sensitivity was 45%. Its specificity was however 100%. Gram staining is an unreliable tool in early decision making in patients requiring urgent surgical drainage and washout.

The Mechanisms of Virulence Regulation by Small Noncoding RNAs in Low GC Gram-Positive Pathogens

Directory of Open Access Journals (Sweden)

Stephanie Pitman

2015-12-01

Full Text Available The discovery of small noncoding regulatory RNAs (sRNAs in bacteria has grown tremendously recently, giving new insights into gene regulation. The implementation of computational analysis and RNA sequencing has provided new tools to discover and analyze potential sRNAs. Small regulatory RNAs that act by base-pairing to target mRNAs have been found to be ubiquitous and are the most abundant class of post-transcriptional regulators in bacteria. The majority of sRNA studies has been limited to E. coli and other gram-negative bacteria. However, examples of sRNAs in gram-positive bacteria are still plentiful although the detailed gene regulation mechanisms behind them are not as well understood. Strict virulence control is critical for a pathogen’s survival and many sRNAs have been found to be involved in that process. This review outlines the targets and currently known mechanisms of trans-acting sRNAs involved in virulence regulation in various gram-positive pathogens. In addition, their shared characteristics such as CU interaction motifs, the role of Hfq, and involvement in two-component regulators, riboswitches, quorum sensing, or toxin/antitoxin systems are described.

Effect of silver/copper and copper oxide nanoparticle powder on growth of Gram-negative and Gram-positive bacteria and their toxicity against the normal human dermal fibroblasts

International Nuclear Information System (INIS)

Peszke, Jerzy; Nowak, Anna; Szade, Jacek; Szurko, Agnieszka; Zygadło, Dorota; Michałowska, Marlena; Krzyściak, Paweł; Zygoń, Patrycja; Ratuszna, Alicja; Ostafin, Marek M.

2016-01-01

Engineered nanomaterials, especially metallic nanoparticles, are the most popular system applied in daily life products. The study of their biological and toxicity properties seems to be indispensable. In this paper, we present results of biological activity of Ag/Cu nanoparticles. These nanoparticles show more promising killing/inhibiting properties on Gram-negative bacteria than for Gram-positive ones. The Gram-negative bacteria show strong effect already at the concentration of 1 ppm after 15 min of incubation. Moreover, in vitro tests of toxicity made on normal human dermal fibroblast cultures showed that after 72 h of incubation with Ag/Cu nanoparticles, they are less toxic then Cu 2 O/CuO nanoparticles.

Effect of silver/copper and copper oxide nanoparticle powder on growth of Gram-negative and Gram-positive bacteria and their toxicity against the normal human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Peszke, Jerzy; Nowak, Anna, E-mail: ana.maria.nowak@gmail.com; Szade, Jacek; Szurko, Agnieszka; Zygadło, Dorota; Michałowska, Marlena [University of Silesia, A. Chelkowski Institute of Physics (Poland); Krzyściak, Paweł [Jagiellonian University Medical College, Department of Mycology Chair of Microbiology (Poland); Zygoń, Patrycja [Czestochowa University of Technology, Institute of Materials Engineering (Poland); Ratuszna, Alicja [University of Silesia, A. Chelkowski Institute of Physics (Poland); Ostafin, Marek M. [Department of Microbiology University of Agriculture (Poland)

2016-12-15

Engineered nanomaterials, especially metallic nanoparticles, are the most popular system applied in daily life products. The study of their biological and toxicity properties seems to be indispensable. In this paper, we present results of biological activity of Ag/Cu nanoparticles. These nanoparticles show more promising killing/inhibiting properties on Gram-negative bacteria than for Gram-positive ones. The Gram-negative bacteria show strong effect already at the concentration of 1 ppm after 15 min of incubation. Moreover, in vitro tests of toxicity made on normal human dermal fibroblast cultures showed that after 72 h of incubation with Ag/Cu nanoparticles, they are less toxic then Cu{sub 2}O/CuO nanoparticles.

Evaluation of Inhibitory and Lethal Effects of Aqueous, Ethanolic and Hydroalcoholic Extracts of Aerial Parts of Salvia chorassanica against Some Gram-negative and Gram-positive Bacteria in Vitro

Directory of Open Access Journals (Sweden)

Azam Mehraban

2016-05-01

Full Text Available Abstract Background and Objectives: Development of bacterial resistance to antibiotics has led to an increased tendency to development of new more effective and non-toxic antimicrobial compounds. In this study, the inhibitory and lethal effects of aqueous, ethanolic, and hydroalcoholic extracts of aerial parts of Salvia chorassanica were evaluated against Listeria monocytogenes, Bacillus cereus, Salmonella typhi, and Escherichia coli O:157. Methods: In this study, Kirby–Bauer disk diffusion method was used to evaluate antimicrobial activity. In this method, bacteria were cultivated as grass culture in Mueller Hinton Agar (MHA media. To determine the minimum inhibitory concentration and minimum bactericidal concentration, micro-dilution method with ELISA and addition of phenyl tetrazolium chloride reagent, were used. Data were analyzed using one-way ANOVA and Duncan’s test at the significance level of p<0.05. Results: The highest diameter of inhibition in agar diffusion method was related to hydroalcoholic extract of aerial parts of Salvia chorassanica against Gram-positive bacteria Bacillus cereus. The amount of calculated MIC of hydro-alcoholic extract for Gram-positive bacteria was 30mg/ml. This amount was the lowest among other measured MIC. Conclusion: Based on the results of this study, Gram-negative bacteria showed more resistance to different extracts of aerial parts of Salvia chorassanica as compared to Gram-positive bacteria, so that Salmonella typhi was found to be the most resistant bacterium among the tested bacteria.

Characterization and Discrimination of Gram-Positive Bacteria Using Raman Spectroscopy with the Aid of Principal Component Analysis

Directory of Open Access Journals (Sweden)

Alia Colniță

2017-09-01

Full Text Available Raman scattering and its particular effect, surface-enhanced Raman scattering (SERS, are whole-organism fingerprinting spectroscopic techniques that gain more and more popularity in bacterial detection. In this work, two relevant Gram-positive bacteria species, Lactobacillus casei (L. casei and Listeria monocytogenes (L. monocytogenes were characterized based on their Raman and SERS spectral fingerprints. The SERS spectra were used to identify the biochemical structures of the bacterial cell wall. Two synthesis methods of the SERS-active nanomaterials were used and the recorded spectra were analyzed. L. casei and L. monocytogenes were successfully discriminated by applying Principal Component Analysis (PCA to their specific spectral data.

Experience With Rapid Microarray-Based Diagnostic Technology and Antimicrobial Stewardship for Patients With Gram-Positive Bacteremia.

Science.gov (United States)

Neuner, Elizabeth A; Pallotta, Andrea M; Lam, Simon W; Stowe, David; Gordon, Steven M; Procop, Gary W; Richter, Sandra S

2016-11-01

OBJECTIVE To describe the impact of rapid diagnostic microarray technology and antimicrobial stewardship for patients with Gram-positive blood cultures. DESIGN Retrospective pre-intervention/post-intervention study. SETTING A 1,200-bed academic medical center. PATIENTS Inpatients with blood cultures positive for Staphylococcus aureus, Enterococcus faecalis, E. faecium, Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S. anginosus, Streptococcus spp., and Listeria monocytogenes during the 6 months before and after implementation of Verigene Gram-positive blood culture microarray (BC-GP) with an antimicrobial stewardship intervention. METHODS Before the intervention, no rapid diagnostic technology was used or antimicrobial stewardship intervention was undertaken, except for the use of peptide nucleic acid fluorescent in situ hybridization and MRSA agar to identify staphylococcal isolates. After the intervention, all Gram-positive blood cultures underwent BC-GP microarray and the antimicrobial stewardship intervention consisting of real-time notification and pharmacist review. RESULTS In total, 513 patients with bacteremia were included in this study: 280 patients with S. aureus, 150 patients with enterococci, 82 patients with stretococci, and 1 patient with L. monocytogenes. The number of antimicrobial switches was similar in the pre-BC-GP (52%; 155 of 300) and post-BC-GP (50%; 107 of 213) periods. The time to antimicrobial switch was significantly shorter in the post-BC-GP group than in the pre-BC-GP group: 48±41 hours versus 75±46 hours, respectively (P<.001). The most common antimicrobial switch was de-escalation and time to de-escalation, was significantly shorter in the post-BC-GP group than in the pre-BC-GP group: 53±41 hours versus 82±48 hours, respectively (P<.001). There was no difference in mortality or hospital length of stay as a result of the intervention. CONCLUSIONS The combination of a rapid microarray diagnostic test with an antimicrobial

Antimicrobial resistance in Gram-positive bacteria from Timorese River Buffalo (Bubalus bubalis) skin microbiota.

Science.gov (United States)

Oliveira, Manuela; Monteiro, José L; Rana, Sílvia; Vilela, Cristina L

2010-06-01

The Timorese River Buffalo (Bubalus bubalis) plays a major role in the East Timor economy, as it is an important source of animal protein in human nutrition. They are widely spread throughout the country and are in direct contact with the populations. In spite of this proximity, information on their microbiota is scarce. This work aimed at characterizing the skin microbiota of the East Timorese River Buffalo and its antimicrobial resistance profile. Skin swab samples were taken from 46 animals in surveys conducted in three farms located in "Suco de Nairete", Lospalos district, during July and August 2006. Bacteria were isolated and identified according to conventional microbiological procedures. A total of 456 isolates were obtained, including Gram-positive (n = 243) and Gram-negative (n = 213) bacteria. Due to their importance as potential pathogens and as vehicles for antimicrobial resistance transmission, Gram-positive cocci (n = 27) and bacilli (n = 77) isolates were further characterized, and their antimicrobial resistance profile determined by the disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines. This study shows the high bacterial diversity of B. bubalis skin microbiota, representing an important first step towards understanding its importance and epidemiologic role in animal health. It also points out the potential role of these animals as vectors of antimicrobial resistant bacteria dissemination and the importance of antimicrobial resistance monitoring in developing countries.

Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium.

Science.gov (United States)

Tsukatani, Tadayuki; Suenaga, Hikaru; Higuchi, Tomoko; Shiga, Masanobu; Noguchi, Katsuya; Matsumoto, Kiyoshi

2011-01-01

Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5µg/ml crystal violet, 5.0 µg/ml daptomycin, and 5.0µg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.

Secretory phospholipase A2 in dromedary tears: a host defense against staphylococci and other gram-positive bacteria.

Science.gov (United States)

Ben Bacha, Abir; Abid, Islem

2013-03-01

The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

Jeotgalibacillus soli sp. nov., a Gram-stain-positive bacterium isolated from soil.

Science.gov (United States)

Cunha, Sofia; Tiago, Igor; Paiva, Gabriel; Nobre, Fernanda; da Costa, Milton S; Veríssimo, António

2012-03-01

A Gram-staining-positive, motile, rod-shaped, spore-forming bacterium, designated P9(T), was isolated from soil in Portugal. This organism was aerobic and catalase- and oxidase-positive. It had an optimum growth temperature of about 35 °C and an optimum growth pH of about 8.0-8.5, and grew in medium with 0-9% (w/v) NaCl. The cell-wall peptidoglycan was of the A1α type, with L-lysine as the diagnostic diamino acid. The major respiratory quinone was menaquinone 7 (MK-7) and the major fatty acids were anteiso-C(15:0) (45.4%), iso-C(15:0) (22.0%) and anteiso-C(17:0) (11.2%). The genomic DNA G+C content was about 39.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain P9(T) was most closely related to Jeotgalibacillus campisalis DSM 18983(T) (96.8%) and Jeotgalibacillus marinus DSM 1297(T) (96.5%). These two recognized species formed a coherent cluster with strain P9(T) that was supported by a bootstrap value of 99%. On the basis of the phylogenetic analysis and physiological and biochemical characteristics, strain P9(T) (=DSM 23228(T)=LMG 25523(T)) represents a novel species of the genus Jeotgalibacillus, for which the name Jeotgalibacillus soli sp. nov. is proposed.

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA.

Directory of Open Access Journals (Sweden)

David Sychantha

2017-10-01

Full Text Available The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.

Streptococcus mutans: a new Gram-positive paradigm?

Science.gov (United States)

Quivey, Robert G.; Koo, Hyun; Abranches, Jacqueline

2013-01-01

Despite the enormous contributions of the bacterial paradigms Escherichia coli and Bacillus subtilis to basic and applied research, it is well known that no single organism can be a perfect representative of all other species. However, given that some bacteria are difficult, or virtually impossible, to cultivate in the laboratory, that some are recalcitrant to genetic and molecular manipulation, and that others can be extremely dangerous to manipulate, the use of model organisms will continue to play an important role in the development of basic research. In particular, model organisms are very useful for providing a better understanding of the biology of closely related species. Here, we discuss how the lifestyle, the availability of suitable in vitro and in vivo systems, and a thorough understanding of the genetics, biochemistry and physiology of the dental pathogen Streptococcus mutans have greatly advanced our understanding of important areas in the field of bacteriology such as interspecies biofilms, competence development and stress responses. In this article, we provide an argument that places S. mutans, an organism that evolved in close association with the human host, as a novel Gram-positive model organism. PMID:23393147

Changes of the Quinolones Resistance to Gram-positive Cocci Isolated during the Past 8 Years in the First Bethune Hospital

Science.gov (United States)

Xu, Jiancheng; Chen, Qihui; Yao, Hanxin; Zhou, Qi

This study was to investigate the quinolones resistance to gram-positive cocci isolated in the First Bethune Hospital during the past 8 years. Disk diffusion test was used to study the antimicrobial resistance. The data were analyzed by WHONET 5 software according to Clinical and Laboratory Standards Institute (CLSI). The rates of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococci (MRCNS) were 50.8%∼83.3% and 79.4%∼81.5%during the past 8 years, respectively. In recent 8 years, the quinolones resistance to gram-positive cocci had increased. Monitoring of the quinolones resistance to gram-positive cocci should be strengthened. The change of the antimicrobial resistance should be investigated in order to guide rational drug usage in the clinic and prevent bacterial strain of drug resistance from being transmitted.

A Trojan-Horse Strategy Including a Bacterial Suicide Action for the Efficient Use of a Specific Gram-Positive Antibiotic on Gram-Negative Bacteria.

Science.gov (United States)

Schalk, Isabelle J

2018-05-10

In the alarming context of rising bacterial antibiotic resistance, there is an urgent need to discover new antibiotics or increase and/or enlarge the activity of those currently in use. The need for new antibiotics is even more urgent in the case of Gram-negative bacteria, such as Acinetobacter, Pseudomonas, and Enterobacteria, which have become resistant to many antibiotics and have an outer membrane with very low permeability to drugs. Vectorization of antibiotics using siderophores may be a solution to bypass such a bacterial wall: the drugs use the iron transporters of the outer membrane as gates to enter bacteria in a Trojan-horse strategy. Designing siderophore-antibiotics that can cross outer membranes has become almost routine, but their transport across the inner membrane is still a limiting step, as well as a strategy that allows dissociation of the antibiotic from the siderophore once inside the bacteria. Liu et al. ( J. Med. Chem. 2018 , DOI: 10.1021/acs.jmedchem.8b00218 ) report the synthesis of a siderophore-cephalosporin compound and demonstrate that β-lactams, such as cephalosporins, can serve as β-lactamase-triggered releasable linkers to allow intracellular delivery of Gram-positive antibiotics to Gram-negative bacteria.

Antibacterial activity and interactions of plant essential oil combinations against Gram-positive and Gram-negative bacteria

Directory of Open Access Journals (Sweden)

Cristina Anamaria Semeniuc

2017-04-01

Full Text Available The aim of this study was to compare the antibacterial effects of several essential oils (EOs alone and in combination against different Gram-positive and Gram-negative bacteria associated with food products. Parsley, lovage, basil, and thyme EOs, as well as their mixtures (1:1, v/v, were tested against Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella typhimurium. The inhibitory effects ranged from strong (thyme EO against E. coli to no inhibition (parsley EO against P. aeruginosa. Thyme EO exhibited strong (against E. coli, moderate (against S. typhimurium and B. cereus, or mild inhibitory effects (against P. aeruginosa and S. aureus, and basil EO showed mild (against E. coli and B. cereus or no inhibitory effects (against S. typhimurium, P. aeruginosa, and S. aureus. Parsley and lovage EOs revealed no inhibitory effects against all tested strains. Combinations of lovage/thyme and basil/thyme EOs displayed antagonistic effects against all bacteria, parsley/thyme EOs against B. cereus, S. aureus, P. aeruginosa, and E. coli, and lovage/basil EOs against B. cereus and E. coli. Combinations of parsley/lovage and parsley/basil EOs exhibited indifferent effects against all bacteria. The combination of lovage/basil EO showed indifferent effect against S. aureus, P. aeruginosa, and S. typhimurium, and the combination parsley/thyme EO against S. typhimurium. Thyme EO has the highest percentage yield and antibacterial potential from all tested formulations; its combination with parsley, lovage, and basil EOs determines a reduction of its antibacterial activity. Hence, it is recommended to be used alone as the antibacterial agent.

Determination of the irradiation dose for the inhibition (D-10 radiation doses) of some gram negative and gram positive bacteria in peptone saline water

International Nuclear Information System (INIS)

Ayhan, H.; Tutluer, H.

1994-01-01

Determination of the irradiation dose for the inhibition of some pathogenic bacteria which cause food poisoning and spoilage were aimed. For this purpose, Salmonella typhi, Salmonella typhimurium,Salmonella enteridits,Klebsiella pneumonia, Pseudomonas fluorescence,Proteus vulgaris, Aeromonas hydrophila ,(gram-negative bacteria) and Bacillus cereus, Staphylococcus aureus strain 24,Staphylococcus aureus ATCC 6538 P,Staphylococcus epidermidis strain 115 and Clostridium perfringens A4TTK,(gram-positive bacteria) were used.Sensitivity of above mentioned bacteria to gamma rays (source Cs-137) was examined in saline with 0.1% peptone at different temperatures.Survivor plots (log.10 number of survivors versus dose) were determined by regression analysis of the data.Decimal reduction doses (D values in kGy) were calculated as the slope obtained from the regression analysis

Evaluation of the LightCycler Staphylococcus M GRADE kits on positive blood cultures that contained gram-positive cocci in clusters.

Science.gov (United States)

Shrestha, Nabin K; Tuohy, Marion J; Padmanabhan, Ravindran A; Hall, Gerri S; Procop, Gary W

2005-12-01

We evaluated the Roche LightCycler Staphylococcus M(GRADE) kits to differentiate between Staphylococcus aureus and coagulase-negative staphylococci in blood cultures growing clusters of gram-positive cocci. Testing 100 bottles (36 containing S. aureus), the assay was 100% sensitive and 98.44% specific for S. aureus and 100% sensitive and specific for coagulase-negative staphylococci.

Widespread abundance of functional bacterial amyloid in Mycolata and other Gram-positive bacteria

DEFF Research Database (Denmark)

Jordal, Peter Bruun; Dueholm, Morten Simonsen; Larsen, Poul

2009-01-01

extracellular fibrils were also produced. In three cases, FuBA was only revealed after extensive removal of extracellular material by saponification, indicating an integrated attachment within the cellular envelope. Spores from species within the genera Streptomyces, Bacillus and Nocardia were all coated...... analysis. We conclude that amyloid is widespread among Gram-positive bacteria and may in many species constitute a hitherto overlooked integral part of the spores and the cellular envelope....

Limitations in the use of Drosophila melanogaster as a model host for gram-positive bacterial infection

DEFF Research Database (Denmark)

Jensen, Rikke Lind; Pedersen, K.S.; Loeschcke, V

2007-01-01

resistance respectively, were subjected to infection by L. monocytogenes, S. aureus and E. coli. Mortality rates were comparable with that of the Oregon R strain. Conclusions: Use of the injection method shows the limitation of D. melanogaster as a model host for gram-positive bacteria as opportunistic......Aims: To examine sensitivities of various Drosophila melanogaster strains towards human pathogenic and nonpathogenic gram-positive bacteria. Methods and Results: The D. melanogaster Oregon R strain was infected by injecting the thorax with a needle containing Escherichia coli (negative control...... with the negative control. Infection with L. innocua, B. subtilis or C. maltaromaticum also resulted in a high fly mortality, whereas Lact. plantarum and P. acidilactici resulted in a slightly increased mortality. Four additional D. melanogaster lines, three of which had been selected for heat, cold and desiccation...

Characterization of an Hfq dependent antisense sRNA in the Gram-positive human pathogen Listeria monocytogenes

DEFF Research Database (Denmark)

Nielsen, Jesper Sejrup; Lei Kristensen, Lisbeth; Hanghøj Chrisitansen, Mie

between sRNA and target mRNA rely on the RNA chaperone Hfq. Hfq is a ubiquitous protein found in almost all genres of bacterial life. However, so far its role as an RNA chaperone has only been described in Gram-negative species such as Escherichia coli and Salmonella (Vogel, J. 2009). We previously...... identified several Hfq-binding sRNAs in the Gram-positive human pathogen L. monocytogenes (Christiansen et al 2006). Through bioinformatics, we have identified a number of candidate targets for one of these sRNAs (LhrA). Here, we present the characterization of one of these targets. Our results suggest...

Gram-positive bacterial lipoglycans based on a glycosylated diacylglycerol lipid anchor are microbe-associated molecular patterns recognized by TLR2.

Directory of Open Access Journals (Sweden)

Landry Blanc

Full Text Available Innate immune recognition is the first line of host defense against invading microorganisms. It is a based on the detection, by pattern recognition receptors (PRRs, of invariant molecular signatures that are unique to microorganisms. TLR2 is a PRR that plays a major role in the detection of Gram-positive bacteria by recognizing cell envelope lipid-linked polymers, also called macroamphiphiles, such as lipoproteins, lipoteichoic acids and mycobacterial lipoglycans. These microbe-associated molecular patterns (MAMPs display a structure based on a lipid anchor, being either an acylated cysteine, a glycosylated diacylglycerol or a mannosyl-phosphatidylinositol respectively, and having in common a diacylglyceryl moiety. A fourth class of macroamphiphile, namely lipoglycans, whose lipid anchor is made, as for lipoteichoic acids, of a glycosylated diacylglycerol unit rather than a mannosyl-phosphatidylinositol, is found in Gram-positive bacteria and produced by certain Actinobacteria, including Micrococcus luteus, Stomatococcus mucilaginosus and Corynebacterium glutamicum. We report here that these alternative lipoglycans are also recognized by TLR2 and that they stimulate TLR2-dependant cytokine production, including IL-8, TNF-α and IL-6, and cell surface co-stimulatory molecule CD40 expression by a human macrophage cell line. However, they differ by their co-receptor requirement and the magnitude of the innate immune response they elicit. M. luteus and S. mucilaginosus lipoglycans require TLR1 for recognition by TLR2 and induce stronger responses than C. glutamicum lipoglycan, sensing of which by TLR2 is dependent on TLR6. These results expand the repertoire of MAMPs recognized by TLR2 to lipoglycans based on a glycosylated diacylglycerol lipid anchor and reinforce the paradigm that macroamphiphiles based on such an anchor, including lipoteichoic acids and alternative lipoglycans, induce TLR2-dependant innate immune responses.

Breaking barriers: expansion of the use of endolysins as novel antibacterials against Gram-negative bacteria.

Science.gov (United States)

Briers, Yves; Lavigne, Rob

2015-01-01

The emergence and spread of antibiotic-resistant bacteria drives the search for novel classes of antibiotics to replenish our armamentarium against bacterial infections. This is particularly critical for Gram-negative pathogens, which are intrinsically resistant to many existing classes of antibiotics due to the presence of a protective outer membrane. In addition, the antibiotics development pipeline is mainly oriented to Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus. A promising novel class of antibacterials is endolysins. These enzymes encoded by bacterial viruses hydrolyze the peptidoglycan layer with high efficiency, resulting in abrupt osmotic lysis and cell death. Their potential as novel antibacterials to treat Gram-positive bacteria has been extensively demonstrated; however, the Gram-negative outer membrane has presented a formidable barrier for the use of endolysins against Gram-negatives until recently. This review reports on the most recent advances in the development of endolysins to kill Gram-negative species with a special focus on endolysin-engineered Artilysins(®).

CRP-ductin, the mouse homologue of gp-340/deleted in malignant brain tumors 1 (DMBT1), binds gram-positive and gram-negative bacteria and interacts with lung surfactant protein D

DEFF Research Database (Denmark)

Madsen, Jens; Tornøe, Ida; Nielsen, Ole

2003-01-01

CRP-ductin is a protein expressed mainly by mucosal epithelial cells in the mouse. Sequence homologies indicate that CRP-ductin is the mouse homologue of human gp-340, a glycoprotein that agglutinates microorganisms and binds the lung mucosal collectin surfactant protein-D (SP-D). Here we report...... that purified CRP-ductin binds human SP-D in a calcium-dependent manner and that the binding is not inhibited by maltose. The same properties have previously been observed for gp-340 binding of SP-D. CRP-ductin also showed calcium-dependent binding to both gram-positive and -negative bacteria. A polyclonal...... antibody raised against gp-340 reacted specifically with CRP-ductin in Western blots. Immunoreactivity to CRP-ductin was found in the exocrine pancreas, in epithelial cells throughout the gastrointestinal tract and in the parotid ducts. A panel of RNA preparations from mouse tissues was screened for CRP...

New and improved? A review of novel antibiotics for Gram-positive bacteria.

Science.gov (United States)

Abbas, M; Paul, M; Huttner, A

2017-10-01

The number of antibiotics in the pipeline targeting Gram-positive pathogens has increased in recent years. This narrative review aims to provide a summary of existing evidence on efficacy, microbiological spectrum and safety of novel systemic antibiotics that have either recently been licensed or completed phase III trials, and possess activity predominantly against Gram-positive organisms. A review of the published literature via the MEDLINE database was performed. In addition, ongoing trials were identified through a search of the clinical trial registration platform clinicaltrials.gov, and when necessary, pharmaceutical companies responsible for the development of the drug were contacted for further information. Data on development, microbiological spectrum, pharmacokinetic/pharmacodynamic properties, clinical efficacy, safety and cost are presented for the new cephalosporins ceftaroline and ceftobiprole; the lipoglycopeptides dalbavancin, oritavancin and telavancin; the fluoroquinolones delafloxacin, nemonoxacin and zabofloxacin; the dihydrofolate-reductase inhibitor iclaprim; the pleuromutilin lefamulin; and the tetracycline omadacycline. Although promising, these new antibiotics have so far been tested in non-severe infections whose treatment is generally uncomplicated and whose aetiologies were not predominantly multidrug-resistant pathogens. None of the new antibiotics have shown superiority to standard care, and none have been investigated for patient-relevant outcomes. Safety and pharmacokinetic data continue to be lacking. How these new drugs are to be integrated into the current armamentarium remains to be established. Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Antibacterial properties of biosurfactants against selected Gram-positive and -negative bacteria.

Science.gov (United States)

Díaz De Rienzo, Mayri A; Stevenson, Paul; Marchant, Roger; Banat, Ibrahim M

2016-01-01

The antibacterial properties and ability to disrupt biofilms of biosurfactants (rhamnolipids, sophorolipids) and sodium dodecyl sulphate (SDS) in the presence and absence of selected organic acids were investigated. Pseudomonas aeruginosa PAO1 was inhibited by sophorolipids and SDS at concentrations >5% v/v, and the growth of Escherichia coli NCTC 10418 was also inhibited by sophorolipids and SDS at concentrations >5% and 0.1% v/v, respectively. Bacillus subtilis NCTC 10400 was inhibited by rhamnolipids, sophorolipids and SDS at concentrations >0.5% v/v of all three; the same effect was observed with Staphylococcus aureus ATCC 9144. The ability to attach to surfaces and biofilm formation of P. aeruginosa PAO1, E. coli NCTC 10418 and B. subtilis NCTC 10400 was inhibited by sophorolipids (1% v/v) in the presence of caprylic acid (0.8% v/v). In the case of S. aureus ATCC 9144, the best results were obtained using caprylic acid on its own. It was concluded that sophorolipids are promising compounds for the inhibition/disruption of biofilms formed by Gram-positive and Gram-negative microorganisms and this activity can be enhanced by the presence of booster compounds such as caprylic acid. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Automated Interpretation of Blood Culture Gram Stains by Use of a Deep Convolutional Neural Network.

Science.gov (United States)

Smith, Kenneth P; Kang, Anthony D; Kirby, James E

2018-03-01

Microscopic interpretation of stained smears is one of the most operator-dependent and time-intensive activities in the clinical microbiology laboratory. Here, we investigated application of an automated image acquisition and convolutional neural network (CNN)-based approach for automated Gram stain classification. Using an automated microscopy platform, uncoverslipped slides were scanned with a 40× dry objective, generating images of sufficient resolution for interpretation. We collected 25,488 images from positive blood culture Gram stains prepared during routine clinical workup. These images were used to generate 100,213 crops containing Gram-positive cocci in clusters, Gram-positive cocci in chains/pairs, Gram-negative rods, or background (no cells). These categories were targeted for proof-of-concept development as they are associated with the majority of bloodstream infections. Our CNN model achieved a classification accuracy of 94.9% on a test set of image crops. Receiver operating characteristic (ROC) curve analysis indicated a robust ability to differentiate between categories with an area under the curve of >0.98 for each. After training and validation, we applied the classification algorithm to new images collected from 189 whole slides without human intervention. Sensitivity and specificity were 98.4% and 75.0% for Gram-positive cocci in chains and pairs, 93.2% and 97.2% for Gram-positive cocci in clusters, and 96.3% and 98.1% for Gram-negative rods. Taken together, our data support a proof of concept for a fully automated classification methodology for blood-culture Gram stains. Importantly, the algorithm was highly adept at identifying image crops with organisms and could be used to present prescreened, classified crops to technologists to accelerate smear review. This concept could potentially be extended to all Gram stain interpretive activities in the clinical laboratory. Copyright © 2018 American Society for Microbiology.

Rapid, highly sensitive detection of Gram-negative bacteria with lipopolysaccharide based disposable aptasensor.

Science.gov (United States)

Zhang, Jian; Oueslati, Rania; Cheng, Cheng; Zhao, Ling; Chen, Jiangang; Almeida, Raul; Wu, Jayne

2018-07-30

Gram-negative bacteria are one of the most common microorganisms in the environment. Their differential detection and recognition from Gram-positive bacteria has been attracting much attention over the years. Using Escherichia coli (E. coli) as a model, we demonstrated on-site detection of Gram-negative bacteria by an AC electrokinetics-based capacitive sensing method using commercial microelectrodes functionalized with an aptamer specific to lipopolysaccharides. Dielectrophoresis effect was utilized to enrich viable bacteria to the microelectrodes rapidly, achieving a detection limit of 10 2 cells/mL within a 30 s' response time. The sensor showed a negligible response to Staphylococcus aureus (S. aureus), a Gram-positive species. The developed sensor showed significant advantages in sensitivity, selectivity, cost, operation simplicity, and response time. Therefore, this sensing method has shown great application potential for environmental monitoring, food safety, and real-time diagnosis. Copyright © 2018 Elsevier B.V. All rights reserved.

Antimicrobial Photodynamic Therapy to Kill Gram-negative Bacteria

Science.gov (United States)

Sperandio, Felipe F; Huang, Ying-Ying; Hamblin, Michael R

2013-01-01

Antimicrobial photodynamic therapy (PDT) or photodynamic inactivation (PDI) is a new promising strategy to eradicate pathogenic microorganisms such as Gram-positive and Gram-negative bacteria, yeasts and fungi. The search for new approaches that can kill bacteria but do not induce the appearance of undesired drug-resistant strains suggests that PDT may have advantages over traditional antibiotic therapy. PDT is a non-thermal photochemical reaction that involves the simultaneous presence of visible light, oxygen and a dye or photosensitizer (PS). Several PS have been studied for their ability to bind to bacteria and efficiently generate reactive oxygen species (ROS) upon photostimulation. ROS are formed through type I or II mechanisms and may inactivate several classes of microbial cells including Gram-negative bacteria such as Pseudomonas aeruginosa, which are typically characterized by an impermeable outer cell membrane that contains endotoxins and blocks antibiotics, dyes, and detergents, protecting the sensitive inner membrane and cell wall. This review covers significant peer-reviewed articles together with US and World patents that were filed within the past few years and that relate to the eradication of Gram-negative bacteria via PDI or PDT. It is organized mainly according to the nature of the PS involved and includes natural or synthetic food dyes; cationic dyes such as methylene blue and toluidine blue; tetrapyrrole derivatives such as phthalocyanines, chlorins, porphyrins, chlorophyll and bacteriochlorophyll derivatives; functionalized fullerenes; nanoparticles combined with different PS; other formulations designed to target PS to bacteria; photoactive materials and surfaces; conjugates between PS and polycationic polymers or antibodies; and permeabilizing agents such as EDTA, PMNP and CaCl2. The present review also covers the different laboratory animal models normally used to treat Gram-negative bacterial infections with antimicrobial PDT. PMID

A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria.

Science.gov (United States)

Yang, Yunpeng; Zhang, Lu; Huang, He; Yang, Chen; Yang, Sheng; Gu, Yang; Jiang, Weihong

2017-01-24

Catabolite control protein A (CcpA) is the master regulator in Gram-positive bacteria that mediates carbon catabolite repression (CCR) and carbon catabolite activation (CCA), two fundamental regulatory mechanisms that enable competitive advantages in carbon catabolism. It is generally regarded that CcpA exerts its regulatory role by binding to a typical 14- to 16-nucleotide (nt) consensus site that is called a catabolite response element (cre) within the target regions. However, here we report a previously unknown noncanonical flexible architecture of the CcpA-binding site in solventogenic clostridia, providing new mechanistic insights into catabolite regulation. This novel CcpA-binding site, named cre var , has a unique architecture that consists of two inverted repeats and an intervening spacer, all of which are variable in nucleotide composition and length, except for a 6-bp core palindromic sequence (TGTAAA/TTTACA). It was found that the length of the intervening spacer of cre var can affect CcpA binding affinity, and moreover, the core palindromic sequence of cre var is the key structure for regulation. Such a variable architecture of cre var shows potential importance for CcpA's diverse and fine regulation. A total of 103 potential cre var sites were discovered in solventogenic Clostridium acetobutylicum, of which 42 sites were picked out for electrophoretic mobility shift assays (EMSAs), and 30 sites were confirmed to be bound by CcpA. These 30 cre var sites are associated with 27 genes involved in many important pathways. Also of significance, the cre var sites are found to be widespread and function in a great number of taxonomically different Gram-positive bacteria, including pathogens, suggesting their global role in Gram-positive bacteria. In Gram-positive bacteria, the global regulator CcpA controls a large number of important physiological and metabolic processes. Although a typical consensus CcpA-binding site, cre, has been identified, it remains

In vitro ciprofloxacin resistance patterns of gram positive bacteria isolated from clinical specimens in a teaching hospital in Saudi Arabia

International Nuclear Information System (INIS)

Akhtar, N.; Alzahrani, A.; Obeid, O.El-Treify; Dassal, D.

2009-01-01

Over the last few decades the ever-increasing level of bacterial resistance to antimicrobials has been a cause of worldwide concern. Fluoroquinolones, particularly ciprofloxacin has been used indiscriminately for both gram-positive and gram-negative bacterial infections. The increased use of ciprofloxacin has led to a progressive loss of bacterial susceptibility to this antibiotic. Therefore it is necessary to have update knowledge of resistance pattern of bacteria to this antibiotic so that alternate appropriate antibiotics can be used for ciprofloxacin-resistant bacterial infections. Objective: To evaluate the trends of ciprofloxacin resistance pattern in commonly isolated gram positive bacteria over time in a Saudi Arabian teaching hospital. Methods: A retrospective analysis was carried out for ciprofloxacin susceptibility patterns of 5534 isolates of gram-positive bacteria isolated from clinical specimens submitted to microbiology laboratories at King Fahd Hospital of the University (KFHU), Al-Khobar, Saudi Arabia during the period from January 2002 to August 2005. Results: Increase in ciprofloxacin resistance rates with some fluctuations, among these isolates, were observed. For Staphylococcus aureus, it varied from 4.62, 1.83, 7.01 and 3.98%, methicillin resistant Staphylococcus aureus (MRSA) 97.92, 97.75, 87.01 and 88.26%, Streptococcus pyogenes 5.35, 4.47, 14.44 and 3.53% during the years 2002, 2003, 2004 and 2005 respectively. Cirprofloxacin resistance during the years 2002, 2004 and 2005 for other isolates was as follows: Streptococcus pneumoniae, 30.23, 23.02 and 26.47%; enterococcus group D, 43.05, 20.68 and 57.03% and non-enterococcus group D, 62.96, 76.92 and 87.50% respectively. Conclusion: Ciprofloxacin resistance in gram positive bacterial clinical isolates particularly Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA) enterococcus group D, and non-enterococcus group D, has greatly increased and ciprofloxacin no more remains

Brazilian experience in EU-CORE: daptomycin registry and treatment of serious Gram-positive infections

Directory of Open Access Journals (Sweden)

Artur Timerman

Full Text Available OBJECTIVES: To collect data about non-controlled prescribing use of daptomycin and its impact among Brazilian patients with serious Gram positive bacterial infection, as well as the efficacy and safety outcomes. MATERIALS AND METHODS: This is a multi-center, retrospective, non-interventional registry (August 01, 2009 to June 30, 2011 to collect data on 120 patients (44 patients in the first year and 76 patients in the second year who had received at least one dose of commercial daptomycin in Brazil for the treatment of serious Gram-positive bacterial infection. RESULTS: Right-sided endocarditis (15.8%, complicated skin and soft tissue infections (cSSTIwound (15.0% and bacteremia-catheter-related (14.2% were the most frequent primary infections; lung (21.7% was the most common site for infection. Daptomycin was used empirically in 76 (63.3% patients, and methicillin-resistant Staphylococcus aureus (MRSA was the most common suspected pathogen (86.1%. 82.5% of the cultures were obtained prior to or shortly after initiation of daptomycin therapy. Staphylococcus spp. - coagulase negative, MRSA, and methicillin-susceptible S. aureus were the most frequently identified pathogens (23.8%, 23.8% and 12.5%, respectively. The most common daptomycin dose administered for bacteremia and cSSTI was 6 mg/kg (30.6% and 4 mg/kg (51.7%, respectively. The median duration of inpatient daptomycin therapy was 14 days. Most patients (57.1% did not receive daptomycin while in intensive care unit. Carbapenem (22.5% was the most commonly used antibiotic concomitantly. The patients showed clinical improvement after two days (median following the start of daptomycin therapy. The clinical success rate was 80.8% and the overall rate of treatment failure was 10.8%. The main reasons for daptomycin discontinuation were successful end of therapy (75.8%, switched therapy (11.7%, and treatment failure (4.2%. Daptomycin demonstrated a favorable safety and tolerability profile

Microbiological method for radiation sterilization (II). Identification procedure of gram positive bacteria by using BBL CRYSTAL GP identification kit

International Nuclear Information System (INIS)

Koshikawa, Tomihiko

2004-01-01

The part II in this title series describes details of the commercially available BBL CRYSTAL GP Identification Kit with the software (Becton, Dickinson and Co., Ltd.), by which identification of Gram positive bacteria as well as their number becoming easier in the radiation sterilization of medical devices. Isolation of a bacterium has to be confirmed by microscopy and its Gram positive property, by the Gram staining. The exponentially growing bacteria are to be inoculated in the Kit and cultured for 18-24 hr at 37 deg C with the lid attached by substrates for identification. Reactions to substrates are to be judged by CRYSTAL auto-reader, which is further to be searched by the computer software (code-book) for final identification. For possible misidentification, re-isolation of the bacterium, prolonged culture, concentrated inoculation and re-consideration for ranking of identification the software provides are necessary as well as other identification approaches. Representative bacteria as the bioburden are spp. of Bacilli, Corynebacteria, Staphylococci and Micrococci. (N.I.)

Veillonella, Firmicutes: Microbes disguised as Gram negatives

DEFF Research Database (Denmark)

Vesth, Tammi Camilla; Ozen, Asli; Andersen, Sandra Christine

2013-01-01

Negativicutes, including the genus Veillonella, stain Gram negative. Veillonella are among the most abundant organisms of the oral and intestinal microflora of animals and humans, in spite of being strict anaerobes. In this work, the genomes of 24 Negativicutes, including eight Veillonella spp., are compared......, with the exception of a shared LPS biosynthesis pathway. The clade within the class Negativicutes to which the genus Veillonella belongs exhibits unique properties, most of which are in common with Gram-positives and some with Gram negatives. They are only distantly related to Clostridia, but are even less closely...... related to Gram-negative species. Though the Negativicutes stain Gram-negative and possess two membranes, the genome and proteome analysis presented here confirm their place within the (mainly) Gram positive phylum of the Firmicutes. Further studies are required to unveil the evolutionary history...

Two investigational glycylcyclines, DMG-DMDOT and DMG-MINO. Antimicrobial activity studies against gram-positive species.

Science.gov (United States)

Johnson, D M; Jones, R N

1996-01-01

DMG-DMDOT (CL-331,002 OR CL-331,928) and DMG-MINO (CL-329,998 or CL-344,677) are two new semisynthetic tetracyclines called glycylcyclines, with a broad spectrum of activity and includes Enterobacteriaceae, Gram-positive cocci, JK diphtheroids, and Bacillus cereus. Potent activity was demonstrated against all Streptococcus spp. strains [minimum inhibitory concentrations] (MIC90S) 0.06-0.25 micrograms/ml) and staphylococci (oxacillin susceptible ans resistant; MIC90S 0.12-2 micrograms/ml). Both glycylcyclines (MIC90, 0.06 micrograms/ml) were more potent than minocycline (MIC90 8 micrograms/ml) against Enterococcus faecium, many of which were vancomycin resistant (116 strains). Organisms with minocycline MICs at > or = 8 micrograms/ml (Staphylococcus aureus, enterococci, beta-hemolytic streptococci, and pneumococci) had glycylcycline MIC results ranging from 0.06 to 0.5 micrograms/ml (e.g., apparent use against existing tetracycline-resistance phenotypes). Drugs in this class appear promising for therapy of infections caused by Gram-positive species now testing resistant to contemporary antimicrobial agents, and further development of compounds in this class is encouraged.

Environmental gram-positive mastitis treatment: in vitro sensitivity and bacteriologic cure.

Science.gov (United States)

Cattell, M B; Dinsmore, R P; Belschner, A P; Carmen, J; Goodell, G

2001-09-01

A clinical trial was conducted in a large dairy herd to determine the efficacy of intramammary pirlimycin hydrochloride administration during lactation for bacteriologic clearance of gram-positive environmental clinical and subclinical mastitis infections. Quarters infected with environmental streptococci that received pirlimycin therapy (13/28) were 1.8 times more likely to resolve infection than untreated quarters (5/14). The small numbers of quarters infected with coagulase-negative staphylococci resulted in inadequate power to assess treatment differences in cure rate. Although the association was not statistically significant, quarters from cows with sensitive environmental streptococci isolates from composite samples (8/13) resolved infection with treatment at approximately twice the rate of treated quarters with resistant isolates (3/10).

Evaluation of the Verigene Gram-positive blood culture nucleic acid test for rapid detection of bacteria and resistance determinants.

Science.gov (United States)

Wojewoda, Christina M; Sercia, Linda; Navas, Maria; Tuohy, Marion; Wilson, Deborah; Hall, Geraldine S; Procop, Gary W; Richter, Sandra S

2013-07-01

Rapid identification of pathogens from blood cultures can decrease lengths of stay and improve patient outcomes. We evaluated the accuracy of the Verigene Gram-positive blood culture (BC-GP) nucleic acid test for investigational use only (Nanosphere, Inc., Northbrook, IL) for the identification of Gram-positive bacteria from blood cultures. The detection of resistance genes (mecA in Staphylococcus aureus and Staphylococcus epidermidis and vanA or vanB in Enterococcus faecium and Enterococcus faecalis) by the BC-GP assay also was assessed. A total of 186 positive blood cultures (in BacT/Alert FA bottles) with Gram-positive cocci observed with Gram staining were analyzed using the BC-GP assay. The BC-GP results were compared with the identification and susceptibility profiles obtained with routine methods in the clinical laboratory. Discordant results were arbitrated with additional biochemical, cefoxitin disk, and repeat BC-GP testing. The initial BC-GP organism identification was concordant with routine method results for 94.6% of the blood cultures. Only 40% of the Streptococcus pneumoniae identifications were correct. The detection of the mecA gene for 69 blood cultures with only S. aureus or S. epidermidis was concordant with susceptibility testing results. For 3 of 6 cultures with multiple Staphylococcus spp., mecA detection was reported but was correlated with oxacillin resistance in a species other than S. aureus or S. epidermidis. The detection of vanA agreed with susceptibility testing results for 45 of 46 cultures with E. faecalis or E. faecium. Comparison of the mean times to results for each organism group showed that BC-GP results were available 31 to 42 h earlier than phenotypic identifications and 41 to 50 h earlier than susceptibility results.

Gram-positive bacteria of marine origin: a numerical taxonomic study on Mediterranean isolates.

Science.gov (United States)

Ortigosa, M; Garay, E; Pujalte, M J

1997-12-01

A numerical taxonomic study was performed on 65 Gram-positive wild strains of heterotrophic, aerobic, marine bacteria, and 9 reference strains. The isolates were obtained from oysters and seawater sampled monthly over one year, by direct plating on Marine Agar. The strains were characterized by 96 morphological, biochemical, physiological and nutritional tests. Clustering yielded 13 phena at 0.62 similarity level (Sl coefficient). Only one of the seven phena containing wild isolates could be identified (Bacillus marinus). A pronounced salt requirement was found in most isolates.

High positive predictive value of Gram stain on catheter-drawn blood samples for the diagnosis of catheter-related bloodstream infection in intensive care neonates.

Science.gov (United States)

Deleers, M; Dodémont, M; Van Overmeire, B; Hennequin, Y; Vermeylen, D; Roisin, S; Denis, O

2016-04-01

Catheter-related bloodstream infections (CRBSIs) remain a leading cause of healthcare-associated infections in preterm infants. Rapid and accurate methods for the diagnosis of CRBSIs are needed in order to implement timely and appropriate treatment. A retrospective study was conducted during a 7-year period (2005-2012) in the neonatal intensive care unit of the University Hospital Erasme to assess the value of Gram stain on catheter-drawn blood samples (CDBS) to predict CRBSIs. Both peripheral samples and CDBS were obtained from neonates with clinically suspected CRBSI. Gram stain, automated culture and quantitative cultures on blood agar plates were performed for each sample. The paired quantitative blood culture was used as the standard to define CRBSI. Out of 397 episodes of suspected CRBSIs, 35 were confirmed by a positive ratio of quantitative culture (>5) or a colony count of CDBS culture >100 colony-forming units (CFU)/mL. All but two of the 30 patients who had a CDBS with a positive Gram stain were confirmed as having a CRBSI. Seven patients who had a CDBS with a negative Gram stain were diagnosed as CRBSI. The sensitivity, specificity, positive predictive value and negative predictive value of Gram stain on CDBS were 80, 99.4, 93.3 and 98.1 %, respectively. Gram staining on CDBS is a viable method for rapidly (<1 h) detecting CRBSI without catheter withdrawal.

Silver enhances antibiotic activity against gram-negative bacteria.

Science.gov (United States)

Morones-Ramirez, J Ruben; Winkler, Jonathan A; Spina, Catherine S; Collins, James J

2013-06-19

A declining pipeline of clinically useful antibiotics has made it imperative to develop more effective antimicrobial therapies, particularly against difficult-to-treat Gram-negative pathogens. Silver has been used as an antimicrobial since antiquity, yet its mechanism of action remains unclear. We show that silver disrupts multiple bacterial cellular processes, including disulfide bond formation, metabolism, and iron homeostasis. These changes lead to increased production of reactive oxygen species and increased membrane permeability of Gram-negative bacteria that can potentiate the activity of a broad range of antibiotics against Gram-negative bacteria in different metabolic states, as well as restore antibiotic susceptibility to a resistant bacterial strain. We show both in vitro and in a mouse model of urinary tract infection that the ability of silver to induce oxidative stress can be harnessed to potentiate antibiotic activity. Additionally, we demonstrate in vitro and in two different mouse models of peritonitis that silver sensitizes Gram-negative bacteria to the Gram-positive-specific antibiotic vancomycin, thereby expanding the antibacterial spectrum of this drug. Finally, we used silver and antibiotic combinations in vitro to eradicate bacterial persister cells, and show both in vitro and in a mouse biofilm infection model that silver can enhance antibacterial action against bacteria that produce biofilms. This work shows that silver can be used to enhance the action of existing antibiotics against Gram-negative bacteria, thus strengthening the antibiotic arsenal for fighting bacterial infections.

Use of MALDI-TOF Mass Spectrometry for the Fast Identification of Gram-Positive Fish Pathogens

Science.gov (United States)

Assis, Gabriella B. N.; Pereira, Felipe L.; Zegarra, Alexandra U.; Tavares, Guilherme C.; Leal, Carlos A.; Figueiredo, Henrique C. P.

2017-01-01

Gram-positive cocci, such as Streptococcus agalactiae, Lactococcus garvieae, Streptococcus iniae, and Streptococcus dysgalactiae subsp. dysgalactiae, are found throughout the world, particularly in outbreaks in farmed fish, and are thus associated with high economic losses, especially in the cultivation of Nile Tilapia. The aim of this study was to evaluate the efficacy of matrix-assisted laser desorption ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as an alternative for the diagnosis of these pathogens. One hundred and thirty-one isolates from Brazilian outbreaks assisted by the national authority were identified using a MALDI Biotyper from Bruker Daltonics. The results showed an agreement with respect to identification (Kappa = 1) between this technique and 16S ribosomal RNA gene sequencing for S. agalactiae and L. garvieae. However, for S. iniae and S. dysgalactiae subsp. dysgalactiae, perfect agreement was only achieved after the creation of a custom main spectra profile, as well as further comparisons with 16S ribosomal RNA and multilocus sequence analysis. MALDI-TOF MS was shown to be an efficient technology for the identification of these Gram-positive pathogens, yielding a quick and precise diagnosis. PMID:28848512

Prediction of lipoprotein signal peptides in Gram-negative bacteria.

Science.gov (United States)

Juncker, Agnieszka S; Willenbrock, Hanni; Von Heijne, Gunnar; Brunak, Søren; Nielsen, Henrik; Krogh, Anders

2003-08-01

A method to predict lipoprotein signal peptides in Gram-negative Eubacteria, LipoP, has been developed. The hidden Markov model (HMM) was able to distinguish between lipoproteins (SPaseII-cleaved proteins), SPaseI-cleaved proteins, cytoplasmic proteins, and transmembrane proteins. This predictor was able to predict 96.8% of the lipoproteins correctly with only 0.3% false positives in a set of SPaseI-cleaved, cytoplasmic, and transmembrane proteins. The results obtained were significantly better than those of previously developed methods. Even though Gram-positive lipoprotein signal peptides differ from Gram-negatives, the HMM was able to identify 92.9% of the lipoproteins included in a Gram-positive test set. A genome search was carried out for 12 Gram-negative genomes and one Gram-positive genome. The results for Escherichia coli K12 were compared with new experimental data, and the predictions by the HMM agree well with the experimentally verified lipoproteins. A neural network-based predictor was developed for comparison, and it gave very similar results. LipoP is available as a Web server at www.cbs.dtu.dk/services/LipoP/.

Expression levels of matrix metalloproteinase-9 and gram-negative bacteria in symptomatic and asymptomatic periapical lesions.

Science.gov (United States)

Ahmed, Geraldine M; El-Baz, Alaa A; Hashem, Ahmed Abdel Rahman; Shalaan, Abeer K

2013-04-01

The aim of this study was to test the hypothesis that the expression of matrix metalloproteinase (MMP)-9 is significantly elevated in patients with symptomatic apical periodontitis and to correlate this with the detected amount of gram-negative bacteria. Twenty-six patients with periapical lesions involving at least 2 teeth were included in this study. The patients were divided into 2 groups: the symptomatic (SYM) group included 13 patients expressing pain with periapical lesions, and the asymptomatic (ASYM) group included 13 patients expressing no pain. Root canal treatment was performed followed by endodontic surgery and periapical lesion collection. Periapical lesions were serially cut into 4-μ sections. Some sections were processed for histologic examination using hematoxylin-eosin stain. Other sections were processed for immunohistochemical examination. For MMP-9, the area fraction of the positive cells was measured, and the percentage of the MMP-9-immunopositive area to the total area of the microscopic field was calculated. For gram-negative stain cells, the number of cells showing the pink-red color was counted per microscopic field. The Student's t test was used to compare the SYM and ASYM groups. The Pearson correlation coefficient was used to determine a significant correlation between the number of cells and the MMP-9 level. The significance level was set at P ≤ .05. The SYM group showed a statistically significantly higher mean number of gram-negative cells (P = .001) and MMP-9 area percent (P < .001) than the ASYM group. There was a statistically significant positive (r = .927) correlation between the number of gram-negative cells and the MMP-9 area percent (P< .001). There is good evidence to suspect a significant role of gram-negative bacteria and MMP-9 in symptomatic periapical lesions. Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Crystallization and preliminary X-ray analysis of three dUTPases from Gram-positive bacteria

International Nuclear Information System (INIS)

Li, Gui-Lan; Wang, Juan; Li, Lan-Fen; Su, Xiao-Dong

2009-01-01

All organisms examined to date possess a dUTPase that performs the important function of efficiently hydrolyzing dUTP to dUMP in order to prevent the incorporation of dUTP into DNA. Three putative dUTPases from Gram-positive bacteria have been studied in this work. All organisms examined to date possess a dUTPase that performs the important function of efficiently hydrolyzing dUTP to dUMP in order to prevent the incorporation of dUTP into DNA. Three putative dUTPases from Gram-positive bacteria have been studied in this work. Two dUTPase-encoding genes, yncF and yosS, have been identified in Bacillus subtilis. The gene dut, encoding dUTPase from the dental pathogen Streptococcus mutans, was amplified from S. mutans genomic DNA. The three genes were cloned into expression vectors and overexpressed at high levels in Escherichia coli. Each protein was purified in two steps using chromatographic methods. Crystals of the YosS and YncF proteins and of S. mutans dUTPase were obtained using the vapour-diffusion method. X-ray diffraction data sets were collected from crystals of selenomethionine-labelled YosS and S. mutans dUTPase to resolutions of 2.3 and 1.7 Å, respectively. The crystal of native YncF diffracted to 2.7 Å resolution

Gram staining apparatus for space station applications

Science.gov (United States)

Molina, T. C.; Brown, H. D.; Irbe, R. M.; Pierson, D. L.

1990-01-01

A self-contained, portable Gram staining apparatus (GSA) has been developed for use in the microgravity environment on board the Space Station Freedom. Accuracy and reproducibility of this apparatus compared with the conventional Gram staining method were evaluated by using gram-negative and gram-positive controls and different species of bacteria grown in pure cultures. A subsequent study was designed to assess the performance of the GSA with actual specimens. A set of 60 human and environmental specimens was evaluated with the GSA and the conventional Gram staining procedure. Data obtained from these studies indicated that the GSA will provide the Gram staining capability needed for the microgravity environment of space.

A Flexible Binding Site Architecture Provides New Insights into CcpA Global Regulation in Gram-Positive Bacteria

Directory of Open Access Journals (Sweden)

Yunpeng Yang

2017-01-01

Full Text Available Catabolite control protein A (CcpA is the master regulator in Gram-positive bacteria that mediates carbon catabolite repression (CCR and carbon catabolite activation (CCA, two fundamental regulatory mechanisms that enable competitive advantages in carbon catabolism. It is generally regarded that CcpA exerts its regulatory role by binding to a typical 14- to 16-nucleotide (nt consensus site that is called a catabolite response element (cre within the target regions. However, here we report a previously unknown noncanonical flexible architecture of the CcpA-binding site in solventogenic clostridia, providing new mechanistic insights into catabolite regulation. This novel CcpA-binding site, named crevar, has a unique architecture that consists of two inverted repeats and an intervening spacer, all of which are variable in nucleotide composition and length, except for a 6-bp core palindromic sequence (TGTAAA/TTTACA. It was found that the length of the intervening spacer of crevar can affect CcpA binding affinity, and moreover, the core palindromic sequence of crevar is the key structure for regulation. Such a variable architecture of crevar shows potential importance for CcpA’s diverse and fine regulation. A total of 103 potential crevar sites were discovered in solventogenic Clostridium acetobutylicum, of which 42 sites were picked out for electrophoretic mobility shift assays (EMSAs, and 30 sites were confirmed to be bound by CcpA. These 30 crevar sites are associated with 27 genes involved in many important pathways. Also of significance, the crevar sites are found to be widespread and function in a great number of taxonomically different Gram-positive bacteria, including pathogens, suggesting their global role in Gram-positive bacteria.

Antimicrobial susceptibility trends among gram-positive and -negative clinical isolates collected between 2005 and 2012 in Mexico: results from the Tigecycline Evaluation and Surveillance Trial.

Science.gov (United States)

Morfin-Otero, Rayo; Noriega, Eduardo Rodriguez; Dowzicky, Michael J

2015-12-15

The Tigecycline Evaluation and Surveillance Trial (T.E.S.T) is a global antimicrobial surveillance study of both gram-positive and gram-negative organisms. This report presents data on antimicrobial susceptibility among organisms collected in Mexico between 2005 and 2012 as part of T.E.S.T., and compares rates between 2005-2007 and 2008-2012. Each center in Mexico submitted at least 200 isolates per collection year; including 65 gram-positive isolates and 135 gram-negative isolates. Minimum inhibitory concentrations (MICs) were determined using Clinical Laboratory Standards Institute (CLSI) broth microdilution methodology and antimicrobial susceptibility was established using the 2013 CLSI-approved breakpoints. For tigecycline US Food and Drug Administration (FDA) breakpoints were applied. Isolates of E. coli and K. pneumoniae with a MIC for ceftriaxone of >1 mg/L were screened for ESBL production using the phenotypic confirmatory disk test according to CLSI guidelines. The rates of some key resistant phenotypes changed during this study: vancomycin resistance among Enterococcus faecium decreased from 28.6 % in 2005-2007 to 19.1 % in 2008-2012, while β-lactamase production among Haemophilus influenzae decreased from 37.6 to 18.9 %. Conversely, methicillin-resistant Staphylococcus aureus increased from 38.1 to 47.9 %, meropenem-resistant Acinetobacter spp. increased from 17.7 to 33.0 % and multidrug-resistant Acinetobacter spp. increased from 25.6 to 49.7 %. The prevalence of other resistant pathogens was stable over the study period, including extended-spectrum β-lactamase-positive Escherichia coli (39.0 %) and Klebsiella pneumoniae (25.0 %). The activity of tigecycline was maintained across the study years with MIC90s of ≤2 mg/L against Enterococcus spp., S. aureus, Streptococcus agalactiae, Streptococcus pneumoniae, Enterobacter spp., E. coli, K. pneumoniae, Klebsiella oxytoca, Serratia marcescens, H. influenzae, and Acinetobacter spp. All gram-positive

Veillonella, Firmicutes: Microbes disguised as Gram negatives.

Science.gov (United States)

Vesth, Tammi; Ozen, Aslı; Andersen, Sandra C; Kaas, Rolf Sommer; Lukjancenko, Oksana; Bohlin, Jon; Nookaew, Intawat; Wassenaar, Trudy M; Ussery, David W

2013-12-20

The Firmicutes represent a major component of the intestinal microflora. The intestinal Firmicutes are a large, diverse group of organisms, many of which are poorly characterized due to their anaerobic growth requirements. Although most Firmicutes are Gram positive, members of the class Negativicutes, including the genus Veillonella, stain Gram negative. Veillonella are among the most abundant organisms of the oral and intestinal microflora of animals and humans, in spite of being strict anaerobes. In this work, the genomes of 24 Negativicutes, including eight Veillonella spp., are compared to 20 other Firmicutes genomes; a further 101 prokaryotic genomes were included, covering 26 phyla. Thus a total of 145 prokaryotic genomes were analyzed by various methods to investigate the apparent conflict of the Veillonella Gram stain and their taxonomic position within the Firmicutes. Comparison of the genome sequences confirms that the Negativicutes are distantly related to Clostridium spp., based on 16S rRNA, complete genomic DNA sequences, and a consensus tree based on conserved proteins. The genus Veillonella is relatively homogeneous: inter-genus pair-wise comparison identifies at least 1,350 shared proteins, although less than half of these are found in any given Clostridium genome. Only 27 proteins are found conserved in all analyzed prokaryote genomes. Veillonella has distinct metabolic properties, and significant similarities to genomes of Proteobacteria are not detected, with the exception of a shared LPS biosynthesis pathway. The clade within the class Negativicutes to which the genus Veillonella belongs exhibits unique properties, most of which are in common with Gram-positives and some with Gram negatives. They are only distantly related to Clostridia, but are even less closely related to Gram-negative species. Though the Negativicutes stain Gram-negative and possess two membranes, the genome and proteome analysis presented here confirm their place within the

Type-IVC Secretion System: A Novel Subclass of Type IV Secretion System (T4SS) Common Existing in Gram-Positive Genus Streptococcus

Science.gov (United States)

Chen, Chen; Gao, George F.

2012-01-01

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. Type IV secretion system (T4SS) is one of versatile secretion systems essential for the virulence and even survival of some bacteria species, and they enable the secretion of protein and DNA substrates across the cell envelope. T4SS was once believed to be present only in Gram-negative bacteria. In this study, we present evidence of a new subclass of T4SS, Type-IVC secretion system and indicate its common existence in the Gram-positive bacterial genus Streptococcus. We further identified that VirB1, VirB4, VirB6 and VirD4 are the minimal key components of this system. Using genome comparisons and evolutionary relationship analysis, we proposed that Type-IVC secretion system is movable via transposon factors and mediates the conjugative transfer of DNA, enhances bacterial pathogenicity, and could cause large-scale outbreaks of infections in humans. PMID:23056296

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria

Science.gov (United States)

Quiles-Puchalt, Nuria; Tormo-Más, María Ángeles; Campoy, Susana; Toledo-Arana, Alejandro; Monedero, Vicente; Lasa, Íñigo; Novick, Richard P.; Christie, Gail E.; Penadés, José R.

2013-01-01

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria. PMID:23771138

An extreme-halophile archaebacterium possesses the interlock type of prephenate dehydratase characteristic of the Gram-positive eubacteria

Science.gov (United States)

Jensen, R. A.; d'Amato, T. A.; Hochstein, L. I.

1988-01-01

The focal point of phenylalanine biosynthesis is a dehydratase reaction which in different organisms may be prephenate dehydratase, arogenate dehydratase, or cyclohexadienyl dehydratase. Gram-positive, Gram-negative, and cyanobacterial divisions of the eubacterial kingdom exhibit different dehydratase patterns. A new extreme-halophile isolate, which grows on defined medium and is tentatively designated as Halobacterium vallismortis CH-1, possesses the interlock type of prephenate dehydratase present in Gram-positive bacteria. In addition to the conventional sensitivity to feedback inhibition by L-phenylalanine, the phenomenon of metabolic interlock was exemplified by the sensitivity of prephenate dehydratase to allosteric effects produced by extra-pathway (remote) effectors. Thus, L-tryptophan inhibited activity while L-tyrosine, L-methionine, L-leucine and L-isoleucine activated the enzyme. L-Isoleucine and L-phenylalanine were effective at micromolar levels; other effectors operated at mM levels. A regulatory mutant selected for resistance to growth inhibition caused by beta-2-thienylalanine possessed an altered prephenate dehydratase in which a phenomenon of disproportionately low activity at low enzyme concentration was abolished. Inhibition by L-tryptophan was also lost, and activation by allosteric activators was diminished. Not only was sensitivity to feedback inhibition by L-phenylalanine lost, but the mutant enzyme was now activated by this amino acid (a mutation type previously observed in Bacillus subtilis). It remains to be seen whether this type of prephenate dehydratase will prove to be characteristic of all archaebacteria or of some archaebacterial subgroup cluster.

Alternate gram staining technique using a fluorescent lectin.

Science.gov (United States)

Sizemore, R K; Caldwell, J J; Kendrick, A S

1990-01-01

Fluorescence-labeled wheat germ agglutinin binds specifically to N-acetylglucosamine in the outer peptidoglycan layer of gram-positive bacteria. The peptidoglycan layer of gram-negative bacteria is covered by a membrane and is not labeled by the lectin. By exploiting this phenomenon, an alternative Gram staining technique has been developed. Images PMID:1697149

Antimicrobial Activities of Methanol, Ethanol and Supercritical CO2 Extracts of Philippine Piper betle L. on Clinical Isolates of Gram Positive and Gram Negative Bacteria with Transferable Multiple Drug Resistance.

Directory of Open Access Journals (Sweden)

Demetrio L Valle

Full Text Available Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC and the minimum bactericidal concentrations (MBC of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA, vancomycin-resistant Enterococcus (VRE, extended spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19μg/ml to 1250 μg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 μg/ml of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn. Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant

Antimicrobial Activities of Methanol, Ethanol and Supercritical CO2 Extracts of Philippine Piper betle L. on Clinical Isolates of Gram Positive and Gram Negative Bacteria with Transferable Multiple Drug Resistance.

Science.gov (United States)

Valle, Demetrio L; Cabrera, Esperanza C; Puzon, Juliana Janet M; Rivera, Windell L

2016-01-01

Piper betle L. has traditionally been used in alternative medicine in different countries for various therapeutic purposes, including as an anti-infective agent. However, studies reported in the literature are mainly on its activities on drug susceptible bacterial strains. This study determined the antimicrobial activities of its ethanol, methanol, and supercritical CO2 extracts on clinical isolates of multiple drug resistant bacteria which have been identified by the Infectious Disease Society of America as among the currently more challenging strains in clinical management. Assay methods included the standard disc diffusion method and the broth microdilution method for the determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentrations (MBC) of the extracts for the test microorganisms. This study revealed the bactericidal activities of all the P. betle leaf crude extracts on methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended spectrum β-lactamase-producing Enterobacteriaceae, carbapenem-resistant Enterobacteriaceae, and metallo-β-lactamase-producing Pseudomonas aeruginosa and Acinetobacter baumannii, with minimum bactericidal concentrations that ranged from 19μg/ml to 1250 μg/ml. The extracts proved to be more potent against the Gram positive MRSA and VRE than for the Gram negative test bacteria. VRE isolates were more susceptible to all the extracts than the MRSA isolates. Generally, the ethanol extracts proved to be more potent than the methanol extracts and supercritical CO2 extracts as shown by their lower MICs for both the Gram positive and Gram negative MDRs. MTT cytotoxicity assay showed that the highest concentration (100 μg/ml) of P. betle ethanol extract tested was not toxic to normal human dermal fibroblasts (HDFn). Data from the study firmly established P. betle as an alternative source of anti-infectives against multiple drug resistant bacteria.

Desulfotomaculum spp. and related Gram-positive sulfate-reducing bacteria in deep subsurface environments.

Directory of Open Access Journals (Sweden)

Thomas eAullo

2013-12-01

Full Text Available Gram-positive spore-forming sulfate reducers and particularly members of the genus Desulfotomaculum are commonly found in the subsurface biosphere by culture based and molecular approaches. Due to their metabolic versatility and their ability to persist as endospores. Desulfotomaculum spp. are well adapted for colonizing environments through a slow sedimentation process. Because of their ability to grow autotrophically (H2/CO2 and produce sulfide or acetate, these microorganisms may play key roles in deep lithoautotrophic microbial communities. Available data about Desulfotomaculum spp. and related species from studies carried out from deep freshwater lakes, marine sediments, oligotrophic and organic rich deep geological settings are discussed in this review.

Antimicrobial Resistance in Gram-Negative Rods Causing Bacteremia in Hematopoietic Stem Cell Transplant Recipients

DEFF Research Database (Denmark)

Averbuch, Diana; Tridello, Gloria; Hoek, Jennifer

2017-01-01

Background: This intercontinental study aimed to study gram-negative rod (GNR) resistance in hematopoietic stem cell transplantation (HSCT). Methods: GNR bacteremias occurring during 6 months post-HSCT (February 2014-May 2015) were prospectively collected, and analyzed for rates and risk factors...

Gram staining with an automatic machine.

Science.gov (United States)

Felek, S; Arslan, A

1999-01-01

This study was undertaken to develop a new Gram-staining machine controlled by a micro-controller and to investigate the quality of slides that were stained in the machine. The machine was designed and produced by the authors. It uses standard 220 V AC. Staining, washing, and drying periods are controlled by a timer built in the micro-controller. A software was made that contains a certain algorithm and time intervals for the staining mode. One-hundred and forty smears were prepared from Escherichia coli, Staphylococcus aureus, Neisseria sp., blood culture, trypticase soy broth, direct pus and sputum smears for comparison studies. Half of the slides in each group were stained with the machine, the other half by hand and then examined by four different microbiologists. Machine-stained slides had a higher clarity and less debris than the hand-stained slides (p stained slides, some Gram-positive organisms showed poor Gram-positive staining features (p Gram staining with the automatic machine increases the staining quality and helps to decrease the work load in a busy diagnostic laboratory.

Burdock (Arctium lappa Leaf Extracts Increase the In Vitro Antimicrobial Efficacy of Common Antibiotics on Gram-positive and Gram-negative Bacteria

Directory of Open Access Journals (Sweden)

Pirvu Lucia

2017-04-01

Full Text Available This work aimed to study the potential effects of four Arctii folium extracts, 5 mg gallic [GAE] acid equivalents per 1 mL sample, on six antibiotics (Ampicillin/AM, Tetracycline/TE, Ciprofloxacin/CIP, Sulfamethoxazole-Trimethoprim/SXT, Chloramphenicol/C and Gentamicin/CN tested on four Gram-positive (Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and Staphylococcus epidermidis ATCC 12228 and five Gram-negative (Proteus mirabilis ATCC 29245, Escherichia coli ATCC 35218, E. coli ATCC 11229, E. coli ATCC 8739, and Bacillus cereus ATCC 11778 bacteria. Arctii folium extracts were the whole ethanol extract/W and subsequent ethyl acetate/EA, aqueous/AQ, and chloroform/CHL fractions. Chemical qualitative analysis (HPTLC method emphasized five main polyphenol compounds in Arctii folium polar extracts: chlorogenic acid (Rf≈0.52/0.55 and its isomer, 1,5-di-O-caffeoylquinic acid (Rf≈0.90/0.92, plus cynarin (Rf≈0.77, hyperoside (Rf≈0.68/0.64 and isoquercitrin (Rf≈0.69/0.71. Microbiological screening indicated Arctii folium polar extracts (AQ and W efficacy on S. epidermidis ATCC 12228; the MIC values were in the range of common antibiotics, being 32 and 128 μg GAE per mL sample respectively. The unpredictable effects (stimulatory or inhibitory of Arctii folium extracts in combination with typical antibiotics as well as a potential use of the whole ethanol extract/W for restoring the antimicrobial potency of susceptible antibiotics have also been evidenced.

Is the gram stain useful in the microbiologic diagnosis of VAP? A meta-analysis.

Science.gov (United States)

O'Horo, John C; Thompson, Deb; Safdar, Nasia

2012-08-01

In a meta-analysis examining respiratory specimen Gram stain for diagnosis of ventilator-associated pneumonia, absence of bacteria on Gram stain had a high negative predictive value, but a positive Gram stain correlated poorly with organisms recovered in culture. Rapid and accurate diagnosis of ventilator-associated pneumonia (VAP) is a major challenge and no generally accepted gold standard exists for VAP diagnosis. We conducted a meta-analysis to examine the role of respiratory specimen Gram stain to diagnose VAP, and the correlation with final culture results. In 21 studies, pooled sensitivity of Gram stain for VAP was 0.79 (95% confidence interval [CI], .77-0.81; P Gram stain for a VAP prevalence of 20%-30% was 91%, suggesting that VAP is unlikely with a negative Gram stain but the positive predictive value of Gram stain was only 40%. Pooled kappa was 0.42 for gram-positive organisms and 0.34 for gram-negative organisms, suggesting fair concordance between organisms on Gram stain and recovery by culture. Therefore, a positive Gram stain should not be used to narrow anti-infective therapy until culture results become available.

The Role of Functional Amyloids in Multicellular Growth and Development of Gram-Positive Bacteria

DEFF Research Database (Denmark)

Dragoš, Anna; Kovács, Ákos T.; Claessen, Dennis

2017-01-01

Amyloid fibrils play pivotal roles in all domains of life. In bacteria, these fibrillar structures are often part of an extracellular matrix that surrounds the producing organism and thereby provides protection to harsh environmental conditions. Here, we discuss the role of amyloid fibrils...... in the two distant Gram-positive bacteria, Streptomyces coelicolor and Bacillus subtilis. We describe how amyloid fibrils contribute to a multitude of developmental processes in each of these systems, including multicellular growth and community development. Despite this variety of tasks, we know...... surprisingly little about how their assembly is organized to fulfill all these roles....

Purification Techniques of Bacteriocins from Lactic Acid Bacteria and Other Gram-Positive Bacteria

Science.gov (United States)

Saavedra, Lucila; Sesma, Fernando

The search for new antimicrobial peptides produced by lactic acid ­bacteria and other Gram-positive microorganisms has become an interesting field of research in the past decades. The fact that bacteriocins are active against numerous foodborne and human pathogens, are produced by generally regarded as safe (GRAS) microorganisms, and are readily degraded by proteolytic host systems makes them attractive candidates for biotechnological applications. However, before suggesting or choosing a new bacteriocin for future technology developments, it is necessary to elucidate its biochemical structure and its mode of action, which may be carried out once the bacteriocin is purified to homogeneity. This chapter focuses on describing the main strategies used for the purification of numerous bacteriocins.

Antibacterial activity of oregano (Origanum vulgare Linn.) against gram positive bacteria.

Science.gov (United States)

Saeed, Sabahat; Tariq, Perween

2009-10-01

The present investigation is focused on antibacterial potential of infusion, decoction and essential oil of oregano (Origanum vulgare) against 111 Gram-positive bacterial isolates belonging to 23 different species related to 3 genera. Infusion and essential oil exhibited antibacterial activity against Staphylococcus saprophyticus, S. aureus, Micrococcus roseus, M. kristinae, M. nishinomiyaensis, M. lylae, M. luteus, M. sedentarius, M. varians, Bacillus megaterium, B. thuringiensis, B. alvei, B. circulans, B. brevis, B. coagulans, B. pumilus, B. laterosporus, B. polymyxa, B. macerans, B. subtilis, B. firmus, B. cereus and B. lichiniformis. The infusion exhibited maximum activity against B. laterosporus (17.5 mm mean zone of inhibition+/-1.5 Standard deviation) followed by B. polymyxa (17.0 mm+/-2.0 SD) and essential oil of oregano exhibited maximum activity against S. saprophyticus (16.8 mm+/-1.8 SD) followed by B. circulans (14.5 mm+/-0.5 SD). While all these tested isolates were found resistant to decoction of oregano.

[Usefulness of sputum Gram staining in community-acquired pneumonia].

Science.gov (United States)

Sato, Tadashi; Aoshima, Masahiro; Ohmagari, Norio; Tada, Hiroshi; Chohnabayashi, Naohiko

2002-07-01

To evaluate the usefulness of sputum gram staining in community-acquired pneumonia (CAP), we reviewed 144 cases requiring hospitalization in the last 4 years. The sensitivity was 75.5%, specificity 68.2%, positive predictive value 74.1%, negative predictive value 69.8%, positive likelihood ratio 2.37, negative likelihood ratio 0.36 and accuracy 72.2% in 97 cases. Both sputum gram staining and culture were performed. Concerning bacterial pneumonia (65 cases), we compared the Gram staining group (n = 33), which received initial antibiotic treatment, based on sputum gram staining with the Empiric group (n = 32) that received antibiotics empirically. The success rates of the initial antibiotic treatment were 87.9% vs. 78.1% (P = 0.473); mean hospitalization periods were 9.67 vs. 11.75 days (P = 0.053); and periods of intravenous therapy were 6.73 vs. 7.91 days (P = 0.044), respectively. As for initial treatment, penicillins were used in the Gram staining group more frequently (P gram staining is useful for the shortening of the treatment period and the appropriate selection of initial antibiotics in bacterial pneumonia. We believe, therefore, that sputum gram staining is indispensable as a diagnostic tool CAP.

The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of Gram-positive anaerobic cocci

NARCIS (Netherlands)

Veloo, A. C. M.; de Vries, E D; Jean-Pierre, H.; Justesen, U. S.; Morris, T.; Urban, E.; Wybo, I.; van Winkelhoff, A. J.

OBJECTIVES: Gram-positive anaerobic cocci (GPAC) account for 24-31% of the anaerobic bacteria isolated from human clinical specimens. At present GPAC are underrepresented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the MALDI-TOF

Super-Resolution Imaging of Protein Secretion Systems and the Cell Surface of Gram-Negative Bacteria

Directory of Open Access Journals (Sweden)

Sachith D. Gunasinghe

2017-05-01

Full Text Available Gram-negative bacteria have a highly evolved cell wall with two membranes composed of complex arrays of integral and peripheral proteins, as well as phospholipids and glycolipids. In order to sense changes in, respond to, and exploit their environmental niches, bacteria rely on structures assembled into or onto the outer membrane. Protein secretion across the cell wall is a key process in virulence and other fundamental aspects of bacterial cell biology. The final stage of protein secretion in Gram-negative bacteria, translocation across the outer membrane, is energetically challenging so sophisticated nanomachines have evolved to meet this challenge. Advances in fluorescence microscopy now allow for the direct visualization of the protein secretion process, detailing the dynamics of (i outer membrane biogenesis and the assembly of protein secretion systems into the outer membrane, (ii the spatial distribution of these and other membrane proteins on the bacterial cell surface, and (iii translocation of effector proteins, toxins and enzymes by these protein secretion systems. Here we review the frontier research imaging the process of secretion, particularly new studies that are applying various modes of super-resolution microscopy.

Synergistic effect of Carum copticum and Mentha piperita essential oils with ciprofloxacin, vancomycin, and gentamicin on Gram-negative and Gram-positive bacteria

Science.gov (United States)

Talei, Gholam-Reza; Mohammadi, Mohsen; Bahmani, Mahmoud; Kopaei, Mahmoud Rafieian

2017-01-01

Background: Infectious diseases have always been an important health issue in human communities. In the recent years, much research has been conducted on antimicrobial effects of nature-based compounds because of increased prevalence of antibiotic resistance. The present study was conducted to investigate synergistic effect of Carum copticum and Mentha piperita essential oils with ciprofloxacin, vancomycin, and gentamicin on Gram-negative and Gram-positive bacteria. Materials and Methods: In this experimental study, the synergistic effects of C. copticum and M. piperita essential oils with antibiotics on Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus epidermidis (ATCC 14990), and Listeria monocytogenes (ATCC 7644) were studied according to broth microdilution and the MIC and fractional inhibitory concentration (FIC) of these two essential oils determined. Results: C. copticum essential oil at 30 μg/ml could inhibit S. aureus, and in combination with vancomycin, decreased MIC from 0.5 to 0.12 μg/ml. Moreover, the FIC was derived 0.24 μg/ml which represents a potent synergistic effect with vancomycin against S. aureus growth. C. copticum essential oil alone or combined with other antibiotics is effective in treating bacterial infections. Conclusions: In addition, C. copticum essential oil can strengthen the activities of certain antibiotics, which makes it possible to use this essential oil, especially in drug resistance or to lower dosage or toxicity of the drugs. PMID:28929050

Competitive binding of polyethyleneimine-coated gold nanoparticles to enzymes and bacteria: a key mechanism for low-level colorimetric detection of gram-positive and gram-negative bacteria

International Nuclear Information System (INIS)

Thiramanas, Raweewan; Laocharoensuk, Rawiwan

2016-01-01

The article describes a simple and rapid method for colorimetric detection of bacteria. It is based on competitive binding of positively charged polyethyleneimine-coated gold nanoparticles (PEI-AuNPs) to negatively charged enzymes and bacteria. The PEI-AuNPs are electrostatically attracted by both the bacterial surface and the enzyme β-galactosidase (β-Gal). Binding to the latter results in the inhibition of enzyme activity. However, in the presence of a large number of bacteria, the PEI-AuNPs preferentially bind to bacteria. Hence, the enzyme will not be inhibited and its activity can be colorimetrically determined via hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside (CPRG). The detection limit of this assay is as low as 10 cfu·mL −1 , and the linear range extends from 10 6 to 10 8 cfu·mL −1 . The assay is applicable to both Gram-negative (such as enterotoxigenic Escherichia coli; ETEC) and Gram-positive (Staphylococcus aureus; S. aureus) bacteria. Results are obtained within 10 min using an optical reader, and within 2–3 h by bare-eye detection. The method was applied to the identification of ETEC contamination at a level of 10 cfu·mL −1 in spiked drinking water. Given its low detection limit and rapidity (sample preconcentration is not required), this method holds great promise for on-site detection of total bacterial contamination. (author)

EFFECT OF SILICATE ON GRAM STAINING AND VIABILITY OF PNEUMOCOCCI AND OTHER BACTERIA

Science.gov (United States)

MacLeod, Colin M.; Roe, Amy S.

1956-01-01

Application of silicate solutions to living or heat-killed pneumococci and to certain "viridans" streptococci causes their conversion from a Gram-positive to a Gram-negative state. The original staining properties can be restored by suspending the silicate-treated bacteria in alkaline solutions of various salts but not by simple washing in water. Living pneumococci and the strains of streptococci whose staining properties are similarly affected are killed when suspended in silicate solutions. In other Gram-positive species silicate causes conversion to Gram negativity but restoration to positivity occurs upon washing in water. In a third group of Gram-positive organisms silicate has no effect on the Gram reaction. The viability of organisms in these two groups is unaffected by silicate under the conditions employed. No effect on staining or viability of Gram-negative bacteria has been observed. The effects of silicate on staining and viability are inhibited by nutrient broth or whole serum but not by purified serum albumin. Lecithin, choline, and other substituted ammonium compounds also inhibit the effects of silicate on pneumococci. PMID:13306854

Fermentation of glycolate by a pure culture of a strictly anaerobic gram-positive bacterium belonging to the family Lachnospiraceae.

Science.gov (United States)

Janssen, Peter H; Hugenholtz, Philip

2003-05-01

The component bacteria of a three-membered mixed culture able to ferment glycolate to acetate, propionate and CO(2) were isolated in pure culture. All three strains were strict anaerobes that, on the basis of comparative 16S rRNA gene sequence analysis, belonged to the order Clostridiales in the phylum Firmicutes (low G+C gram-positive bacteria). Two of the strains were not involved in glycolate metabolism. The third, the glycolate-fermenting strain 19gly4 (DSM 11261), was related to members of the family Lachnospiraceae. The cells of strain 19gly4 were oval- to lemon-shaped, 0.85 microm long and 0.65 microm in diameter, occurring singly, in pairs, or in chains of up to 30 cells. Strain 19gly4 fermented glycolate or fumarate to acetate, succinate, and CO(2). Hydrogen was not formed, and strain 19gly4 was able to grow on glycolate in pure culture without any syntrophic hydrogen transfer and without the use of an external electron acceptor. There was no evidence for homoacetogenic metabolism. This bacterium therefore differs in metabolism from previously reported glycolate-utilising anaerobes.

Salirhabdus euzebyi gen. nov., sp. nov., a Gram-positive, halotolerant bacterium isolated from a sea salt evaporation pond.

Science.gov (United States)

Albuquerque, Luciana; Tiago, Igor; Rainey, Fred A; Taborda, Marco; Nobre, M Fernanda; Veríssimo, António; da Costa, Milton S

2007-07-01

A low-G+C, Gram-positive bacterium, designated CVS-14(T), was recovered from a sea salt evaporation pond on the island of Sal in the Cape Verde Archipelago. This organism was catalase- and oxidase-positive. Cells were motile, spore-forming aerobic rods, with an optimum growth temperature of about 35-40 degrees C and optimum pH between 7.0 and 8.5. Optimal growth occurred in media containing 4-6 % (w/v) NaCl, although the organism was able to grow in medium without added NaCl and in medium containing 16 % NaCl. The cell-wall peptidoglycan was of A1 gamma type and the major respiratory quinone was menaquinone 7 (MK-7). Major fatty acids were iso-15 : 0, anteiso-15 : 0, iso-17 : 0 and anteiso-17 : 0. The DNA G+C content was 37.0 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain CVS-14(T) formed a distinct new branch within the radiation of the moderately halophilic bacilli group, forming a separate lineage from species of the genera Salinibacillus, Paucisalibacillus, Oceanobacillus, Lentibacillus and Virgibacillus. Strain CVS-14(T) showed 16S rRNA gene pairwise similarity values of approximately 95 % with species of the genus Salinibacillus. On the basis of morphological, physiological, chemotaxonomic and phylogenetic characteristics, strain CVS-14(T) is considered to represent a novel species in a new genus, for which the name Salirhabdus euzebyi gen. nov., sp. nov. is proposed. The type strain is CVS-14(T) (=LMG 22839(T)=CIP 108577(T)).

Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells.

Science.gov (United States)

Brunati, Cristina; Thomsen, Thomas T; Gaspari, Eleonora; Maffioli, Sonia; Sosio, Margherita; Jabes, Daniela; Løbner-Olesen, Anders; Donadio, Stefano

2018-02-01

To characterize NAI-107 and related lantibiotics for their in vitro activity against Gram-negative pathogens, alone or in combination with polymyxin, and against non-dividing cells or biofilms of Staphylococcus aureus. NAI-107 was also evaluated for its propensity to select or induce self-resistance in Gram-positive bacteria. We used MIC determinations and chequerboard experiments to establish the antibacterial activity of the examined compounds against target microorganisms. Time-kill assays were used to evaluate killing of exponential and stationary-phase cells. The effects on biofilms (growth inhibition and biofilm eradication) were evaluated using biofilm-coated pegs. The frequency of spontaneous resistant mutants was evaluated by either direct plating or by continuous sub-culturing at 0.5 × MIC levels, followed by population analysis profiles. The results showed that NAI-107 and its brominated variant are highly active against Neisseria gonorrhoeae and some other fastidious Gram-negative pathogens. Furthermore, all compounds strongly synergized with polymyxin against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, and showed bactericidal activity. Surprisingly, NAI-107 alone was bactericidal against non-dividing A. baumannii cells. Against S. aureus, NAI-107 and related lantibiotics showed strong bactericidal activity against dividing and non-dividing cells. Activity was also observed against S. aureus biofilms. As expected for a lipid II binder, no significant resistance to NAI-107 was observed by direct plating or serial passages. Overall, the results of the current work, along with previously published results on the efficacy of NAI-107 in experimental models of infection, indicate that this lantibiotic represents a promising option in addressing the serious threat of antibiotic resistance. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial

Effect of Light-Activated Hypocrellin B on the Growth and Membrane Permeability of Gram-Negative Escherichia coli Cells

Directory of Open Access Journals (Sweden)

Yuan Jiang

2014-01-01

Full Text Available Aim. To investigate the effect of light-activated hypocrellin B on the growth and membrane permeability of Gram-negative bacteria. Methods. Escherichia coli (E. coli as a model bacterium of Gram-negative bacteria was incubated with various concentrations of hypocrellin B for 60 min and was subsequently irradiated by blue light with wavelength of 470 nm at the dose of 12 J/cm2. Colony forming units were counted and the growth inhibition rate of E. coli cells was calculated after light-activated hypocrellin B. Membrane permeability was measured using flow cytometry and confocal laser scanning microscopy (CLSM with propidium iodide (PI staining. Bacterial morphology was observed using transmission electron microscopy (TEM. Reactive oxygen species in bacterial cells were measured using flow cytometry with DCFH-DA staining. Results. Significant growth inhibition rate of E. coli cells was observed after photodynamic action of hypocrellin B. Remarkable damage to the ultrastructure of E. coli was also observed by TEM. Flow cytometry and CLSM observation showed that light-activated hypocrellin B markedly increased membrane permeability of E. coli. Flow cytometry showed the intracellular ROS increase in E. coli treated by photodynamic action of hypocrellin B. Conclusion. Light-activated hypocrellin B caused intracellular ROS increase and structural damages and inhibited the growth of Gram-negative E. coli cells.

Decreased mortality associated with prompt Gram staining of blood cultures.

Science.gov (United States)

Barenfanger, Joan; Graham, Donald R; Kolluri, Lavanya; Sangwan, Gaurav; Lawhorn, Jerry; Drake, Cheryl A; Verhulst, Steven J; Peterson, Ryan; Moja, Lauren B; Ertmoed, Matthew M; Moja, Ashley B; Shevlin, Douglas W; Vautrain, Robert; Callahan, Charles D

2008-12-01

Gram stains of positive blood cultures are the most important factor influencing appropriate therapy. The sooner appropriate therapy is initiated, the better. Therefore, it is reasonable to expect that the sooner Gram stains are performed, the better. To determine the value of timely Gram stains and whether improvement in Gram stain turnaround time (TAT) is feasible, we compared data for matched pairs of patients with cultures processed promptly ( or =1 hour TAT) and then monitored TAT by control charting.In 99 matched pairs, average difference in time to detection of positive blood cultures within a pair of patients was less than 0.1 hour. For the less than 1 hour TAT group, the average TAT and crude mortality were 0.1 hour and 10.1%, respectively; for the 1 hour or longer TAT group, they were 3.3 hours and 19.2%, respectively (P Gram stains.

Should gram stains have a role in diagnosing hip arthroplasty infections?

Science.gov (United States)

Johnson, Aaron J; Zywiel, Michael G; Stroh, D Alex; Marker, David R; Mont, Michael A

2010-09-01

The utility of Gram stains in diagnosing periprosthetic infections following total hip arthroplasty has recently been questioned. Several studies report low sensitivity of the test, and its poor ability to either confirm or rule out infection in patients undergoing revision total hip arthroplasty. Despite this, many institutions including that of the senior author continue to perform Gram stains during revision total hip arthroplasty. We assessed the sensitivity, specificity, accuracy, and positive and negative predictive values of Gram stains from surgical-site samplings taken from procedures on patients with both infected and aseptic revision total hip arthroplasties. A review was performed on patients who underwent revision total hip arthroplasty between 2000 and 2007. Eighty-two Gram stains were performed on patients who had infected total hip arthroplasties and underwent revision procedures. Additionally, of the 410 revision total hip arthroplasties performed on patients who were confirmed infection-free, 120 Gram stains were performed. Patients were diagnosed as infected using multiple criteria at the time of surgery. Sensitivity, specificity, positive and negative predictive values, and accuracy were calculated from these Gram stain results. The Gram stain demonstrated a sensitivity and specificity of 9.8% and 100%, respectively. In this series, the Gram stain had a negative predictive value of 62%, a positive predictive value of 100%, and an accuracy of 63%. Gram stains obtained from surgical-site samples had poor sensitivity and poor negative predictive value. Based on these findings, as well as those of other authors, we believe that Gram stains should no longer be considered for diagnosing infections in revision total hip arthroplasty. Level III, diagnostic study. See Guidelines for Authors for a complete description of levels of evidence.

An unusual class of anthracyclines potentiate Gram-positive antibiotics in intrinsically resistant Gram-negative bacteria

Science.gov (United States)

Cox, Georgina; Koteva, Kalinka; Wright, Gerard D.

2014-01-01

Objectives An orthogonal approach taken towards novel antibacterial drug discovery involves the identification of small molecules that potentiate or enhance the activity of existing antibacterial agents. This study aimed to identify natural-product rifampicin adjuvants in the intrinsically resistant organism Escherichia coli. Methods E. coli BW25113 was screened against 1120 actinomycete fermentation extracts in the presence of subinhibitory (2 mg/L) concentrations of rifampicin. The active molecule exhibiting the greatest rifampicin potentiation was isolated using activity-guided methods and identified using mass and NMR spectroscopy. Susceptibility testing and biochemical assays were used to determine the mechanism of antibiotic potentiation. Results The anthracycline Antibiotic 301A1 was isolated from the fermentation broth of a strain of Streptomyces (WAC450); the molecule was shown to be highly synergistic with rifampicin (fractional inhibitory concentration index = 0.156) and moderately synergistic with linezolid (FIC index = 0.25) in both E. coli and Acinetobacter baumannii. Activity was associated with inhibition of efflux and the synergistic phenotype was lost when tested against E. coli harbouring mutations within the rpoB gene. Structure–activity relationship studies revealed that other anthracyclines do not synergize with rifampicin and removal of the sugar moiety of Antibiotic 301A1 abolishes activity. Conclusions Screening only a subsection of our natural product library identified a small-molecule antibiotic adjuvant capable of sensitizing Gram-negative bacteria to antibiotics to which they are ordinarily intrinsically resistant. This result demonstrates the great potential of this approach in expanding antibiotic effectiveness in the face of the growing challenge of resistance in Gram-negatives. PMID:24627312

Recovery of vancomycin-resistant gram-positive cocci from children.

Science.gov (United States)

Green, M; Wadowsky, R M; Barbadora, K

1990-03-01

A cross-sectional survey of vancomycin-resistant gram-positive cocci (VRGPC) in the feces of children was initiated after several bacteremic infections with these organisms occurred at our hospital. A selective medium consisting of colistin-nalidixic acid agar, 5% sheep blood, vancomycin (5 mg/liter), and amphotericin B (8 mg/liter) was developed to isolate VRGPC. A single stool specimen submitted to the clinical microbiology laboratory from each of 48 patients was inoculated onto the medium. Plates were incubated at 35 degrees C with 5% carbon dioxide and examined at 24, 48, and 72 h. Susceptibilities were determined by broth microdilution. A total of 14 isolates from 11 of 48 (22%) children were recovered. The density of growth ranged from a single colony to 2+. The VRGPC were identified as Leuconostoc lactis (n = 2), Lactobacillus confusus (n = 4), Enterococcus species (n = 5), and Lactococcus lactis (n = 3). One strain of Lactobacillus confusus was recovered from both the stool and the blood of one of these patients. The MICs of vancomycin were 4 micrograms/ml for one of the isolates, 8 micrograms/ml for four of the isolates, and more than 16 micrograms/ml for the remaining eight isolates. All isolates were susceptible to both penicillin and ampicillin. Only 1 of the 11 children had received prior treatment with vancomycin. We conclude that low concentrations of VRGPC may be common in the gastrointestinal tracts of children.

Differential count of cells in the milk of cows with subclinical mastitis with the colorations of May-Grünwald Giemsa and Gram

Directory of Open Access Journals (Sweden)

Ana Paula Lopes Marques

2016-11-01

Full Text Available ABSTRACT. Marques A.P.L., Botteon R.C.C.M., Machado C.H., Medeiros B.P., Assis J.D., Barros J.P.N. & Araújo F.L. [Differential count of cells in the milk of cows with subclinical mastitis with the colorations of May-Grünwald Giemsa and Gram.] Contagem diferencial de células no leite de vacas com mastite subclínica com as colorações de May-Grünwald Giemsa e Gram. Revista Brasileira de Medicina Veterinária, 38(Supl.2:123-127, 2016. Programa de Pós-Graduação em Medicina Veterinária, Universidade Federal Rural do Rio de Janeiro, Instituto de Veterinária, Departamento de Medicina e Cirurgia Veterinária, BR 465, Km 7, Seropédica, RJ 23897-970, Brasil. E-mail: marquesapl@ufrrj.br Somatic cell count (SCC determines the amount of leucocytes and epithelial cells present in milk. It is used for monitoring the subclinical mastitis in the herd. Various tests can be used for SCC. For convenience and accuracy there is the electronic counting by flow cytometry. Microscopic slides count is the standard method for the determination of SCC in raw milk with the classic method described by Prescot and Breed (1910. This study aimed to evaluate the differential cell count in milk of cows with subclinical mastitis, by optical microscopy, in slides stained by May Grunwald-Giemsa and Gram. In both staining the cells showed morphological changes. However, the unidentifiable cell percentage did not exceed 10% enhancing the applicability of the differential cell counts, as used in leucocytes in hematologic smears. There were small variations in the counts with the two colorations. There was a predominance of neutrophils (˃60%, followed by lymphocytes (˃25% consistent with the characterization of the samples as coming from rooms with subclinical mastitis. May Grunwald- -Giemsa stain showed similar results to Gram. The main differences between the colorations are based on the amplitude and intensity of staining, the Gram stain which stood out. In Gram

Design, Synthesis and Evaluation of Branched RRWQWR-Based Peptides as Antibacterial Agents Against Clinically Relevant Gram-Positive and Gram-Negative Pathogens

Directory of Open Access Journals (Sweden)

Sandra C. Vega

2018-03-01

Full Text Available Multidrug resistance of pathogenic bacteria has become a public health crisis that requires the urgent design of new antibacterial drugs such as antimicrobial peptides (AMPs. Seeking to obtain new, lactoferricin B (LfcinB-based synthetic peptides as viable early-stage candidates for future development as AMPs against clinically relevant bacteria, we designed, synthesized and screened three new cationic peptides derived from bovine LfcinB. These peptides contain at least one RRWQWR motif and differ by the copy number (monomeric, dimeric or tetrameric and structure (linear or branched of this motif. They comprise a linear palindromic peptide (RWQWRWQWR, a dimeric peptide (RRWQWR2KAhx and a tetrameric peptide (RRWQWR4K2Ahx2C2. They were screened for antibacterial activity against Enterococcus faecalis (ATCC 29212 and ATCC 51575 strains, Pseudomonas aeruginosa (ATCC 10145 and ATCC 27853 strains and clinical isolates of two Gram-positive bacteria (Enterococcus faecium and Staphylococcus aureus and two Gram-negative bacteria (Klebsiella pneumoniae and Pseudomonas aeruginosa. All three peptides exhibited greater activity than did the reference peptide, LfcinB (17–31, which contains a single linear RRWQWR motif. Against the ATCC reference strains, the three new peptides exhibited minimum inhibitory concentration (MIC50 values of 3.1–198.0 μM and minimum bactericidal concentration (MBC values of 25–200 μM, and against the clinical isolates, MIC50 values of 1.6–75.0 μM and MBC values of 12.5–100 μM. However, the tetrameric peptide was also found to be strongly hemolytic (49.1% at 100 μM. Scanning Electron Microscopy (SEM demonstrated that in the dimeric and tetrameric peptides, the RRWQWR motif is exposed to the pathogen surface. Our results may inform the design of new, RRWQWR-based AMPs.

Identification of Multiple Soluble Fe(III Reductases in Gram-Positive Thermophilic Bacterium Thermoanaerobacter indiensis BSB-33

Directory of Open Access Journals (Sweden)

Subrata Pal

2014-01-01

Full Text Available Thermoanaerobacter indiensis BSB-33 has been earlier shown to reduce Fe(III and Cr(VI anaerobically at 60°C optimally. Further, the Gram-positive thermophilic bacterium contains Cr(VI reduction activity in both the membrane and cytoplasm. The soluble fraction prepared from T. indiensis cells grown at 60°C was found to contain the majority of Fe(III reduction activity of the microorganism and produced four distinct bands in nondenaturing Fe(III reductase activity gel. Proteins from each of these bands were partially purified by chromatography and identified by mass spectrometry (MS with the help of T. indiensis proteome sequences. Two paralogous dihydrolipoamide dehydrogenases (LPDs, thioredoxin reductase (Trx, NADP(H-nitrite reductase (Ntr, and thioredoxin disulfide reductase (Tdr were determined to be responsible for Fe(III reductase activity. Amino acid sequence and three-dimensional (3D structural similarity analyses of the T. indiensis Fe(III reductases were carried out with Cr(VI reducing proteins from other bacteria. The two LPDs and Tdr showed very significant sequence and structural identity, respectively, with Cr(VI reducing dihydrolipoamide dehydrogenase from Thermus scotoductus and thioredoxin disulfide reductase from Desulfovibrio desulfuricans. It appears that in addition to their iron reducing activity T. indiensis LPDs and Tdr are possibly involved in Cr(VI reduction as well.

Evolution of Recognition of Ligands from Gram-Positive Bacteria: Similarities and Differences in the TLR2-Mediated Response between Mammalian Vertebrates and Teleost Fish

NARCIS (Netherlands)

Ribeiro, Carla M. S.; Hermsen, Trudi; Taverne-Thiele, Anja J.; Savelkoul, Huub F. J.; Wiegertjes, Geert F.

2010-01-01

We investigated the role of the TLR2 receptor in the recognition of ligands from Gram-positive bacteria in fish. Comparative sequence analysis showed a highly conserved Toll/IL-1 receptor domain. Although the leucine-rich repeat domain was less conserved, the position of the critical peptidoglycan

A Comparison of Heat versus Methanol Fixation for Gram Staining Bacteria

Science.gov (United States)

Minnerath, Jeanne M.; Roland, Jenna M.; Rossi, Lucas C.; Weishalla, Steven R.; Wolf, Melissa M.

2009-01-01

Gram staining bacteria is a fundamental technique introduced in general biology and microbiology laboratory courses. Two common problems students encounter when Gram staining bacteria are (1) having a difficult time locating bacterial cells on the microscope slide and (2) over-decolorizing bacterial cells during the staining procedure such that…

Use of the Lactococcal nisA Promoter To Regulate Gene Expression in Gram-Positive Bacteria : Comparison of Induction Level and Promoter Strength

NARCIS (Netherlands)

Eichenbaum, Zehava; Federle, Michael J.; Marra, Diana; Vos, Willem M. de; Kuipers, Oscar P.; Kleerebezem, Michiel; Scott, June R.

1998-01-01

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and

The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of Gram-positive anaerobic cocci

DEFF Research Database (Denmark)

Veloo, A C M; de Vries, E D; Jean-Pierre, H

2016-01-01

Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix-assisted......Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix......-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) database for the identification of GPAC. Main spectral profiles (MSPs) were created for 108 clinical GPAC isolates. Identity was confirmed using 16S rRNA gene sequencing. Species identification was considered to be reliable...... if the sequence similarity with its closest relative was ≥98.7%. The optimized database was validated using 140 clinical isolates. The 16S rRNA sequencing identity was compared with the MALDI-TOF MS result. MSPs were added from 17 species that were not yet represented in the MALDI-TOF MS database or were under...

Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction

Directory of Open Access Journals (Sweden)

Kim Sang-Hoon

2012-02-01

Full Text Available Abstract Background The genome of the Gram-positive, metal-reducing, dehalorespiring Desulfitobacterium hafniense DCB-2 was sequenced in order to gain insights into its metabolic capacities, adaptive physiology, and regulatory machineries, and to compare with that of Desulfitobacterium hafniense Y51, the phylogenetically closest strain among the species with a sequenced genome. Results The genome of Desulfitobacterium hafniense DCB-2 is composed of a 5,279,134-bp circular chromosome with 5,042 predicted genes. Genome content and parallel physiological studies support the cell's ability to fix N2 and CO2, form spores and biofilms, reduce metals, and use a variety of electron acceptors in respiration, including halogenated organic compounds. The genome contained seven reductive dehalogenase genes and four nitrogenase gene homologs but lacked the Nar respiratory nitrate reductase system. The D. hafniense DCB-2 genome contained genes for 43 RNA polymerase sigma factors including 27 sigma-24 subunits, 59 two-component signal transduction systems, and about 730 transporter proteins. In addition, it contained genes for 53 molybdopterin-binding oxidoreductases, 19 flavoprotein paralogs of the fumarate reductase, and many other FAD/FMN-binding oxidoreductases, proving the cell's versatility in both adaptive and reductive capacities. Together with the ability to form spores, the presence of the CO2-fixing Wood-Ljungdahl pathway and the genes associated with oxygen tolerance add flexibility to the cell's options for survival under stress. Conclusions D. hafniense DCB-2's genome contains genes consistent with its abilities for dehalogenation, metal reduction, N2 and CO2 fixation, anaerobic respiration, oxygen tolerance, spore formation, and biofilm formation which make this organism a potential candidate for bioremediation at contaminated sites.

Antimicrobial-Resistance Genetic Markers in Potentially Pathogenic Gram Positive Cocci Isolated from Brazilian Soft Cheese.

Science.gov (United States)

Resende, Juliana Alves; Fontes, Cláudia Oliveira; Ferreira-Machado, Alessandra Barbosa; Nascimento, Thiago César; Silva, Vânia Lúcia; Diniz, Cláudio Galuppo

2018-02-01

Although most Brazilian dairy products meet high technological standards, there are quality issues regarding milk production, which may reduce the final product quality. Several microbial species may contaminate milk during manufacture and handling. If antimicrobial usage remains uncontrolled in dairy cattle, the horizontal transfer of antimicrobial resistance genes in foodstuffs may be of particular concern for both food producers and dairy industry. This study focused on the evaluation of putative Gram positive cocci in Minas cheese and of antimicrobial and biocide resistance genes among the isolated bacteria. Representative samples of 7 different industrially trademarked Minas cheeses (n = 35) were processed for selective culture and isolation of Gram positive cocci. All isolated bacteria were identified by DNA sequencing of the 16S rRNA gene. Antimicrobial resistance genes were screened by PCR. Overall, 208 strains were isolated and identified as follows: Enterococcus faecalis (47.6%), Macrococcus caseolyticus (18.3%), Enterococcus faecium (11.5%), Enterococcus caseliflavus (7.7%), Staphylococcus haemolyticus (7.2%), Staphylococcus aureus (4.3%), Staphylococcus epidermidis (2.9%), and Enterococcus hirae (0.5%). The genetic markers mecA (78.0%) and smr (71.4%) were the most prevalent, but others were also detected, such as blaZ (65.2%), msrA (60.9%), msrB (46.6%), linA (54.7%), and aacA-aphD (47.6%). The occurrence of opportunist pathogenic bacteria harboring antimicrobial resistance markers in the cheese samples are of special concern, since these bacteria are not considered harmful contaminating agents according to the Brazilian sanitary regulations. However, they are potentially pathogenic bacteria and the cheese may be considered a reservoir for antimicrobial resistance genes available for horizontal transfer through the food chain, manufacturing personnel and consumers. © 2018 Institute of Food Technologists®.

An unusual class of anthracyclines potentiate Gram-positive antibiotics in intrinsically resistant Gram-negative bacteria.

Science.gov (United States)

Cox, Georgina; Koteva, Kalinka; Wright, Gerard D

2014-07-01

An orthogonal approach taken towards novel antibacterial drug discovery involves the identification of small molecules that potentiate or enhance the activity of existing antibacterial agents. This study aimed to identify natural-product rifampicin adjuvants in the intrinsically resistant organism Escherichia coli. E. coli BW25113 was screened against 1120 actinomycete fermentation extracts in the presence of subinhibitory (2 mg/L) concentrations of rifampicin. The active molecule exhibiting the greatest rifampicin potentiation was isolated using activity-guided methods and identified using mass and NMR spectroscopy. Susceptibility testing and biochemical assays were used to determine the mechanism of antibiotic potentiation. The anthracycline Antibiotic 301A(1) was isolated from the fermentation broth of a strain of Streptomyces (WAC450); the molecule was shown to be highly synergistic with rifampicin (fractional inhibitory concentration index = 0.156) and moderately synergistic with linezolid (FIC index = 0.25) in both E. coli and Acinetobacter baumannii. Activity was associated with inhibition of efflux and the synergistic phenotype was lost when tested against E. coli harbouring mutations within the rpoB gene. Structure-activity relationship studies revealed that other anthracyclines do not synergize with rifampicin and removal of the sugar moiety of Antibiotic 301A(1) abolishes activity. Screening only a subsection of our natural product library identified a small-molecule antibiotic adjuvant capable of sensitizing Gram-negative bacteria to antibiotics to which they are ordinarily intrinsically resistant. This result demonstrates the great potential of this approach in expanding antibiotic effectiveness in the face of the growing challenge of resistance in Gram-negatives. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Lack of clinical utility of urine gram stain for suspected urinary tract infection in pediatric patients.

Science.gov (United States)

Cantey, Joseph B; Gaviria-Agudelo, Claudia; McElvania TeKippe, Erin; Doern, Christopher D

2015-04-01

Urinary tract infection (UTI) is one of the most common infections in children. Urine culture remains the gold standard for diagnosis, but the utility of urine Gram stain relative to urinalysis (UA) is unclear. We reviewed 312 pediatric patients with suspected UTI who had urine culture, UA, and urine Gram stain performed from a single urine specimen. UA was considered positive if ≥10 leukocytes per oil immersion field were seen or if either nitrates or leukocyte esterase testing was positive. Urine Gram stain was considered positive if any organisms were seen. Sensitivity, specificity, and positive and negative predictive values were calculated using urine culture as the gold standard. Thirty-seven (12%) patients had a culture-proven UTI. Compared to urine Gram stain, UA had equal sensitivity (97.3% versus 97.5%) and higher specificity (85% versus 74%). Empirical therapy was prescribed before the Gram stain result was known in 40 (49%) patients and after in 42 (51%) patients. The antibiotics chosen did not differ between the two groups (P=0.81), nor did they differ for patients with Gram-negative rods on urine Gram stain compared to those with Gram-positive cocci (P=0.67). From these data, we conclude that UA has excellent negative predictive value that is not enhanced by urine Gram stain and that antibiotic selection did not vary based on the urine Gram stain result. In conclusion, the clinical utility of urine Gram stain does not warrant the time or cost it requires. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The use of Gram stain and matrix-assisted laser desorption ionization time-of-flight mass spectrometry on positive blood culture: synergy between new and old technology.

Science.gov (United States)

Fuglsang-Damgaard, David; Nielsen, Camilla Houlberg; Mandrup, Elisabeth; Fuursted, Kurt

2011-10-01

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is promising as an alternative to more costly and cumbersome methods for direct identifications in blood cultures. We wanted to evaluate a simplified pre-treatment method for using MALDI-TOF-MS directly on positive blood cultures using BacT/Alert blood culture system, and to test an algorithm combining the result of the initial microscopy with the result suggested by MALDI-TOF-MS. Using the recommended cut-off score of 1.7 the best results were obtained among Gram-negative rods with correct identifications in 91% of Enterobacteriaceae, 83% in aerobic/non-fermentative Gram-negative rods, whereas results were more modest among Gram-positive cocci with correct identifications in 52% of Staphylococci, 54% in Enterococci and only 20% in Streptococci. Combining the results of Gram stain with the top reports by MALDI-TOF-MS, increased the sensitivity from 91% to 93% in the score range from 1.5 to 1.7 and from 48% to 85% in the score range from 1.3 to 1.5. Thus, using this strategy and accepting a cut-off at 1.3 instead of the suggested 1.7, overall sensitivity could be increased from 88.1% to 96.3%. MALDI-TOF-MS is an efficient method for direct routine identification of bacterial isolates in blood culture, especially when combined with the result of the Gram stain. © 2011 The Authors. APMIS © 2011 APMIS.

1-variation in cell morphology and gram-staining property of bacilli under different salt stresses and media composition

International Nuclear Information System (INIS)

Shuaib, I.; Mehmood, U.; Hasnain, S.

2004-01-01

Hs-3, Hs-4 and Az-9 are soil tolerant strains, which show Gram negative to Gram-variable staining behavior under varying environmental conditions. These strains were grown in different media composition (lowry, nutrient, pennassy and M-9 minimal both in broth cultures and agar media) and salt stresses (Mg SO/sub 4/, KCl, KNO/sub 3/) supplemented with 0.1 and 1M of NaCl at 37 deg. Centi grade for 4, 8, 16 and 24 hours. Media composition and various salts stress manifested great variation in staining behavior and cell morphology. Az-9 exhibited maximum variation in staining and morphology in rich medium. Hs-3 showed maximum filamentation under KCl stress in pennassy medium. KCl and KNO/sub 3/ stresses caused filamentation in all strains while spore formation was pronounced under MgSO/sub 4/ and NaCl stress in Az-9 in nutrient agar. Potassium salt caused adverse affects on cell morphology by degeneration or lysis of cells with passage of time. (author)

Therapeutic Potential of a Scorpion Venom-Derived Antimicrobial Peptide and Its Homologs Against Antibiotic-Resistant Gram-Positive Bacteria

Directory of Open Access Journals (Sweden)

Gaomin Liu

2018-05-01

Full Text Available The alarming rise in the prevalence of antibiotic resistance among pathogenic bacteria poses a unique challenge for the development of effective therapeutic agents. Antimicrobial peptides (AMPs have attracted a great deal of attention as a possible solution to the increasing problem of antibiotic-resistant bacteria. Marcin-18 was identified from the scorpion Mesobuthus martensii at both DNA and protein levels. The genomic sequence revealed that the marcin-18 coding gene contains a phase-I intron with a GT-AG splice junction located in the DNA region encoding the N-terminal part of signal peptide. The peptide marcin-18 was also isolated from scorpion venom. A protein sequence homology search revealed that marcin-18 shares extremely high sequence identity to the AMPs meucin-18 and megicin-18. In vitro, chemically synthetic marcin-18 and its homologs (meucin-18 and megicin-18 showed highly potent inhibitory activity against Gram-positive bacteria, including some clinical antibiotic-resistant strains. Importantly, in a mouse acute peritonitis model, these peptides significantly decreased the bacterial load in ascites and rescued nearly all mice heavily infected with clinical methicillin-resistant Staphylococcus aureus from lethal bacteremia. Peptides exerted antimicrobial activity via a bactericidal mechanism and killed bacteria through membrane disruption. Taken together, marcin-18 and its homologs have potential for development as therapeutic agents for treating antibiotic-resistant, Gram-positive bacterial infections.

Positive role of peptidoglycan breaks in lactococcal biofilm formation

NARCIS (Netherlands)

Mercier, C; Durrieu, C; Briandet, R; Domakova, E; Tremblay, J; Buist, G; Kulakauskas, S

2002-01-01

Bacterial attachment to solid matrices depends on adhesive molecules present on the cell surface. Here we establish a positive correlation between peptidoglycan (PG) breaks, rather than particular molecules, and biofilm-forming capacity in the Gram-positive bacterium Lactococcus lactis. The L.

The Na+ transport in gram-positive bacteria defect in the Mrp antiporter complex measured with 23Na nuclear magnetic resonance.

Science.gov (United States)

Górecki, Kamil; Hägerhäll, Cecilia; Drakenberg, Torbjörn

2014-01-15

(23)Na nuclear magnetic resonance (NMR) has previously been used to monitor Na(+) translocation across membranes in gram-negative bacteria and in various other organelles and liposomes using a membrane-impermeable shift reagent to resolve the signals resulting from internal and external Na(+). In this work, the (23)Na NMR method was adapted for measurements of internal Na(+) concentration in the gram-positive bacterium Bacillus subtilis, with the aim of assessing the Na(+) translocation activity of the Mrp (multiple resistance and pH) antiporter complex, a member of the cation proton antiporter-3 (CPA-3) family. The sodium-sensitive growth phenotype observed in a B. subtilis strain with the gene encoding MrpA deleted could indeed be correlated to the inability of this strain to maintain a lower internal Na(+) concentration than an external one. Copyright © 2013 Elsevier Inc. All rights reserved.

Multiplex identification of sepsis-causing Gram-negative pathogens from the plasma of infected blood.

Science.gov (United States)

Chung, Boram; Park, Chulmin; Cho, Sung-Yeon; Shin, Juyoun; Shin, Sun; Yim, Seon-Hee; Lee, Dong-Gun; Chung, Yeun-Jung

2018-02-01

Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram-negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram-negative pathogens and also validated whether our system can identify Gram-negative pathogens with the cell-free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram-negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX-M group 1 to identify the ESBL producing Gram-negative pathogens. All six target-specific peaks were clearly separated without any non-specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram-negative clinical isolates, all of them were successfully identified without any non-specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis-causing, drug-resistant Gram-negative pathogens and also the major ESBL producing Gram-negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram-negative pathogens for sepsis patients, which is very crucial for better treatment outcomes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ClubSub-P: Cluster-Based Subcellular Localization Prediction for Gram-Negative Bacteria and Archaea

Science.gov (United States)

Paramasivam, Nagarajan; Linke, Dirk

2011-01-01

The subcellular localization (SCL) of proteins provides important clues to their function in a cell. In our efforts to predict useful vaccine targets against Gram-negative bacteria, we noticed that misannotated start codons frequently lead to wrongly assigned SCLs. This and other problems in SCL prediction, such as the relatively high false-positive and false-negative rates of some tools, can be avoided by applying multiple prediction tools to groups of homologous proteins. Here we present ClubSub-P, an online database that combines existing SCL prediction tools into a consensus pipeline from more than 600 proteomes of fully sequenced microorganisms. On top of the consensus prediction at the level of single sequences, the tool uses clusters of homologous proteins from Gram-negative bacteria and from Archaea to eliminate false-positive and false-negative predictions. ClubSub-P can assign the SCL of proteins from Gram-negative bacteria and Archaea with high precision. The database is searchable, and can easily be expanded using either new bacterial genomes or new prediction tools as they become available. This will further improve the performance of the SCL prediction, as well as the detection of misannotated start codons and other annotation errors. ClubSub-P is available online at http://toolkit.tuebingen.mpg.de/clubsubp/ PMID:22073040

Use of magnetic beads for Gram staining of bacteria in aqueous suspension.

Science.gov (United States)

Yazdankhah, S P; Sørum, H; Larsen, H J; Gogstad, G

2001-12-01

A Gram staining technique was developed using monodisperse magnetic beads in concentrating bacteria in suspension for downstream application. The technique does not require heat fixation of organisms, electrical power, or a microscope. Gram-negative and Gram-positive bacteria were identified macroscopically based on the colour of the suspension. The bacteria concentrated on magnetic beads may also be identified microscopically.

Gram-typing of mastitis bacteria in milk samples using flow cytometry

DEFF Research Database (Denmark)

Langerhuus, Sine Nygaard; Ingvartsen, Klaus Lønne; Bennedsgaard, Torben Werner

2013-01-01

Fast identification of pathogenic bacteria in milk samples from cows with clinical mastitis is central to proper treatment. In Denmark, time to bacterial diagnosis is typically 24 to 48 h when using traditional culturing methods. The PCR technique provides a faster and highly sensitive identifica......Fast identification of pathogenic bacteria in milk samples from cows with clinical mastitis is central to proper treatment. In Denmark, time to bacterial diagnosis is typically 24 to 48 h when using traditional culturing methods. The PCR technique provides a faster and highly sensitive...... cytometry-based method, which can detect and distinguish gram-negative and gram-positive bacteria in mastitis milk samples. The differentiation was based on bacterial fluorescence intensities upon labeling with biotin-conjugated wheat germ agglutinin and acridine orange. Initially 19 in-house bacterial...... characteristic curves for the 19 bacterial cultures. The method was then tested on 53 selected mastitis cases obtained from the department biobank (milk samples from 6 gram-negative and 47 gram-positive mastitis cases). Gram-negative bacteria in milk samples were detected with a sensitivity of 1...

Incidence of carbapenem-resistant gram negatives in Italian transplant recipients: a nationwide surveillance study.

Science.gov (United States)

Lanini, Simone; Costa, Alessandro Nanni; Puro, Vincenzo; Procaccio, Francesco; Grossi, Paolo Antonio; Vespasiano, Francesca; Ricci, Andrea; Vesconi, Sergio; Ison, Michael G; Carmeli, Yehuda; Ippolito, Giuseppe

2015-01-01

Bacterial infections remain a challenge to solid organ transplantation. Due to the alarming spread of carbapenem-resistant gram negative bacteria, these organisms have been frequently recognized as cause of severe infections in solid organ transplant recipients. Between 15 May and 30 September 2012 we enrolled 887 solid organ transplant recipients in Italy with the aim to describe the epidemiology of gram negative bacteria spreading, to explore potential risk factors and to assess the effect of early isolation of gram negative bacteria on recipients' mortality during the first 90 days after transplantation. During the study period 185 clinical isolates of gram negative bacteria were reported, for an incidence of 2.39 per 1000 recipient-days. Positive cultures for gram negative bacteria occurred early after transplantation (median time 26 days; incidence rate 4.33, 1.67 and 1.14 per 1,000 recipient-days in the first, second and third month after SOT, respectively). Forty-nine of these clinical isolates were due to carbapenem-resistant gram negative bacteria (26.5%; incidence 0.63 per 1000 recipient-days). Carbapenems resistance was particularly frequent among Klebsiella spp. isolates (49.1%). Recipients with longer hospital stay and those who received either heart or lung graft were at the highest risk of testing positive for any gram negative bacteria. Moreover recipients with longer hospital stay, lung recipients and those admitted to hospital for more than 48h before transplantation had the highest probability to have culture(s) positive for carbapenem-resistant gram negative bacteria. Forty-four organ recipients died (0.57 per 1000 recipient-days) during the study period. Recipients with at least one positive culture for carbapenem-resistant gram negative bacteria had a 10.23-fold higher mortality rate than those who did not. The isolation of gram-negative bacteria is most frequent among recipient with hospital stays >48 hours prior to transplant and in those

Incidence of carbapenem-resistant gram negatives in Italian transplant recipients: a nationwide surveillance study.

Directory of Open Access Journals (Sweden)

Simone Lanini

Full Text Available Bacterial infections remain a challenge to solid organ transplantation. Due to the alarming spread of carbapenem-resistant gram negative bacteria, these organisms have been frequently recognized as cause of severe infections in solid organ transplant recipients.Between 15 May and 30 September 2012 we enrolled 887 solid organ transplant recipients in Italy with the aim to describe the epidemiology of gram negative bacteria spreading, to explore potential risk factors and to assess the effect of early isolation of gram negative bacteria on recipients' mortality during the first 90 days after transplantation. During the study period 185 clinical isolates of gram negative bacteria were reported, for an incidence of 2.39 per 1000 recipient-days. Positive cultures for gram negative bacteria occurred early after transplantation (median time 26 days; incidence rate 4.33, 1.67 and 1.14 per 1,000 recipient-days in the first, second and third month after SOT, respectively. Forty-nine of these clinical isolates were due to carbapenem-resistant gram negative bacteria (26.5%; incidence 0.63 per 1000 recipient-days. Carbapenems resistance was particularly frequent among Klebsiella spp. isolates (49.1%. Recipients with longer hospital stay and those who received either heart or lung graft were at the highest risk of testing positive for any gram negative bacteria. Moreover recipients with longer hospital stay, lung recipients and those admitted to hospital for more than 48h before transplantation had the highest probability to have culture(s positive for carbapenem-resistant gram negative bacteria. Forty-four organ recipients died (0.57 per 1000 recipient-days during the study period. Recipients with at least one positive culture for carbapenem-resistant gram negative bacteria had a 10.23-fold higher mortality rate than those who did not.The isolation of gram-negative bacteria is most frequent among recipient with hospital stays >48 hours prior to transplant

Comparative in vitro activities of trovafloxacin (CP-99,219) against 445 gram-positive isolates from patients with endocarditis and those with other bloodstream infections

NARCIS (Netherlands)

H.P. Endtz (Hubert); J.W. Mouton (Johan); J.G. den Hollander (Jan); N.P.W.C.J. van den Braak (Nicole); H.A. Verbrugh (Henri)

1997-01-01

textabstractThe in vitro activity of trovafloxacin (CP-99,219), a new fluoroquinolone, was compared with the in vitro activities of other commonly used quinolones and other antimicrobial agents against 445 gram-positive microorganisms isolated between 1986 and 1995 from

Overcoming function annotation errors in the Gram-positive pathogen Streptococcus suis by a proteomics-driven approach

Directory of Open Access Journals (Sweden)

Bárcena José A

2008-12-01

Full Text Available Abstract Background Annotation of protein-coding genes is a key step in sequencing projects. Protein functions are mainly assigned on the basis of the amino acid sequence alone by searching of homologous proteins. However, fully automated annotation processes often lead to wrong prediction of protein functions, and therefore time-intensive manual curation is often essential. Here we describe a fast and reliable way to correct function annotation in sequencing projects, focusing on surface proteomes. We use a proteomics approach, previously proven to be very powerful for identifying new vaccine candidates against Gram-positive pathogens. It consists of shaving the surface of intact cells with two proteases, the specific cleavage-site trypsin and the unspecific proteinase K, followed by LC/MS/MS analysis of the resulting peptides. The identified proteins are contrasted by computational analysis and their sequences are inspected to correct possible errors in function prediction. Results When applied to the zoonotic pathogen Streptococcus suis, of which two strains have been recently sequenced and annotated, we identified a set of surface proteins without cytoplasmic contamination: all the proteins identified had exporting or retention signals towards the outside and/or the cell surface, and viability of protease-treated cells was not affected. The combination of both experimental evidences and computational methods allowed us to determine that two of these proteins are putative extracellular new adhesins that had been previously attributed a wrong cytoplasmic function. One of them is a putative component of the pilus of this bacterium. Conclusion We illustrate the complementary nature of laboratory-based and computational methods to examine in concert the localization of a set of proteins in the cell, and demonstrate the utility of this proteomics-based strategy to experimentally correct function annotation errors in sequencing projects. This

Monograph: In vitro efficacy of 30 ethnomedicinal plants used by Indian aborigines against 6 multidrug resistant Gram-positive pathogenic bacteria

Directory of Open Access Journals (Sweden)

Mahesh Chandra Sahu

2015-02-01

Full Text Available Objective: To monitor in vitro antibacterial activities of leaf extracts of 30 common and noncommon plants used by aborigines in Kalahandi district, Odisha, against 6 clinically isolated multidrug resistant (MDR Gram-positive bacteria of 3 genera, Staphylococcus, Streptococcus, and Enterococcus. Methods: The antibiotic sensitivity patterns of 6 bacterial strains were studied with the diskdiffusion method with 1 7 antibiotics belonging to 8 classes. Monitored plants have ethnomedicinal use and several are used as traditional medicines. Antibacterial properties were studied with the agar-well diffusion method. Minimum inhibitory concentration and minimum bactericidal concentration values of ethanolic and aqueous extracts of plants were determined by the microbroth-dilution method. Results: Ethanolic plant-extracts had the better antibacterial potencies in comparison to their corresponding aqueous extracts. Plants with most conspicuous antibacterial properties in controlling MDR strains of Gram-positive bacteria were aqueous and ethanolic extracts of plants, Ixora coccinea, Nyctanthes arbor-tristis, Polycythaemia rubra, Pongamia pinnata and Syzygium cumini, Carthamus tinctorius, Cucurbita maxima, Murraya koenigii, Leucas aspera, Plumbago indica and Psidium guajava. Ethanolic extracts of most plants had phytochemicals, alkaloids, glycosides, terpenoids, reducing sugars, saponins, tannins, flavonoids and steroids. Conclusions: These plants could be used further for the isolation of pure compounds to be used as complementary non-microbial antimicrobial medicines.

Identification of the sigmaB regulon of Bacillus cereus and conservation of sigmaB-regulated genes in low-GC-content gram-positive bacteria

NARCIS (Netherlands)

Schaik, van W.; Voort, van der M.; Molenaar, D.; Moezelaar, R.; Vos, de W.M.; Abee, T.

2007-01-01

The alternative sigma factor B has an important role in the acquisition of stress resistance in many gram-positive bacteria, including the food-borne pathogen Bacillus cereus. Here, we describe the identification of the set of B-regulated genes in B. cereus by DNA microarray analysis of the

Antimicrobial susceptibility of quinupristin/dalfopristin tested against gram-positive cocci from Latin America: results from the Global SMART (GSMART surveillance study

Directory of Open Access Journals (Sweden)

Sader Helio S.

2001-01-01

Full Text Available Gram-positive cocci are important causes of both nosocomial and community-acquired infections, and antimicrobial resistance among these pathogens has become an important problem worldwide. Since resistance among these organisms can vary substantially by geographic location, we conducted a multicenter surveillance study with isolates from five Latin American countries (15 medical centers. Quinupristin/dalfopristin (formerly RP-59500 is a novel streptogramin combination with focused activity against Gram-positive cocci, many exhibiting emerging resistance. The in vitro activity of quinupristin/dalfopristin and 12 other antimicrobial agents were evaluated against 1,948 strains including Staphylococcus aureus (747 strains, coagulase-negative staphylococci (CoNS; 446 strains, enterococci (429 strains, and various Streptococcus spp. (326 strains. Oxacillin resistance was observed in 41% of S. aureus (MIC, or = 13 mm and 40% of CoNS (MIC, or = 18 mm. Vancomycin, teicoplanin, and quinupristin/dalfopristin (MIC90, 0.25 - 1 mug/ml remained effective against all strains, but cross-resistance was high among other tested drugs. The quinupristin/dalfopristin MIC50 for Streptococcus pneumoniae and other streptococci was only 0.5 mug/ml (13% to 28% were penicillin-resistant; 12% to 22% were macrolide-resistant. Enterococci demonstrated variable inhibition by quinupristin/dalfopristin depending upon identification and the susceptibility testing method used. The demonstrated quinupristin/dalfopristin activity against Enterococcus faecium was confirmed, but potential species identification errors with various commercial systems continue to confuse susceptibility statistics, even though some strains of E. faecium confirmed by PCR-based or other molecular identification techniques did have quinupristin/dalfopristin MICs of > or = 4 mug/mL. Most important, glycopeptide-resistant enterococci are rapidly emerging in Latin America, and quinupristin/dalfopristin appears

A molecular gram stain using broad range PCR and pyrosequencing technology: a potentially useful tool for diagnosing orthopaedic infections.

Science.gov (United States)

Kobayashi, Naomi; Bauer, Thomas W; Togawa, Daisuke; Lieberman, Isador H; Sakai, Hiroshige; Fujishiro, Takaaki; Tuohy, Marion J; Procop, Gary W

2005-06-01

The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the "molecular Gram stain" were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.

Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

Energy Technology Data Exchange (ETDEWEB)

Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

2014-01-01

A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

Complete genome sequence of Paenibacillus riograndensis SBR5(T), a Gram-positive diazotrophic rhizobacterium.

Science.gov (United States)

Brito, Luciana Fernandes; Bach, Evelise; Kalinowski, Jörn; Rückert, Christian; Wibberg, Daniel; Passaglia, Luciane M; Wendisch, Volker F

2015-08-10

Paenibacillus riograndensis is a Gram-positive rhizobacterium which exhibits plant growth promoting activities. It was isolated from the rhizosphere of wheat grown in the state of Rio Grande do Sul, Brazil. Here we announce the complete genome sequence of P. riograndensis strain SBR5(T). The genome of P. riograndensis SBR5(T) consists of a circular chromosome of 7,893,056bps. The genome was finished and fully annotated, containing 6705 protein coding genes, 87 tRNAs and 27 rRNAs. The knowledge of the complete genome helped to explain why P. riograndensis SBR5(T) can grow with the carbon sources arabinose and mannitol, but not myo-inositol, and to explain physiological features such as biotin auxotrophy and antibiotic resistances. The genome sequence will be valuable for functional genomics and ecological studies as well as for application of P. riograndensis SBR5(T) as plant growth-promoting rhizobacterium. Copyright © 2015 Elsevier B.V. All rights reserved.

ClubSub-P: Cluster-based subcellular localization prediction for Gram-negative bacteria and Archaea.

Directory of Open Access Journals (Sweden)

Nagarajan eParamasivam

2011-11-01

Full Text Available The subcellular localization of proteins provides important clues to their function in a cell. In our efforts to predict useful vaccine targets against Gram-negative bacteria, we noticed that misannotated start codons frequently lead to wrongly assigned subcellular localizations. This and other problems in subcellular localization prediction, such as the relatively high false positive and false negative rates of some tools, can be avoided by applying multiple prediction tools to groups of homologous proteins. Here we present ClubSub-P, an online database that combines existing subcellular localization prediction tools into a consensus pipeline from more than 600 proteomes of fully sequenced microorganisms. On top of the consensus prediction at the level of single sequences, the tool uses clusters of homologous proteins from Gram-negative bacteria and from Archaea to eliminate false positive and false negative predictions. ClubSub-P can assign the subcellular localization of proteins from Gram-negative bacteria and Archaea with high precision. The database is searchable, and can easily be expanded using either new bacterial genomes or new prediction tools as they become available. This will further improve the performance of the subcellular localization prediction, as well as the detection of misannotated start codons and other annotation errors. ClubSub-P is available online at http://toolkit.tuebingen.mpg.de/clubsubp/

Potential strategies for the eradication of multidrug-resistant Gram-negative bacterial infections.

Science.gov (United States)

Huwaitat, Rawan; McCloskey, Alice P; Gilmore, Brendan F; Laverty, Garry

2016-07-01

Antimicrobial resistance is one of the leading threats to society. The increasing burden of multidrug-resistant Gram-negative infection is particularly concerning as such bacteria are demonstrating resistance to nearly all currently licensed therapies. Various strategies have been hypothesized to treat multidrug-resistant Gram-negative infections including: targeting the Gram-negative outer membrane; neutralization of lipopolysaccharide; inhibition of bacterial efflux pumps and prevention of protein folding. Silver and silver nanoparticles, fusogenic liposomes and nanotubes are potential strategies for extending the activity of licensed, Gram-positive selective, antibiotics to Gram-negatives. This may serve as a strategy to fill the current void in pharmaceutical development in the short term. This review outlines the most promising strategies that could be implemented to solve the threat of multidrug-resistant Gram-negative infections.

The usefulness of changing focus during examination using Gram staining as initial diagnostic clue for infective tuberculosis.

Science.gov (United States)

Atsukawa, Yoshiko; Kawakami, Sayoko; Asahara, Miwa; Ishigaki, Shinobu; Tanaka, Takashi; Ono, Yasuo; Nishiya, Hajime; Fujisaki, Ryuichi; Koga, Ichiro; Ota, Yasuo; Miyazawa, Yukihisa

2011-08-01

Gram staining is a useful technique for detecting bacteria but is highly questionable in detecting Mycobacterium tuberculosis. Its detection generally requires special staining, such as Ziehl-Neelsen staining. We experienced three cases in which tuberculosis was first suggested by Gram staining of sputum or pus, confirmed by Ziehl-Neelsen staining, and diagnosed by polymerase chain reaction or culture. To find colorless tubercle bacilli in clinical samples with various organisms, varying the focus to slightly longer and shorter during study of the slides is indispensable. We present criteria for detecting infective pulmonary tuberculosis in Gram staining. First, in the ordinary focus, weakly stained, thin, gram-positive bacilli are found; second, with a slightly longer focus distance, the thin, cord-like, conspicuous gram-positive bacilli can be observed; and third, with a shorter focus distance, the gram-positive bacilli have changed into the brightened, colorless, or ghost ones. Four laboratory technologists each evaluated 20 Gram-stained samples after being lectured on the criteria, with no prior information about the sample. They accurately evaluated the presence of the bacilli in Gram-stained preparations in more than 90% of samples containing 3+ bacilli on Ziehl-Neelsen staining. Gram staining is available as an easy and rapid initial clue to recognize highly infective tuberculosis.

Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture.

Science.gov (United States)

Turner, Robert D; Hurd, Alexander F; Cadby, Ashley; Hobbs, Jamie K; Foster, Simon J

2013-01-01

Cellular integrity and morphology of most bacteria is maintained by cell wall peptidoglycan, the target of antibiotics essential in modern healthcare. It consists of glycan strands, cross-linked by peptides, whose arrangement determines cell shape, prevents lysis due to turgor pressure and yet remains dynamic to allow insertion of new material, and hence growth. The cellular architecture and insertion pattern of peptidoglycan have remained elusive. Here we determine the peptidoglycan architecture and dynamics during growth in rod-shaped Gram-negative bacteria. Peptidoglycan is made up of circumferentially oriented bands of material interspersed with a more porous network. Super-resolution fluorescence microscopy reveals an unexpected discontinuous, patchy synthesis pattern. We present a consolidated model of growth via architecture-regulated insertion, where we propose only the more porous regions of the peptidoglycan network that are permissive for synthesis.

Rationalizing the permeation of polar antibiotics into Gram-negative bacteria

International Nuclear Information System (INIS)

Scorciapino, Mariano Andrea; Acosta-Gutierrez, Silvia; Benkerrou, Dehbia; D’Agostino, Tommaso; Malloci, Giuliano; Samanta, Susruta; Bodrenko, Igor; Ceccarelli, Matteo

2017-01-01

The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI

Rationalizing the permeation of polar antibiotics into Gram-negative bacteria

Science.gov (United States)

Scorciapino, Mariano Andrea; Acosta-Gutierrez, Silvia; Benkerrou, Dehbia; D'Agostino, Tommaso; Malloci, Giuliano; Samanta, Susruta; Bodrenko, Igor; Ceccarelli, Matteo

2017-03-01

The increasing level of antibiotic resistance in Gram-negative bacteria, together with the lack of new potential drug scaffolds in the pipeline, make the problem of infectious diseases a global challenge for modern medicine. The main reason that Gram-negative bacteria are particularly challenging is the presence of an outer cell-protecting membrane, which is not present in Gram-positive species. Such an asymmetric bilayer is a highly effective barrier for polar molecules. Several protein systems are expressed in the outer membrane to control the internal concentration of both nutrients and noxious species, in particular: (i) water-filled channels that modulate the permeation of polar molecules and ions according to concentration gradients, and (ii) efflux pumps to actively expel toxic compounds. Thus, besides expressing specific enzymes for drugs degradation, Gram-negative bacteria can also resist by modulating the influx and efflux of antibiotics, keeping the internal concentration low. However, there are no direct and robust experimental methods capable of measuring the permeability of small molecules, thus severely limiting our knowledge of the molecular mechanisms that ultimately control the permeation of antibiotics through the outer membrane. This is the innovation gap to be filled for Gram-negative bacteria. This review is focused on the permeation of small molecules through porins, considered the main path for the entry of polar antibiotics into Gram-negative bacteria. A fundamental understanding of how these proteins are able to filter small molecules is a prerequisite to design/optimize antibacterials with improved permeation. The level of sophistication of modern molecular modeling algorithms and the advances in new computer hardware has made the simulation of such complex processes possible at the molecular level. In this work we aim to share our experience and perspectives in the context of a multidisciplinary extended collaboration within the IMI

Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

Science.gov (United States)

Mars, Ruben A. T.; Nicolas, Pierre; Denham, Emma L.

2016-01-01

SUMMARY Bacteria can employ widely diverse RNA molecules to regulate their gene expression. Such molecules include trans-acting small regulatory RNAs, antisense RNAs, and a variety of transcriptional attenuation mechanisms in the 5′ untranslated region. Thus far, most regulatory RNA research has focused on Gram-negative bacteria, such as Escherichia coli and Salmonella. Hence, there is uncertainty about whether the resulting insights can be extrapolated directly to other bacteria, such as the Gram-positive soil bacterium Bacillus subtilis. A recent study identified 1,583 putative regulatory RNAs in B. subtilis, whose expression was assessed across 104 conditions. Here, we review the current understanding of RNA-based regulation in B. subtilis, and we categorize the newly identified putative regulatory RNAs on the basis of their conservation in other bacilli and the stability of their predicted secondary structures. Our present evaluation of the publicly available data indicates that RNA-mediated gene regulation in B. subtilis mostly involves elements at the 5′ ends of mRNA molecules. These can include 5′ secondary structure elements and metabolite-, tRNA-, or protein-binding sites. Importantly, sense-independent segments are identified as the most conserved and structured potential regulatory RNAs in B. subtilis. Altogether, the present survey provides many leads for the identification of new regulatory RNA functions in B. subtilis. PMID:27784798

Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression.

Science.gov (United States)

Mars, Ruben A T; Nicolas, Pierre; Denham, Emma L; van Dijl, Jan Maarten

2016-12-01

Bacteria can employ widely diverse RNA molecules to regulate their gene expression. Such molecules include trans-acting small regulatory RNAs, antisense RNAs, and a variety of transcriptional attenuation mechanisms in the 5' untranslated region. Thus far, most regulatory RNA research has focused on Gram-negative bacteria, such as Escherichia coli and Salmonella. Hence, there is uncertainty about whether the resulting insights can be extrapolated directly to other bacteria, such as the Gram-positive soil bacterium Bacillus subtilis. A recent study identified 1,583 putative regulatory RNAs in B. subtilis, whose expression was assessed across 104 conditions. Here, we review the current understanding of RNA-based regulation in B. subtilis, and we categorize the newly identified putative regulatory RNAs on the basis of their conservation in other bacilli and the stability of their predicted secondary structures. Our present evaluation of the publicly available data indicates that RNA-mediated gene regulation in B. subtilis mostly involves elements at the 5' ends of mRNA molecules. These can include 5' secondary structure elements and metabolite-, tRNA-, or protein-binding sites. Importantly, sense-independent segments are identified as the most conserved and structured potential regulatory RNAs in B. subtilis. Altogether, the present survey provides many leads for the identification of new regulatory RNA functions in B. subtilis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A thiol-disulfide oxidoreductase of the Gram-positive pathogen Corynebacterium diphtheriae is essential for viability, pilus assembly, toxin production and virulence.

Science.gov (United States)

Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Jooya, Neda; Chang, Chungyu; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

2015-12-01

The Gram-positive pathogen Corynebacterium diphtheriae exports through the Sec apparatus many extracellular proteins that include the key virulence factors diphtheria toxin and the adhesive pili. How these proteins attain their native conformations after translocation as unfolded precursors remains elusive. The fact that the majority of these exported proteins contain multiple cysteine residues and that several membrane-bound oxidoreductases are encoded in the corynebacterial genome suggests the existence of an oxidative protein-folding pathway in this organism. Here we show that the shaft pilin SpaA harbors a disulfide bond in vivo and alanine substitution of these cysteines abrogates SpaA polymerization and leads to the secretion of degraded SpaA peptides. We then identified a thiol-disulfide oxidoreductase (MdbA), whose structure exhibits a conserved thioredoxin-like domain with a CPHC active site. Remarkably, deletion of mdbA results in a severe temperature-sensitive cell division phenotype. This mutant also fails to assemble pilus structures and is greatly defective in toxin production. Consistent with these defects, the ΔmdbA mutant is attenuated in a guinea pig model of diphtheritic toxemia. Given its diverse cellular functions in cell division, pilus assembly and toxin production, we propose that MdbA is a component of the general oxidative folding machine in C. diphtheriae. © 2015 John Wiley & Sons Ltd.

The accuracy of Gram stain of respiratory specimens in excluding Staphylococcus aureus in ventilator-associated pneumonia.

Science.gov (United States)

Gottesman, Tamar; Yossepowitch, Orit; Lerner, Evgenia; Schwartz-Harari, Orna; Soroksky, Arie; Yekutieli, Daniel; Dan, Michael

2014-10-01

To evaluate the Gram stain of deep tracheal aspirate as a tool to direct empiric antibiotic therapy, and more specifically as a tool to exclude the need for empiric antibiotic coverage against Staphylococcus aureus in ventilator-associated pneumonia (VAP). A prospective, single-center, observational, cohort study. All wards at a community hospital. Adult patients requiring mechanical ventilation, identified as having VAP in a 54-month prospective surveillance database. Sampling of lower airway secretions by deep endotracheal aspiration was taken from each patient who developed VAP. Samples were sent immediately for Gram stain and qualitative bacterial cultures. Demographic and relevant clinical data were collected; Gram stain, culture, and antibiotic susceptibility results were documented; and outcome was followed prospectively. The analysis included 114 consecutive patients with 115 episodes of VAP from June 2007 to January 2012. Sensitivity of Gram stain compared with culture was 90.47% for gram-positive cocci, 69.6% for gram-negative rods, and 50% for sterile cultures. Specificity was 82.5%, 77.8%, and 79%, respectively. Negative predictive value was high for gram-positive cocci (97%) and sterile cultures (96%) but low for gram-negative rods (20%). Acinetobacter baumanii (45%) and Pseudomonas aeruginosa (38 %) were the prevailing isolates. S aureus was found in 18.3% of the patients. Most isolates were multiresistant. Absence of gram-positive bacteria on Gram stain had a high negative predictive value. These data can be used to narrow the initial empiric antibiotic regimen and to avoid unnecessary exposure of patients to vancomycin and other antistaphyloccocal agents. Copyright © 2014 Elsevier Inc. All rights reserved.

[Comparison of the quick Gram stain method to the B&M modified and favor methods].

Science.gov (United States)

Osawa, Kayo; Kataoka, Nobumasa; Maruo, Toshio

2011-01-01

The Gram stain is an established method for bacterial identification, but the time needed to carry out this stain is 2-3 min. We attempted to shorten this time and stained a total of 70 clinical specimens isolated from using the Bartholomew & Mittwer (B&M) modified or Favor methods with a 3 s duration for washing and staining steps. Results were plotted and analyzed using a Hue Saturation Intensity (HSI) model. The range based on a plot of the two methods with the HSI model was presented as a reference interval. Our results indicated that 100% (35/35) of strains were Gram positive and 97.1% (34/35) were Gram negative for the quick B&M modified method. In the quick Favor method, 80.0% (28/35) were Gram positive and 68.6% (24/35) of strains were Gram negative. We propose that the quick B&M modified method is equivalent to the standard Gram staining method and is superior to the quick Favor method.

Changing epidemiology of central venous catheter-related bloodstream infections: increasing prevalence of Gram-negative pathogens.

Science.gov (United States)

Marcos, Miguel; Soriano, Alex; Iñurrieta, Amaia; Martínez, José A; Romero, Alberto; Cobos, Nazaret; Hernández, Cristina; Almela, Manel; Marco, Francesc; Mensa, Josep

2011-09-01

Gram-positive microorganisms have been the predominant pathogens in central venous catheter-related bloodstream infections (CRBSIs). Recent guidelines recommend empirical therapy according to this and restrict coverage for Gram-negatives to specific circumstances. This study aimed to analyse the epidemiological changes in CRBSIs over the 1991-2008 period and to analyse predictors of Gram-negative CRBSIs. A prospectively collected cohort of patients with confirmed CRBSIs was analysed. Strains isolated and antimicrobial susceptibility, as well as clinical and demographic variables were recorded. Differences observed during the study period were analysed by means of a χ² trend test and factors associated with Gram-negative CRBSIs by means of multivariable analysis. Between 1991 and 2008, 1129 episodes of monomicrobial CRBSIs were recorded. There was an increase in the incidence of CRBSIs, from 0.10 (1991-92) to 0.31 (2007-08) episodes/1000 patient-days. A significant increase in the number of Gram-negative strains among the total isolates was also found, from 3 (4.7%) in 1991-92 to 70 (40.23%) in 2007-08, with a parallel decrease in the percentage of Gram-positives. Solid organ transplantation, prior use of penicillins and hospital stay longer than 11 days were independently associated with a significantly higher risk of Gram-negative CRBSIs, while cirrhosis, diabetes and use of quinolones were associated with a higher risk of Gram-positives. Gram-negative strains are an increasing cause of CRBSIs, reaching a prevalence of 40% in the 2007-08 period in our hospital. If this trend is confirmed in other centres, a broad-spectrum empirical therapy should be considered in managing these infections.

Development of a Multiplexed Microsphere PCR for Culture-Free Detection and Gram-Typing of Bacteria in Human Blood Samples.

Science.gov (United States)

Liang, Fang; Browne, Daniel J; Gray, Megan J; Gartlan, Kate H; Smith, David D; Barnard, Ross T; Hill, Geoffrey R; Corrie, Simon R; Markey, Kate A

2018-05-11

Bloodstream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected bloodstream infection remain centered around blood culture (highly variable timing, in the order of hours to days to become positive), and empiric use of broad-spectrum antibiotics is therefore employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g., combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives); however, current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5 h time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labeled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labeled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.

Case of Mycobacterium tuberculosis meningitis: Gram staining as a useful initial diagnostic clue for tuberculous meningitis.

Science.gov (United States)

Kawakami, Sayoko; Kawamura, Yasuyosi; Nishiyama, Kyouhei; Hatanaka, Hiroki; Fujisaki, Ryuichi; Ono, Yasuo; Miyazawa, Yukihisa; Nishiya, Hajime

2012-12-01

A 32-year-old man was admitted to our hospital because of fever, headache, and loss of consciousness. Four days before admission, he had had difficulty speaking. On the day of admission, his colleague had found him to be unconscious and lying on his back. He was admitted to our hospital. The temperature at the eardrum was 35.2°C. Neurologic evaluation was negative. Computed tomography (CT) scan of the brain showed slight ventricular enlargement bilaterally. An X-ray film of the chest showed no abnormality. On the second hospital day, neck stiffness was noted. The cerebrospinal fluid (CSF) contained 870 white cells/μl, most of which were neutrophils; the glucose level in the CSF was 10 mg/dl, and the protein level was 140 mg/dl. Stained smears of the CSF, including Gram staining and India-ink preparations, disclosed no microorganisms. Capsular antigen tests for several bacteria were negative. Antimicrobial agents were started. However, by changing the microscope focus slightly while viewing Gram stains of the CSF, we could see brightened and Gram-positive bacilli that had been phagocytosed by neutrophils. This finding suggested the presence of Mycobacterium tuberculosis. Ziehl-Neelsen staining of the CSF and gastric juice revealed anti-acid bacilli. Polymerase chain reaction for M. tuberculosis in the gastric juice was positive. This case showed that Gram staining could be useful as an initial adjunct for the diagnosis of tuberculous meningitis, particularly when the CSF shows predominantly neutrocytic pleocytosis, but no other evidence of bacterial meningitis.

A New Family of Capsule Polymerases Generates Teichoic Acid-Like Capsule Polymers in Gram-Negative Pathogens.

Science.gov (United States)

Litschko, Christa; Oldrini, Davide; Budde, Insa; Berger, Monika; Meens, Jochen; Gerardy-Schahn, Rita; Berti, Francesco; Schubert, Mario; Fiebig, Timm

2018-05-29

Group 2 capsule polymers represent crucial virulence factors of Gram-negative pathogenic bacteria. They are synthesized by enzymes called capsule polymerases. In this report, we describe a new family of polymerases that combine glycosyltransferase and hexose- and polyol-phosphate transferase activity to generate complex poly(oligosaccharide phosphate) and poly(glycosylpolyol phosphate) polymers, the latter of which display similarity to wall teichoic acid (WTA), a cell wall component of Gram-positive bacteria. Using modeling and multiple-sequence alignment, we showed homology between the predicted polymerase domains and WTA type I biosynthesis enzymes, creating a link between Gram-negative and Gram-positive cell wall biosynthesis processes. The polymerases of the new family are highly abundant and found in a variety of capsule-expressing pathogens such as Neisseria meningitidis , Actinobacillus pleuropneumoniae , Haemophilus influenzae , Bibersteinia trehalosi , and Escherichia coli with both human and animal hosts. Five representative candidates were purified, their activities were confirmed using nuclear magnetic resonance (NMR) spectroscopy, and their predicted folds were validated by site-directed mutagenesis. IMPORTANCE Bacterial capsules play an important role in the interaction between a pathogen and the immune system of its host. During the last decade, capsule polymerases have become attractive tools for the production of capsule polymers applied as antigens in glycoconjugate vaccine formulations. Conventional production of glycoconjugate vaccines requires the cultivation of the pathogen and thus the highest biosafety standards, leading to tremendous costs. With regard to animal husbandry, where vaccines could avoid the extensive use of antibiotics, conventional production is not sufficiently cost-effective. In contrast, enzymatic synthesis of capsule polymers is pathogen-free and fast, offers high stereo- and regioselectivity, and works with high efficacy

Numerically Stable Evaluation of Moments of Random Gram Matrices With Applications

KAUST Repository

Elkhalil, Khalil; Kammoun, Abla; Al-Naffouri, Tareq Y.; Alouini, Mohamed-Slim

2017-01-01

This paper focuses on the computation of the positive moments of one-side correlated random Gram matrices. Closed-form expressions for the moments can be obtained easily, but numerical evaluation thereof is prone to numerical stability, especially in high-dimensional settings. This letter provides a numerically stable method that efficiently computes the positive moments in closed-form. The developed expressions are more accurate and can lead to higher accuracy levels when fed to moment based-approaches. As an application, we show how the obtained moments can be used to approximate the marginal distribution of the eigenvalues of random Gram matrices.

Numerically Stable Evaluation of Moments of Random Gram Matrices With Applications

KAUST Repository

Elkhalil, Khalil

2017-07-31

This paper focuses on the computation of the positive moments of one-side correlated random Gram matrices. Closed-form expressions for the moments can be obtained easily, but numerical evaluation thereof is prone to numerical stability, especially in high-dimensional settings. This letter provides a numerically stable method that efficiently computes the positive moments in closed-form. The developed expressions are more accurate and can lead to higher accuracy levels when fed to moment based-approaches. As an application, we show how the obtained moments can be used to approximate the marginal distribution of the eigenvalues of random Gram matrices.

Fast Mechanically Driven Daughter Cell Separation Is Widespread in Actinobacteria

Directory of Open Access Journals (Sweden)

Xiaoxue Zhou

2016-08-01

Full Text Available Dividing cells of the coccoid Gram-positive bacterium Staphylococcus aureus undergo extremely rapid (millisecond daughter cell separation (DCS driven by mechanical crack propagation, a strategy that is very distinct from the gradual, enzymatically driven cell wall remodeling process that has been well described in several rod-shaped model bacteria. To determine if other bacteria, especially those in the same phylum (Firmicutes or with similar coccoid shapes as S. aureus, might use a similar mechanically driven strategy for DCS, we used high-resolution video microscopy to examine cytokinesis in a phylogenetically wide range of species with various cell shapes and sizes. We found that fast mechanically driven DCS is rather rare in the Firmicutes (low G+C Gram positives, observed only in Staphylococcus and its closest coccoid relatives in the Macrococcus genus, and we did not observe this division strategy among the Gram-negative Proteobacteria. In contrast, several members of the high-G+C Gram-positive phylum Actinobacteria (Micrococcus luteus, Brachybacterium faecium, Corynebacterium glutamicum, and Mycobacterium smegmatis with diverse shapes ranging from coccoid to rod all undergo fast mechanical DCS during cell division. Most intriguingly, similar fast mechanical DCS was also observed during the sporulation of the actinobacterium Streptomyces venezuelae.

Gram staining of protected pulmonary specimens in the early diagnosis of ventilator-associated pneumonia.

Science.gov (United States)

Mimoz, O; Karim, A; Mazoit, J X; Edouard, A; Leprince, S; Nordmann, P

2000-11-01

We evaluated prospectively the use of Gram staining of protected pulmonary specimens to allow the early diagnosis of ventilator-associated pneumonia (VAP), compared with the use of 60 bronchoscopic protected specimen brushes (PSB) and 126 blinded plugged telescopic catheters (PTC) obtained from 134 patients. Gram stains were from Cytospin slides; they were studied for the presence of microorganisms in 10 and 50 fields by two independent observers and classified according to their Gram stain morphology. Quantitative cultures were performed after serial dilution and plating on appropriate culture medium. A final diagnosis of VAP, based on a culture of > or = 10(3) c.f.u. ml-1, was established after 81 (44%) samplings. When 10 fields were analysed, a strong relationship was found between the presence of bacteria on Gram staining and the final diagnosis of VAP (for PSB and PTC respectively: sensitivity 74 and 81%, specificity 94 and 100%, positive predictive value 91 and 100%, negative predictive value 82 and 88%). The correlation was less when we compared the morphology of microorganisms observed on Gram staining with those of bacteria obtained from quantitative cultures (for PSB and PTC respectively: sensitivity 54 and 69%, specificity 86 and 89%, positive predictive value 72 and 78%, negative predictive value 74 and 84%). Increasing the number of fields read to 50 was associated with a slight decrease in specificity and positive predictive value of Gram staining, but with a small increase in its sensitivity and negative predictive value. The results obtained by the two observers were similar to each other for both numbers of fields analysed. Gram staining of protected pulmonary specimens performed on 10 fields predicted the presence of VAP and partially identified (using Gram stain morphology) the microorganisms growing at significant concentrations, and could help in the early choice of the treatment of VAP. Increasing the number of fields read or having the Gram

Impact of round-the-clock CSF Gram stain on empirical therapy for suspected central nervous system infections.

Science.gov (United States)

Tissot, F; Prod'hom, G; Manuel, O; Greub, G

2015-09-01

The impact of round-the-clock cerebrospinal fluid (CSF) Gram stain on overnight empirical therapy for suspected central nervous system (CNS) infections was investigated. All consecutive overnight CSF Gram stains between 2006 and 2011 were included. The impact of a positive or a negative test on empirical therapy was evaluated and compared to other clinical and biological indications based on institutional guidelines. Bacterial CNS infection was documented in 51/241 suspected cases. Overnight CSF Gram stain was positive in 24/51. Upon validation, there were two false-positive and one false-negative results. The sensitivity and specificity were 41 and 99 %, respectively. All patients but one had other indications for empirical therapy than Gram stain alone. Upon obtaining the Gram result, empirical therapy was modified in 7/24, including the addition of an appropriate agent (1), addition of unnecessary agents (3) and simplification of unnecessary combination therapy (3/11). Among 74 cases with a negative CSF Gram stain and without formal indication for empirical therapy, antibiotics were withheld in only 29. Round-the-clock CSF Gram stain had a low impact on overnight empirical therapy for suspected CNS infections and was associated with several misinterpretation errors. Clinicians showed little confidence in CSF direct examination for simplifying or withholding therapy before definite microbiological results.

Bacteremias in liver transplant recipients: shift toward gram-negative bacteria as predominant pathogens.

Science.gov (United States)

Singh, Nina; Wagener, Marilyn M; Obman, Asia; Cacciarelli, Thomas V; de Vera, Michael E; Gayowski, Timothy

2004-07-01

During the 1990s, gram-positive bacteria emerged as major pathogens after liver transplantation. We sought to determine whether the pathogens associated with bacteremias in liver transplant recipients have changed. Patients included 233 liver transplant recipients transplanted between 1989 and 2003. The proportion of all infections due to bacteremias increased significantly over time (P gram-negatives increased from 25% in the period of 1989-1993 to 51.8% in 1998-03, that of gram-positive bacteria decreased from 75% in the period of 1989-93 to 48.2% in the period of 1998-2003. Methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, and Pseudomonas aeruginosa were the most frequent pathogens in bacteremic patients. The incidence of bacteremias due to MRSA and Pseudomonas aeruginosa has remained unchanged (P gram-negative bacteria, particularly Klebsiella pneumoniae has increased (P =.02). Klebsiella pneumoniae isolates in the current quartile were not clonally related. In conclusion, bacteremias as a proportion of all infections in liver transplant recipients have increased significantly over time, due in part to a decline in infections due to other major pathogens, e.g., fungi, primarily Candida species, and CMV. Gram-negative bacteria have emerged as predominant pathogens in bacteremic liver transplant recipients.

n-Gram-Based Text Compression

Science.gov (United States)

Duong, Hieu N.; Snasel, Vaclav

2016-01-01

We propose an efficient method for compressing Vietnamese text using n-gram dictionaries. It has a significant compression ratio in comparison with those of state-of-the-art methods on the same dataset. Given a text, first, the proposed method splits it into n-grams and then encodes them based on n-gram dictionaries. In the encoding phase, we use a sliding window with a size that ranges from bigram to five grams to obtain the best encoding stream. Each n-gram is encoded by two to four bytes accordingly based on its corresponding n-gram dictionary. We collected 2.5 GB text corpus from some Vietnamese news agencies to build n-gram dictionaries from unigram to five grams and achieve dictionaries with a size of 12 GB in total. In order to evaluate our method, we collected a testing set of 10 different text files with different sizes. The experimental results indicate that our method achieves compression ratio around 90% and outperforms state-of-the-art methods. PMID:27965708

n-Gram-Based Text Compression

Directory of Open Access Journals (Sweden)

Vu H. Nguyen

2016-01-01

Full Text Available We propose an efficient method for compressing Vietnamese text using n-gram dictionaries. It has a significant compression ratio in comparison with those of state-of-the-art methods on the same dataset. Given a text, first, the proposed method splits it into n-grams and then encodes them based on n-gram dictionaries. In the encoding phase, we use a sliding window with a size that ranges from bigram to five grams to obtain the best encoding stream. Each n-gram is encoded by two to four bytes accordingly based on its corresponding n-gram dictionary. We collected 2.5 GB text corpus from some Vietnamese news agencies to build n-gram dictionaries from unigram to five grams and achieve dictionaries with a size of 12 GB in total. In order to evaluate our method, we collected a testing set of 10 different text files with different sizes. The experimental results indicate that our method achieves compression ratio around 90% and outperforms state-of-the-art methods.

Lactic Acid Bacteria Inducing a Weak Interleukin-12 and Tumor Necrosis Alpha Response in Human Dendritic Cells Inhibit Strongly Stimulating Lactic Acid Bacteria but Act Synergistically with Gram-Negative Bacteria

DEFF Research Database (Denmark)

Zeuthen, Louise Hjerrild; Christensen, Hanne Risager; Frøkiær, Hanne

2006-01-01

The development and maintenance of immune homeostasis indispensably depend on signals from the gut flora. Lactic acid bacteria (LAB), which are gram-positive (G+) organisms, are plausible significant players and have received much attention. Gram-negative (G-) commensals, such as members...

Phenyl thiazolyl urea and carbamate derivatives as new inhibitors of bacterial cell-wall biosynthesis.

Science.gov (United States)

Francisco, Gerardo D; Li, Zhong; Albright, J Donald; Eudy, Nancy H; Katz, Alan H; Petersen, Peter J; Labthavikul, Pornpen; Singh, Guy; Yang, Youjun; Rasmussen, Beth A; Lin, Yang-I; Mansour, Tarek S

2004-01-05

Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including MRSA, VRE and PRSP. 3,4-Difluorophenyl 5-cyanothiazolylurea (3p) with clog P of 2.64 demonstrated antibacterial activity against both gram-positive and gram-negative bacteria.

Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens.

Science.gov (United States)

Hendrick, William A; Orr, Mona W; Murray, Samantha R; Lee, Vincent T; Melville, Stephen B

2017-05-15

The Gram-positive pathogen Clostridium perfringens possesses type IV pili (TFP), which are extracellular fibers that are polymerized from a pool of pilin monomers in the cytoplasmic membrane. Two proteins that are essential for pilus functions are an assembly ATPase (PilB) and an inner membrane core protein (PilC). Two homologues each of PilB and PilC are present in C. perfringens , called PilB1/PilB2 and PilC1/PilC2, respectively, along with four pilin proteins, PilA1 to PilA4. The gene encoding PilA2, which is considered the major pilin based on previous studies, is immediately downstream of the pilB2 and pilC2 genes. Purified PilB2 had ATPase activity, bound zinc, formed hexamers even in the absence of ATP, and bound the second messenger molecule cyclic di-GMP (c-di-GMP). Circular dichroism spectroscopy of purified PilC2 indicated that it retained its predicted degree of alpha-helical secondary structure. Even though no direct interactions between PilB2 and PilC2 could be detected in vivo or in vitro even in the presence of c-di-GMP, high levels of expression of a diguanylate cyclase from C. perfringens (CPE1788) stimulated polymerization of PilA2 in a PilB2- and PilC2-dependent manner. These results suggest that PilB2 activity is controlled by c-di-GMP levels in vivo but that PilB2-PilC2 interactions are either transitory or of low affinity, in contrast to results reported previously from in vivo studies of the PilB1/PilC1 pair in which PilC1 was needed for polar localization of PilB1. This is the first biochemical characterization of a c-di-GMP-dependent assembly ATPase from a Gram-positive bacterium. IMPORTANCE Type IV pili (TFP) are protein fibers involved in important bacterial functions, including motility, adherence to surfaces and host cells, and natural transformation. All clostridia whose genomes have been sequenced show evidence of the presence of TFP. The genetically tractable species Clostridium perfringens was used to study proteins involved in

Microbiology of liver abscesses and the predictive value of abscess gram stain and associated blood cultures.

Science.gov (United States)

Chemaly, Roy F; Hall, Gerri S; Keys, Thomas F; Procop, Gary W

2003-08-01

Although rare, pyogenic liver abscesses are potentially fatal. We evaluated the predictive value of Gram stain of liver abscess aspirates and temporally associated blood cultures. Gram stains detected bacteria in 79% of the liver abscesses tested. The sensitivity and specificity of Gram stain of the liver abscesses were 90% and 100% for Gram-positive cocci (GPC) and 52% and 94% for Gram-negative bacilli (GNB). The sensitivities of the blood cultures for any GPC and GNB present in the liver abscess were 30% and 39%, respectively. Although, Gram stains and blood cultures offer incomplete detection of the microbial contents of pyogenic liver abscesses, both tests should always accompany liver abscess cultures.

Will new antimicrobials overcome resistance among Gram-negatives?

Science.gov (United States)

Bassetti, Matteo; Ginocchio, Francesca; Mikulska, Małgorzata; Taramasso, Lucia; Giacobbe, Daniele Roberto

2011-10-01

The spread of resistance among Gram-positive and Gram-negative bacteria represents a growing challenge for the development of new antimicrobials. The pace of antibiotic drug development has slowed during the last decade and, especially for Gram-negatives, clinicians are facing a dramatic shortage in the availability of therapeutic options to face the emergency of the resistance problem throughout the world. In this alarming scenario, although there is a shortage of compounds reaching the market in the near future, antibiotic discovery remains one of the keys to successfully stem and maybe overcome the tide of resistance. Analogs of already known compounds and new agents belonging to completely new classes of antimicrobials are in early stages of development. Novel and promising anti-Gram-negative antimicrobials belong both to old (cephalosporins, carbapenems, β-lactamase inhibitors, monobactams, aminoglycosides, polymyxin analogues and tetracycline) and completely new antibacterial classes (boron-containing antibacterial protein synthesis inhibitors, bis-indoles, outer membrane synthesis inhibitors, antibiotics targeting novel sites of the 50S ribosomal subunit and antimicrobial peptides). However, all of these compounds are still far from being introduced into clinical practice. Therefore, infection control policies and optimization in the use of already existing molecules are still the most effective approaches to reduce the spread of resistance and preserve the activity of antimicrobials.

Gram and acridine orange staining for diagnosis of septic arthritis in different patient populations.

Science.gov (United States)

Cunningham, Gregory; Seghrouchni, Khalid; Ruffieux, Etienne; Vaudaux, Pierre; Gayet-Ageron, Angèle; Cherkaoui, Abdessalam; Godinho, Eduardo; Lew, Daniel; Hoffmeyer, Pierre; Uçkay, Ilker

2014-06-01

The sensitivity of Gram staining is known to be suboptimal for the diagnosis of native joint septic arthritis. We lack information about the accuracy of Gram compared to other microscopic staining techniques for predicting infection in different patient populations. This was a cohort study with cost evaluations at the Orthopaedic Service of Geneva University Hospitals (January 1996-October 2012). Among 500 episodes of arthritis (196 with immunosuppression, 227 with underlying arthroplasties and 69 with gout or other crystals in synovial fluid), Gram staining revealed pathogens in 146 episodes (146/500, 29 %) or in 146 of the 400 culture-positive episodes (37 %). Correlation between the Gram and acridine staining of the same sample was good (Spearman 0.85). Overall, the sensitivity, specificity, positive predictive value and negative predictive value of Gram stain for rapid diagnosis of septic arthritis was 0.37, 0.99, 0.99 and 0.28, respectively, compared to microbiological cultures. Quite similar values were recorded across the different patient subpopulations, in particular for sensitivity values that were 0.33 for patients with prosthetic joint infections, 0.40 for immunosuppressed patients, 0.36 for patients under antibiotic administration and 0.52 for patients with concomitant crystalline disease. The sensitivity of Gram or acridine orange staining for a rapid diagnosis of episodes of septic arthritis is suboptimal compared to microbiological culture, regardless of underlying conditions, immunosuppression or antibiotic therapy. The sensitivity in the presence of synovial fluid crystals is moderate. Acridine orange and Gram stains are equivalent.

Blood culture gram stain, acridine orange stain and direct sensitivity-based antimicrobial therapy of bloodstream infection in patients with trauma.

Science.gov (United States)

Behera, B; Mathur, P; Gupta, B

2010-01-01

The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early and appropriate treatment in trauma patients with clinical suspicion of sepsis. The study also aimed to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic-organism combination had more discrepancies. Findings from consecutive episodes of blood stream infection at an Apex Trauma centre over a 12-month period are summarized. A total of 509 consecutive positive blood cultures were subjected to Gram staining. AO staining was done in BacT/ALERT-positive Gram-stain negative blood cultures. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining. Results of direct sensitivity were compared to conventional sensitivity for errors. No 'very major' discrepancy was found in this study. About 5.2 and 1.8% minor error rates were noted in gram-positive and gram-negative bacteria, respectively, while comparing the two methods. Most of the discrepancies in gram-negative bacteria were noted in beta lactam - beta lactamase inhibitor combinations. Direct sensitivity testing was not reliable for reporting of methicillin and vancomycin resistance in Staphylococci. Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. Gram staining and AO staining proved particularly helpful in the early detection of candidaemia.

[Yearly changes in antibacterial activities of cefozopran against various clinical isolates between 1996 and 2000--I. Gram-positive bacteria].

Science.gov (United States)

Suzuki, Yumiko; Nishinari, Chisato; Endo, Harumi; Tamura, Chieko; Jinbo, Keiko; Hiramatsu, Nobuyoshi; Akiyama, Kazumitsu; Koyama, Tsuneo

2002-04-01

The in vitro antibacterial activities of cefozopran (CZOP), an agent of cephems, against various clinical isolates obtained between 1996 and 2000 were yearly evaluated and compared with those of other cephems, oxacephems, carbapenems, and penicillins. Fifteen species, 1,062 strains, of Gram-positive bacteria were isolated from the clinical materials annually collected from January to December, and consisted of methicillin-susceptible Staphylococcus aureus (MSSA; n = 127), methicillin-resistant Staphylococcus aureus (MRSA; n = 123), Staphylococcus epidermidis (n = 104), Staphylococcus haemolyticus (n = 58), Streptococcus pyogenes (n = 100), Streptococcus agalactiae (n = 50), Streptococcus pneumoniae (n = 125), Enterococcus faecalis (n = 150), Enterococcus faecium (n = 50), Enterococcus avium (n = 50), and Peptostreptococcus spp. (P. anaerobius, P. asaccharolyticus, P. magnus, P. micros, P. prevotii; n = 125). CZOP possessed stable antibacterial activities against all strains tested throughout 5 years. The MIC90 of CZOP against MRSA and S. haemolyticus tended to decrease while against S. pneumoniae and Peptostreptococcus spp., tended to increase year by year. However, the MIC90 just changed a little and were consistent with the results from the studies performed until the new drug application approval. Increases in the MIC90 against S. pneumoniae were also observed with cefpirome (CPR), cefepime (CFPM), flomoxef (FMOX), sulbactam/cefoperazone (SBT/CPZ), and imipenem (IPM). Increases in the MIC90 against Peptostreptococcus spp. were also observed with ceftazidime (CAZ), CPR, CFPM, FMOX, SBT/CPZ, and IPM. The decreases in the sensitivities were not always considered to depend upon generation of resistant bacteria because the annual MIC range of each antibacterial agent was almost generally wide every year and the annual sensitivity of each strain to the agents extremely varied. In conclusion, the annual antibacterial activities of CZOP against the Gram-positive

A Sortase A-Immobilized Mesoporous Hollow Carbon Sphere-Based Biosensor for Detection of Gram-Positive Bacteria

Science.gov (United States)

Wang, Hongsu; Luo, Ruiping; Chen, Yang; Si, Qi; Niu, Xiaodi

2018-05-01

A sensor based on mesoporous carbon materials immobilized with sortase A (SrtA) for determination of Staphylococcus aureus (S. aureus) is reported. To prepare the biosensor, we first synthesized carboxyl-functionalized mesoporous hollow carbon spheres, then applied them as carriers for immobilization of SrtA. Based on the catalytic mechanism of SrtA, a highly sensitive, inexpensive, and rapid method was developed for S. aureus detection. The sensor showed a linear response in the bacterial concentration range of 0.125 × 102 colony-forming units (CFU) mL-1 to 2.5 × 102 CFU mL-1, with detection limit as low as 9.0 CFU mL-1. The method was successfully used for quantitative detection of S. aureus in whole milk samples, giving results similar to experimental results obtained from the plate counting method. This biosensor could also be used to detect other Gram-positive bacteria that secrete SrtA.

Endophthalmitis caused by gram-positive bacteria resistant to vancomycin: Clinical settings, causative organisms, antimicrobial susceptibilities, and treatment outcomes

Directory of Open Access Journals (Sweden)

Hegde Sharat Shivaramaiah

2018-06-01

Full Text Available Purpose: To report the clinical settings, causative organisms, antimicrobial susceptibilities, and treatment outcomes of patients with endophthalmitis caused by gram-positive bacteria resistant to vancomycin. Methods: Retrospective case series of all patients with culture-proven endophthalmitis caused by gram-positive bacteria resistant to vancomycin between January 2010 and December 2016 in LV Prasad Eye Institute, Visakhapatnam, India. Results: The current study included 14 patients. The clinical settings were post-cataract surgery in 8/14 (57.1% and open globe injury in 6/14 (42.8%. Primary intervention for all patients included tap and intravitreal antibiotic injection. During subsequent follow-up, pars plana vitrectomy was performed in 6 patients and one patient underwent penetrating keratoplasty. Mean number of intravitreal antibiotic injections performed were 3.4 per patient. The most common organisms isolated were coagulase-negative Staphylococci in 6/14 (42.8%, Staphylococcus aureus in 5/14 (35.7%, Streptococcus sp in 2/14 (14.2% and Bacillus sp in 1/14 (7.14%. In addition to vancomycin, resistance to multiple drugs (three or more groups of antibiotics was found in all 14 cases. Antimicrobial susceptibility results showed susceptibility to amikacin in 7/14 (50.0%, gatifloxacin in 6/14 (42.8%, moxifloxacin in 3/13 (23.0%, cefazoline in 5/14 (35.7%, cefuroxime in 3/14 (21.4%, ciprofloxacin in 2/14 (14.2% and linezolid in 5/5 (100%. The mean duration of follow-up was 30.7 weeks (6 weeks–90 weeks. At last follow-up, visual acuity (VA of 20/200 or better was recorded in 7/14 (50% and VA < 5/200 occurred in 7/14 (50%. Conclusion and importance: Antimicrobial susceptibility testing may help in selection of suitable antimicrobial agents for repeat intravitreal injection. Inspite of retreatment with intravitreal antibiotics, these patients generally had poor VA outcomes. Keywords: Coagulase-negative Staphylococci, Endophthalmitis

Sediminibacillus massiliensis sp. nov., a moderately halophilic, Gram-positive bacterium isolated from a stool sample of a young Senegalese man.

Science.gov (United States)

Senghor, Bruno; Bassène, Hubert; Khelaifia, Saber; Robert, Catherine; Fournier, Pierre-Edouard; Ruimy, Raymond; Sokhna, Cheikh; Raoult, Didier; Lagier, Jean-Christophe

2018-07-01

A Gram-positive, moderately halophilic bacterium, referred to as strain Marseille-P3518 T , was isolated from a stool sample with 2% NaCl concentration from a healthy 15-year-old male living in Dielmo, a village in Senegal. Cells are aerobic, rod-shaped and motile and display endospore formation. Strain Marseille-P3518 T can grow in a medium with 0-20% (w/v) sodium chloride (optimally at 5-7.5% w/v). The major fatty acids were 12-methyl-tetradecanoic acid (45.8%), 13-methyl-tetradecanoic acid (26.9%) and 12-methyl-tridecanoic acid (12.8%). The genome is 4,347,479 bp long with 42.1% G+C content. It contains 4282 protein-coding and 107 RNA genes. Phylogenetic analysis based on 16S rRNA gene sequence comparisons showed that strain Marseille-P3518 T is a member of the Bacillaceae family and is closely related to Sediminibacillus albus (97.4% gene sequence similarity). Strain Marseille-P3518 T was clearly differentiated from its phylogenetic neighbors on the basis of phenotypic and genotypic features. Strain Marseille-P3518 T is, therefore, considered to be a novel representative of the genus Sediminibacillus, for which the name Sediminibacillus massiliensis sp. nov. is proposed, and the type strain is Marseille-P3518 T (CSUR P3518T, DSM69894).

Time-to-positivity, type of culture media and oxidase test performed on positive blood culture vials to predict Pseudomonas aeruginosa in patients with Gram-negative bacilli bacteraemia.

Science.gov (United States)

Cobos-Triguero, N; Zboromyrska, Y; Morata, L; Alejo, I; De La Calle, C; Vergara, A; Cardozo, C; Arcas, M P; Soriano, A; Marco, F; Mensa, J; Almela, M; Martínez, J A

2017-02-01

The aim of this study was to determine the usefulness of oxidase test and time-to-positivity (TTP) in aerobic and anaerobic blood culture vials to detect the presence of Pseudomonas aeruginosa in patients with Gram-negative bacilli (GNB) bacteraemia. TTP was recorded for each aerobic and anaerobic blood culture vial of monomicrobial bacteraemia due to GNB. Oxidase test was performed in a pellet of the centrifuged content of the positive blood culture. An algorithm was developed in order to perform the oxidase test efficiently taking into account TTP and type of vial. A total of 341 episodes of GNB bacteraemia were analysed. Sensitivity, specificity, positive predictive value and negative predictive value of the oxidase test performed on positive vials with GNB to predict P. aeruginosa were 95%, 99%, 91%, and 99%, respectively. When growth was first or exclusively detected in anaerobic vials, P. aeruginosa was never identified hence the performance of the oxidase test could be avoided. When growth was only or first detected in aerobic vials, a TTP≥8h predicted P. aeruginosa in 37% or cases (63 of 169), therefore oxidase test is highly recommended. Oxidase test performed onto positive blood culture vials previously selected by TTP and type of vials is an easy and inexpensive way to predict P. aeruginosa. In most cases, this can lead to optimization of treatment in less than 24 hours.

Fast Mechanically Driven Daughter Cell Separation Is Widespread in Actinobacteria.

Science.gov (United States)

Zhou, Xiaoxue; Halladin, David K; Theriot, Julie A

2016-08-30

Dividing cells of the coccoid Gram-positive bacterium Staphylococcus aureus undergo extremely rapid (millisecond) daughter cell separation (DCS) driven by mechanical crack propagation, a strategy that is very distinct from the gradual, enzymatically driven cell wall remodeling process that has been well described in several rod-shaped model bacteria. To determine if other bacteria, especially those in the same phylum (Firmicutes) or with similar coccoid shapes as S. aureus, might use a similar mechanically driven strategy for DCS, we used high-resolution video microscopy to examine cytokinesis in a phylogenetically wide range of species with various cell shapes and sizes. We found that fast mechanically driven DCS is rather rare in the Firmicutes (low G+C Gram positives), observed only in Staphylococcus and its closest coccoid relatives in the Macrococcus genus, and we did not observe this division strategy among the Gram-negative Proteobacteria In contrast, several members of the high-G+C Gram-positive phylum Actinobacteria (Micrococcus luteus, Brachybacterium faecium, Corynebacterium glutamicum, and Mycobacterium smegmatis) with diverse shapes ranging from coccoid to rod all undergo fast mechanical DCS during cell division. Most intriguingly, similar fast mechanical DCS was also observed during the sporulation of the actinobacterium Streptomyces venezuelae Much of our knowledge on bacterial cytokinesis comes from studying rod-shaped model organisms such as Escherichia coli and Bacillus subtilis Less is known about variations in this process among different bacterial species. While cell division in many bacteria has been characterized to some extent genetically or biochemically, few species have been examined using video microscopy to uncover the kinetics of cytokinesis and daughter cell separation (DCS). In this work, we found that fast (millisecond) DCS is exhibited by species in two independent clades of Gram-positive bacteria and is particularly prevalent

Comparative Proteomic Analysis of Desulfotomaculum reducens MI-1: Insights into the Metabolic Versatility of a Gram-positive Sulfate and Metal-reducing Bacterium

Directory of Open Access Journals (Sweden)

Anne Elyse Otwell

2016-02-01

Full Text Available The proteomes of the metabolically versatile and poorly characterized Gram-positive bacterium Desulfotomaculum reducens MI-1 were compared across four cultivation conditions including sulfate reduction, soluble Fe(III reduction, insoluble Fe(III reduction, and pyruvate fermentation. Collectively across conditions, we observed at high confidence ~38% of genome-encoded proteins. Here, we focus on proteins that display significant differential abundance on conditions tested. To the best of our knowledge, this is the first full-proteome study focused on a Gram-positive organism grown either on sulfate or metal-reducing conditions. Several proteins with uncharacterized function encoded within heterodisulfide reductase (hdr-containing loci were upregulated on either sulfate (Dred_0633-4, Dred_0689-90, and Dred_1325-30 or Fe(III-citrate-reducing conditions (Dred_0432-3 and Dred_1778-84. Two of these hdr-containing loci display homology to recently described flavin-based electron bifurcation (FBEB pathways (Dred_1325-30 and Dred_1778-84. Additionally, we propose that a cluster of proteins, which is homologous to a described FBEB lactate dehydrogenase (LDH complex, is performing lactate oxidation in D. reducens (Dred_0367-9. Analysis of the putative sulfate reduction machinery in D. reducens revealed that most of these proteins are constitutively expressed across cultivation conditions tested. In addition, peptides from the single multiheme c-type cytochrome (MHC in the genome were exclusively observed on the insoluble Fe(III condition, suggesting that this MHC may play a role in reduction of insoluble metals.

Two Cases of Endogenous Endophthalmitis Caused by Gram-Positive Bacteria with Good Visual Outcome

Directory of Open Access Journals (Sweden)

Machiko Itoh

2010-09-01

Full Text Available Background: Endogenous endophthalmitis is a rare disease and its visual prognosis is poor. Case Reports: We present two patients, a 60-year-old man and a 53-year-old man, who developed endogenous endophthalmitis caused byGram-positive organismsbut recovered good vision after antibiotics and vitrectomy. Results: The first patient complained of ocular pain and visual decrease in his right eye. Ophthalmoscopy showed inflammation in the anterior chamber and vitreous opacities. Antibiotic was administrated systemically, and blood culture detected Streptococcus anginosus. He underwent successful heart surgery for endocarditis and total dental extraction for severe gingivitis. Vitrectomy was performed 36 days after the onset and vision improved from 0.02 to 0.7. The second patient was referred for acute visual decrease in his left eye. Severe iritis and vitreous opacities were observed, and systemic examination showed acute pyelitis and prostatic abscesses. Blood cultures detected Staphylococcus sp., and systemic antibiotics were given. Vitrectomy was performed 12 days after the onset, and vision improved from 0.06 to 1.2. Conclusions: We conclude that the rapid treatment with systemic antibiotics for the organisms at the primary site, and the vitrectomy, even though delayed, can lead to a good recovery of vision.

A New Class of Quorum Quenching Molecules from Staphylococcus Species Affects Communication and Growth of Gram-Negative Bacteria

Science.gov (United States)

Chu, Ya-Yun; Nega, Mulugeta; Wölfle, Martina; Plener, Laure; Grond, Stephanie; Jung, Kirsten; Götz, Friedrich

2013-01-01

The knowledge that many pathogens rely on cell-to-cell communication mechanisms known as quorum sensing, opens a new disease control strategy: quorum quenching. Here we report on one of the rare examples where Gram-positive bacteria, the ‘Staphylococcus intermedius group’ of zoonotic pathogens, excrete two compounds in millimolar concentrations that suppress the quorum sensing signaling and inhibit the growth of a broad spectrum of Gram-negative beta- and gamma-proteobacteria. These compounds were isolated from Staphylococcus delphini. They represent a new class of quorum quenchers with the chemical formula N-[2-(1H-indol-3-yl)ethyl]-urea and N-(2-phenethyl)-urea, which we named yayurea A and B, respectively. In vitro studies with the N-acyl homoserine lactone (AHL) responding receptor LuxN of V. harveyi indicated that both compounds caused opposite effects on phosphorylation to those caused by AHL. This explains the quorum quenching activity. Staphylococcal strains producing yayurea A and B clearly benefit from an increased competitiveness in a mixed community. PMID:24098134

Antimicrobial resistance in gram-positive pathogens isolated in the UK between October 1996 and January 1997.

Science.gov (United States)

Andrews, J; Ashby, J; Jevons, G; Lines, N; Wise, R

1999-05-01

Antimicrobial resistance in gram-positive pathogens from 30 centres in the UK (ten Teaching, ten Associate Teaching and ten District General Hospitals) was studied over a 4 month period between October 1996 and January 1997. High-level resistance (HLR) and low-level resistance (LLR) to penicillin amongst pneumococci was 3.3% and 3.4%, respectively. However, considerable variation in resistance rates was observed depending on geographical location (LLR range 0-15.4% and HLR range 0-30.8%). Considerable variation in resistance rates was also observed for Staphylococcus aureus to methicillin, with rates ranging from 0% to 56.7% depending on locality. Using conventional MIC methodology, none of the isolates of S. aureus was considered as having reduced sensitivity to vancomycin. However, eight isolates grew on Brain Heart Infusion Agar containing vancomycin (4 mg/L) after prolonged incubation and are therefore worthy of further investigation by electron microscopy. With Enterococcus faecalis, resistance rates were similar between the three types of hospital and only four isolates were considered resistant to glycopeptide antibiotics (one vanA and three vanB phenotype).

A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris*

Science.gov (United States)

Reardon-Robinson, Melissa E.; Osipiuk, Jerzy; Chang, Chungyu; Wu, Chenggang; Jooya, Neda; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

2015-01-01

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria. PMID:26170452

A Disulfide Bond-forming Machine Is Linked to the Sortase-mediated Pilus Assembly Pathway in the Gram-positive Bacterium Actinomyces oris.

Science.gov (United States)

Reardon-Robinson, Melissa E; Osipiuk, Jerzy; Chang, Chungyu; Wu, Chenggang; Jooya, Neda; Joachimiak, Andrzej; Das, Asis; Ton-That, Hung

2015-08-28

Export of cell surface pilins in Gram-positive bacteria likely occurs by the translocation of unfolded precursor polypeptides; however, how the unfolded pilins gain their native conformation is presently unknown. Here, we present physiological studies to demonstrate that the FimA pilin of Actinomyces oris contains two disulfide bonds. Alanine substitution of cysteine residues forming the C-terminal disulfide bridge abrogates pilus assembly, in turn eliminating biofilm formation and polymicrobial interaction. Transposon mutagenesis of A. oris yielded a mutant defective in adherence to Streptococcus oralis, and revealed the essential role of a vitamin K epoxide reductase (VKOR) gene in pilus assembly. Targeted deletion of vkor results in the same defects, which are rescued by ectopic expression of VKOR, but not a mutant containing an alanine substitution in its conserved CXXC motif. Depletion of mdbA, which encodes a membrane-bound thiol-disulfide oxidoreductase, abrogates pilus assembly and alters cell morphology. Remarkably, overexpression of MdbA or a counterpart from Corynebacterium diphtheriae, rescues the Δvkor mutant. By alkylation assays, we demonstrate that VKOR is required for MdbA reoxidation. Furthermore, crystallographic studies reveal that A. oris MdbA harbors a thioredoxin-like fold with the conserved CXXC active site. Consistently, each MdbA enzyme catalyzes proper disulfide bond formation within FimA in vitro that requires the catalytic CXXC motif. Because the majority of signal peptide-containing proteins encoded by A. oris possess multiple Cys residues, we propose that MdbA and VKOR constitute a major folding machine for the secretome of this organism. This oxidative protein folding pathway may be a common feature in Actinobacteria. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Bacterial Cell Mechanics.

Science.gov (United States)

Auer, George K; Weibel, Douglas B

2017-07-25

Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the β-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

Long live the liver: immunohistochemical and stereological study of hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells of male and female rats throughout ageing.

Science.gov (United States)

Marcos, Ricardo; Correia-Gomes, Carla

2016-12-01

Male/female differences in enzyme activity and gene expression in the liver are known to be attenuated with ageing. Nevertheless, the effect of ageing on liver structure and quantitative cell morphology remains unknown. Male and female Wistar rats aged 2, 6, 12 and 18 months were examined by means of stereological techniques and immunohistochemical tagging of hepatocytes (HEP), liver sinusoidal endothelial cells (LSEC), Kupffer cells (KC) and hepatic stellate cells (HSC) in order to assess the total number and number per gram of these cells throughout life. The mean cell volume of HEP and HSC, the lobular position and the collagen content of the liver were also evaluated with stereological techniques. The number per gram of HSC was similar for both genders and was maintained throughout ageing. The mean volume of HSC was also conserved but differences in the cell body and lobular location were observed. Statistically significant gender differences in HEP were noted in young rats (females had smaller and more binucleated HEP) but were attenuated with ageing. The same occurred for KC and LSEC, since the higher number per gram in young females disappeared in older animals. Liver collagen increased with ageing but only in males. Thus, the numbers of these four cell types are related throughout ageing, with well-defined cell ratios. The shape and lobular position of HSC change with ageing in both males and females. Gender dimorphism in HEP, KC and LSEC of young rat liver disappears with ageing.

Self-generated covalent cross-links in the cell-surface adhesins of Gram-positive bacteria.

Science.gov (United States)

Baker, Edward N; Squire, Christopher J; Young, Paul G

2015-10-01

The ability of bacteria to adhere to other cells or to surfaces depends on long, thin adhesive structures that are anchored to their cell walls. These structures include extended protein oligomers known as pili and single, multi-domain polypeptides, mostly based on multiple tandem Ig-like domains. Recent structural studies have revealed the widespread presence of covalent cross-links, not previously seen within proteins, which stabilize these domains. The cross-links discovered so far are either isopeptide bonds that link lysine side chains to the side chains of asparagine or aspartic acid residues or ester bonds between threonine and glutamine side chains. These bonds appear to be formed by spontaneous intramolecular reactions as the proteins fold and are strategically placed so as to impart considerable mechanical strength. © 2015 Authors; published by Portland Press Limited.

Transcriptional attenuation controls macrolide inducible efflux and resistance in Streptococcus pneumoniae and in other Gram-positive bacteria containing mef/mel(msr(D)) elements.

Science.gov (United States)

Chancey, Scott T; Bai, Xianhe; Kumar, Nikhil; Drabek, Elliott F; Daugherty, Sean C; Colon, Thomas; Ott, Sandra; Sengamalay, Naomi; Sadzewicz, Lisa; Tallon, Luke J; Fraser, Claire M; Tettelin, Hervé; Stephens, David S

2015-01-01

Macrolide resistance, emerging in Streptococcus pneumoniae and other Gram-positive bacteria, is increasingly due to efflux pumps encoded by mef/mel(msr) operons found on discrete mobile genetic elements. The regulation of mef/mel(msr) in these elements is not well understood. We identified the mef(E)/mel transcriptional start, localized the mef(E)/mel promoter, and demonstrated attenuation of transcription as a mechanism of regulation of macrolide-inducible mef-mediated macrolide resistance in S. pneumoniae. The mef(E)/mel transcriptional start site was a guanine 327 bp upstream of mef(E). Consensus pneumococcal promoter -10 (5'-TATACT-3') and -35 (5'-TTGAAC-3') boxes separated by 17 bp were identified 7 bp upstream of the start site. Analysis of the predicted secondary structure of the 327 5' region identified four pairs of inverted repeats R1-R8 predicted to fold into stem-loops, a small leader peptide [MTASMRLR, (Mef(E)L)] required for macrolide induction and a Rho-independent transcription terminator. RNA-seq analyses provided confirmation of transcriptional attenuation. In addition, expression of mef(E)L was also influenced by mef(E)L-dependent mRNA stability. The regulatory region 5' of mef(E) was highly conserved in other mef/mel(msr)-containing elements including Tn1207.1 and the 5612IQ complex in pneumococci and Tn1207.3 in Group A streptococci, indicating a regulatory mechanism common to a wide variety of Gram-positive bacteria containing mef/mel(msr) elements.

Numerical Gram-Schmidt orthonormalization

International Nuclear Information System (INIS)

Werneth, Charles M; Dhar, Mallika; Maung, Khin Maung; Sirola, Christopher; Norbury, John W

2010-01-01

A numerical Gram-Schmidt orthonormalization procedure is presented for constructing an orthonormal basis function set from a non-orthonormal set, when the number of basis functions is large. This method will provide a pedagogical illustration of the Gram-Schmidt procedure and can be presented in classes on numerical methods or computational physics.

Novel Penicillin-Type Analogues Bearing a Variable Substituted 2-Azetidinone Ring at Position 6: Synthesis and Biological Evaluation

Directory of Open Access Journals (Sweden)

Margherita De Rosa

2015-12-01

Full Text Available The synthesis and the biological activity of novel semi-synthetic β-lactam compounds containing an azetidinone moiety joined to the amino-nitrogen of the (+-6-aminopenicillanic acid (6-APA as new antibacterial agents is reported. The synthesized compounds were screened for their in vitro antimicrobial activity against a panel of Gram positive and Gram negative pathogens and environmental bacteria. Tested compounds displayed good antimicrobial activity against all tested Gram positive bacteria and for Staphylococcus aureus and Staphylococcus epidermidis antimicrobial activity resulted higher than that of the reference antibiotic. Additionally, in vitro cytotoxic screening was also carried out indicating that the compounds do not cause a cell vitality reduction effective at concentration next to and above those shown to be antimicrobial.

Novel Penicillin-Type Analogues Bearing a Variable Substituted 2-Azetidinone Ring at Position 6: Synthesis and Biological Evaluation.

Science.gov (United States)

De Rosa, Margherita; Vigliotta, Giovanni; Palma, Giuseppe; Saturnino, Carmela; Soriente, Annunziata

2015-12-10

The synthesis and the biological activity of novel semi-synthetic β-lactam compounds containing an azetidinone moiety joined to the amino-nitrogen of the (+)-6-aminopenicillanic acid (6-APA) as new antibacterial agents is reported. The synthesized compounds were screened for their in vitro antimicrobial activity against a panel of Gram positive and Gram negative pathogens and environmental bacteria. Tested compounds displayed good antimicrobial activity against all tested Gram positive bacteria and for Staphylococcus aureus and Staphylococcus epidermidis antimicrobial activity resulted higher than that of the reference antibiotic. Additionally, in vitro cytotoxic screening was also carried out indicating that the compounds do not cause a cell vitality reduction effective at concentration next to and above those shown to be antimicrobial.

Osteopontin adsorption to Gram-positive cells reduces adhesion forces and attachment to surfaces under flow

DEFF Research Database (Denmark)

Kristensen, M F; Zeng, G; Neu, T R

2017-01-01

caries or medical device-related infections. It further investigated if OPN's effect on adhesion is caused by blocking the accessibility of glycoconjugates on bacterial surfaces. Bacterial adhesion was determined in a shear-controlled flow cell system in the presence of different concentrations of OPN......The bovine milk protein osteopontin (OPN) may be an efficient means to prevent bacterial adhesion to dental tissues and control biofilm formation. This study sought to determine to what extent OPN impacts adhesion forces and surface attachment of different bacterial strains involved in dental......, and interaction forces of single bacteria were quantified using single-cell force spectroscopy before and after OPN exposure. Moreover, the study investigated OPN's effect on the accessibility of cell surface glycoconjugates through fluorescence lectin-binding analysis. OPN strongly affected bacterial adhesion...

Surface-Exposed Lipoproteins: An Emerging Secretion Phenomenon in Gram-Negative Bacteria.

Science.gov (United States)

Wilson, Marlena M; Bernstein, Harris D

2016-03-01

Bacterial lipoproteins are hydrophilic proteins that are anchored to a cell membrane by N-terminally linked fatty acids. It is widely believed that nearly all lipoproteins produced by Gram-negative bacteria are either retained in the inner membrane (IM) or transferred to the inner leaflet of the outer membrane (OM). Lipoproteins that are exposed on the cell surface have also been reported but are generally considered to be rare. Results from a variety of recent studies, however, now suggest that the prevalence of surface-exposed lipoproteins has been underestimated. In this review we describe the evidence that the surface exposure of lipoproteins in Gram-negative bacteria is a widespread phenomenon and discuss possible mechanisms by which these proteins might be transported across the OM. Published by Elsevier Ltd.

Relative roles of the cellular and humoral responses in the Drosophila host defense against three gram-positive bacterial infections.

Directory of Open Access Journals (Sweden)

Nadine T Nehme

2011-03-01

Full Text Available Two NF-kappaB signaling pathways, Toll and immune deficiency (imd, are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense.In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus, we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival--independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response.Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen.

Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Analysis of Gram-Positive, Catalase-Negative Cocci Not Belonging to the Streptococcus or Enterococcus Genus and Benefits of Database Extension

DEFF Research Database (Denmark)

Christensen, Jens Jørgen; Dargis, Rimtas; Hammer, Monja

2012-01-01

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry with a Bruker Daltonics microflex LT system was applied to 90 well-characterized catalase-negative, Gram-positive cocci not belonging to the streptococci or enterococci. Biotyper version 2.0.43.1 software...

Prokaryotic cell division: flexible and diverse

NARCIS (Netherlands)

den Blaauwen, T.

2013-01-01

Gram-negative rod-shaped bacteria have different approaches to position the cell division initiating Z-ring at the correct moment in their cell division cycle. The subsequent maturation into a functional division machine occurs in vastly different species in two steps with appreciable time in

Gram-negative folliculitis. A rare problem or is it underdiagnosed? Case report and literature review

Directory of Open Access Journals (Sweden)

Sierra-Téllez Daniela, Ponce-Olivera Rosa María, Tirado-Sánchez Andrés

2011-07-01

Full Text Available AbstractGram-negative folliculitis may be the result of prolonged antibacterial treatments in patients with acne and rosacea. It is caused by alteration of facial skin flora and the nasal mucous, a decrease of Gram-positive bacteria and a proliferation of Gram-negative bacteria (for example Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, Klebsiella sp. and Proteus mirabilis. It should be considered in patients with acne who have not had a clinical improvement after 3-6 months of treatment with tetracyclines. The disease is underestimated, probably because bacteriological studies are rarely requested and the increased use of oral isotretinoin for acne management. One of the most effective treatments for Gram-negative folliculitis is oral isotretinoin (0.5-1 mg / kg / day for 4-5 months. We report the case of Gram negative folliculitis successfully treated with oral isotretinoin.

CD34-positive cells as stem cell support after high dose therapy

International Nuclear Information System (INIS)

Kvalheim, G.; Pharo, A.; Holte, H.

1996-01-01

Six patients, five with breast cancer and one with non-Hodgkin's lymphoma, were mobilized by chemotherapy and G-CSF. CD34-positive cells were isolated by means of immunomagnetic beads and Isolex 300 Cell Separator. Mean purity of isolated CD34-positive cells was 97% and mean yield was 54%. Three patients were treated with high dose therapy followed by reinfusion of CD34-positive cells as stem cell support. Recovery of neutrophils occurred at day 8, 11 and 13 and of platelets at day 9, 14 and 32. It is concluded that immunomagnetic isolated CD34-positive cells give high purity and yield. Although use of CD34-positive cells reduces the content of contaminating tumours cells in the graft, breast cancer cells were still detectable in two out of five CD34-positive cell products. 20 refs., 2 figs., 1 tab

Evaluation of Commercial-off-the-Shelf Materials for the Preservation of Gram Positive Vegetative Cells

Science.gov (United States)

2017-02-01

anthracis vegetative cells that were spotted on two different surfaces. 15. SUBJECT TERMS Bacillus anthracis Microbial cell viability Biosampling...technical capability gap that needs to be addressed because culturing a sample to determine what microorganisms are present remains essential, despite...the end of this incubation, 1 mL of inoculum was spotted on 2 × 2 in. stainless steel or 2 × 2 in. painted concrete surfaces and allowed to dry for

Questions on unusual Mimivirus-like structures observed in human cells [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Elena Angela Lusi

2017-03-01

Full Text Available Background: Mimiviruses or giant viruses that infect amoebas have the ability to retain the Gram stain, which is usually used to colour bacteria. There is some evidence suggesting that Mimiviruses can also infect human cells. Guided by these premises, we performed a routine Gram stain on a variety of human specimens to see if we could detect the same Gram positive blue granules that identify Mimiviruses in the amoebas. Methods: We analysed 24 different human specimens (liver, brain, kidney, lymph node and ovary using Gram stain histochemistry, electron microscopy immunogold, high resolution mass spectrometry and protein identification. Results: We detected in the human cells Gram positive granules that were distinct from bacteria. The fine blue granules displayed the same pattern of the Gram positive granules that diagnose Mimiviruses in the cytoplasm of the amoebas. Electron microscopy confirmed the presence of human Mimiviruses-like structures and mass spectrometry identified histone H4 peptides, which had the same footprints as giant viruses. However, some differences were noted: the Mimivirus-like structures identified in the human cells were ubiquitous and manifested a distinct mammalian retroviral antigenicity. Conclusions: Our main hypotheses are that the structures could be either giant viruses having a retroviral antigenicity or ancestral cellular components having a viral origin. However, other possible alternatives have been proposed to explain the nature and function of the newly identified structures.

Rapid Assessment of Resistance to Antibiotic Inhibitors of Protein Synthesis in the Gram-Positive Pathogens, Enterococcus faecalis and Streptococcus pneumoniae, Based on Evaluation of the Lytic Response.

Science.gov (United States)

Otero, Fátima; Tamayo, María; Santiso, Rebeca; Gosálvez, Jaime; Bou, Germán; Fernández, José Luis

2017-04-01

A novel assay for rapid determination of resistance to antibiotic inhibitors of protein synthesis was developed for the gram-positive pathogens, Enterococcus faecalis and Streptococcus pneumoniae. To this purpose, a lytic response was obtained by a brief incubation with lysozyme or a mixture of lysozyme, Triton X-100, and EDTA for E. faecalis (n = 82) and S. pneumoniae (n = 51), respectively. Lysis was quantified by visualizing the released nucleoids. Antibiotic-susceptible bacteria treated with Clinical and Laboratory Standards Institute (CLSI) breakpoint doses of erythromycin, azithromycin, or doxycycline that inhibited protein synthesis demonstrated a large reduction of lysed cells with respect to the control, that is, without antibiotics. However, cell lysis prevention was much lower in nonsusceptible strains, with unsuccessful inhibition of protein synthesis. ROC analysis showed that a reduction value of ≥35.6% and ≥40.4% discriminates susceptible and nonsusceptible strains for erythromycin and for doxycycline, respectively, in E. faecalis, whereas ≥20.0% is adequate for both macrolides and doxycycline in S. pneumoniae. Resistant stains were identified in 90-120 min with sensitivity and specificity between 91.7% and 100%. This is a proof of concept that evaluation of the lytic response may be a rapid and efficient test for determination of resistance to antibiotic inhibitors of protein synthesis.

Cytokeratin-positive folliculo-stellate cells in chicken adenohypophysis.

Science.gov (United States)

Nishimura, Shotaro; Yamashita, Miyu; Kaneko, Takane; Kawabata, Fuminori; Tabata, Shoji

2017-11-01

Folliculo-stellate (FS) cells are non-endocrine cells found in the adenohypophysis and are identified in many animals by the S100 protein marker. Although keratin is another FS marker in several animals, there is no information on localization of keratin in the avian adenohypophysis. In this study, localization of cytokeratin in chicken adenohypophyseal cells was investigated immunohistochemically. Basic cytokeratin (bCK)-positive cells were arranged radially in the cell cords with their cytoplasmic processes reaching the basal lamina. The cell bodies encircled a follicle in the center of the cell cord. Furthermore, the bCK-positive cells were also S100B-positive. Growth hormone, prolactin, adrenocorticotrophic hormone, and luteinizing hormone β-subunit did not co-localize with the bCK-positive cells. In addition, the bCK-positive cells had a laminin-positive area in their cytoplasm. Transmission electron microscopy observed agranular cells equipped with several microvilli that encircled a follicle. These results indicate that bCK-positive cells in the chicken adenohypophysis may be a predominant FS cell population and produce laminin. It is suggested that they function as sustentacular cells to sustain the adjacent endocrine cells and the structure of the cell cords in the chicken adenohypophysis. © 2017 Japanese Society of Animal Science.

Growth and adhesion to HT-29 cells inhibition of Gram-negatives by Bifidobacterium longum BB536 e Lactobacillus rhamnosus HN001 alone and in combination.

Science.gov (United States)

Inturri, R; Stivala, A; Furneri, P M; Blandino, G

2016-12-01

The aim of this study was to test the inhibitory effect of supernatants of broth cultures of Bifidobacterium longum BB536 and Lactobacillus rhamnosus HN001, both individually and in combination, against Gram-negative strains (uropathogens, enteropathogens and a reference strain). Moreover, in vitro protection of B. longum BB536 and L. rhamnosus HN001, both individually and in combination, against pathogen adhesion to HT-29 cell line, was investigated. The inhibitory activity was performed by the agar diffusion test and in vitro antagonistic activity against pathogen adhesion to human epithelial intestinal HT-29 cells was performed using standardized culture techniques. The study showed that B. longum BB536 and L. rhamnosus HN001, individually and in combination have inhibitory activity against the majority of the Gram negative strains tested. Furthermore, the results showed that both probiotic strains have a good capacity to inhibit pathogenic adhesion to HT-29 cells. Moreover, the ability of B. longum BB536 and L. rhamnosus HN001 to inhibit pathogenic adhesion increased when they were used in combination. The combination of B. longum BB536 and L. rhamnosus HN001 showed inhibitory activity against Gram-negatives and an improved ability to reduce their adhesion properties and to compete with them. The simultaneous presence of the two-probiotic strains could promote competitive mechanisms able to reduce the adhesion properties of pathogen strains and have an important ecological role within the highly competitive environment of the human gut.

Trends of Bloodstream Infections in a University Greek Hospital during a Three-Year Period: Incidence of Multidrug-Resistant Bacteria and Seasonality in Gram-negative Predominance.

Science.gov (United States)

Kolonitsiou, Fevronia; Papadimitriou-Olivgeris, Matthaios; Spiliopoulou, Anastasia; Stamouli, Vasiliki; Papakostas, Vasileios; Apostolopoulou, Eleni; Panagiotopoulos, Christos; Marangos, Markos; Anastassiou, Evangelos D; Christofidou, Myrto; Spiliopoulou, Iris

2017-07-06

The aim of the study was to assess the epidemiology, the incidence of multidrug-resistant bacteria and bloodstream infections' (BSIs) seasonality in a university hospital. This retrospective study was carried out in the University General Hospital of Patras, Greece, during 2011-13 y. Blood cultures from patients with clinical presentation suggestive of bloodstream infection were performed by the BacT/ALERT System. Isolates were identified by Vitek 2 Advanced Expert System. Antibiotic susceptibility testing was performed by the disk diffusion method and E-test. Resistance genes (mecA in staphylococci; vanA/vanB/vanC in enterococci; bla KPC /bla VIM /bla NDM in Klebsiella spp.) were detected by PCR. In total, 4607 (9.7%) blood cultures were positive from 47451 sets sent to Department of Microbiology, representing 1732 BSIs. Gram-negative bacteria (52.3%) were the most commonly isolated, followed by Gram-positive (39.5%), fungi (6.6%) and anaerobes bacteria (1.8%). The highest contamination rate was observed among Gram-positive bacteria (42.3%). Among 330 CNS and 150 Staphylococcus aureus, 281 (85.2%) and 60 (40.0%) were mecA-positive, respectively. From 113 enterococci, eight were vanA, two vanB and two vanC-positives. Of the total 207 carbapenem-resistant Klebsiella pneumoniae (73.4%), 202 carried bla KPC , four bla KPC and bla VIM and one bla VIM . A significant increase in monthly BSIs' incidence was shown (R2: 0.449), which may be attributed to a rise of Gram-positive BSIs (R2: 0.337). Gram-positive BSIs were less frequent in spring (P period. The increasing incidence of BSIs can be attributed to an increase of Gram-positive BSI incidence, even though Gram-negative bacteria remained the predominant ones. Seasonality may play a role in the predominance of Gram-negative's BSI.

Pentaclethra macroloba tannins fractions active against methicillin-resistant staphylococcal and Gram-negative strains showing selective toxicity

Directory of Open Access Journals (Sweden)

Ivana Correa Ramos Leal

2011-12-01

Full Text Available The ethanol extract of the vegetal species Pentaclethra macroloba (Willd. Kuntze, Fabaceae, was fractioned and the antibacterial activity was determined. The active ethyl acetate (ea fraction showed activity against Gram-positive (Staphylococcus spp. and Enterococcus spp. and Gram-negative (Pseudomonas aeruginosa, Acinetobacter spp. and Klebsiella pneumoniae multiresistant bacteria. Gallic acid derivatives were identified as the main compounds in inactive subfractions from the ea fraction, while the active one afforded ellagic acid as the major constituent when submitted to acid hydrolysis reaction, which suggests the presence of hydrolysable tannins. The minimum bactericidal concentration analysis showed a bactericide mechanism of action for the tannin subfraction found. The antibacterial mechanism of action of the active tannin subfraction against S. aureus reference strains (ATCC 29213 e 33591 was proposed adopting an in vitro assay of protein synthesis inhibition. For this, bacterial cells were labeled with [35S] methionine in the presence of the subfraction. The protein synthesis inhibition was observed at 256 µg/mL of this subfraction. At this concentration it did not present cytotoxicity in eukaryotic cells by the neutral red technique, suggesting selective toxicity. The present study is the first in vitro investigation of the antibacterial properties of tannin fractions obtained from a polar extract of P. macroloba.

Pentaclethra macroloba tannins fractions active against methicillin-resistant staphylococcal and Gram-negative strains showing selective toxicity

Directory of Open Access Journals (Sweden)

Ivana Correa Ramos Leal

2011-08-01

Full Text Available The ethanol extract of the vegetal species Pentaclethra macroloba (Willd. Kuntze, Fabaceae, was fractioned and the antibacterial activity was determined. The active ethyl acetate (ea fraction showed activity against Gram-positive (Staphylococcus spp. and Enterococcus spp. and Gram-negative (Pseudomonas aeruginosa, Acinetobacter spp. and Klebsiella pneumoniae multiresistant bacteria. Gallic acid derivatives were identified as the main compounds in inactive subfractions from the ea fraction, while the active one afforded ellagic acid as the major constituent when submitted to acid hydrolysis reaction, which suggests the presence of hydrolysable tannins. The minimum bactericidal concentration analysis showed a bactericide mechanism of action for the tannin subfraction found. The antibacterial mechanism of action of the active tannin subfraction against S. aureus reference strains (ATCC 29213 e 33591 was proposed adopting an in vitro assay of protein synthesis inhibition. For this, bacterial cells were labeled with [35S] methionine in the presence of the subfraction. The protein synthesis inhibition was observed at 256 µg/mL of this subfraction. At this concentration it did not present cytotoxicity in eukaryotic cells by the neutral red technique, suggesting selective toxicity. The present study is the first in vitro investigation of the antibacterial properties of tannin fractions obtained from a polar extract of P. macroloba.

A prospective study of the diagnostic utility of sputum Gram stain in pneumonia.

Science.gov (United States)

Anevlavis, Stavros; Petroglou, Niki; Tzavaras, Athanasios; Maltezos, Efstratios; Pneumatikos, Ioannis; Froudarakis, Marios; Anevlavis, Eleftherios; Bouros, Demosthenes

2009-08-01

Sputum Gram stain and culture have been said to be unreliable indicators of the microbiological diagnosis of bacterial pneumonia. The etiological diagnosis of pneumonia is surrounded by great degree of uncertainty. This uncertainty should be and can be calculated and incorporated in the diagnosis and treatment. To determine the diagnostic accuracy and diagnostic value of sputum Gram stain in etiological diagnosis and initial selection of antimicrobial therapy of bacterial community acquired pneumonia (CAP). DESIGN-METHOD: Prospective study of 1390 patients with CAP admitted January 2002-June 2008, to our institutions. Of the 1390 patients, 178 (12.8%) fulfilled the criteria for inclusion into this study (good-quality sputa and presence of the same microorganism in blood and sputum cultures which was used as gold standard for assessing the diagnostic accuracy and diagnostic value of sputum Gram stain). The sensitivity of sputum Gram stain was 0.82 for Pneumococcal pneumonia, 0.76 for Staphylococcal pneumonia, 0.79 for Haemophilus influenzae pneumonia and 0.78 for Gram-negative bacilli pneumonia. The specificity of sputum Gram stain was 0.93 for Pneumococcal pneumonia, 0.96 for Staphylococcal pneumonia, 0.96 for H. influenzae pneumonia and 0.95 for Gram-negative bacilli pneumonia. The positive likelihood ratio (LR+) was 11.58 for Pneumococcal pneumonia, 19.38 for Staphylococcal pneumonia, 16.84 for H. influenzae pneumonia, 14.26 for Gram-negative bacilli pneumonia. The negative likelihood ratio (LR-) was 0.20 for Pneumococcal pneumonia, 0.25 for Staphylococcal pneumonia, 0.22 for H. influenzae pneumonia, and 0.23 for Gram-negative bacilli pneumonia. Sputum Gram stain is a dependable diagnostic test for the early etiological diagnosis of bacterial CAP that helps in choosing orthological and appropriate initial antimicrobial therapy.

Expression levels of the receptor activator of NF-κB ligand and osteoprotegerin and the number of gram-negative bacteria in symptomatic and asymptomatic periapical lesions.

Science.gov (United States)

Carneiro, E; Parolin, A B; Wichnieski, C; Rosa, E A R; Silva Neto, U X; Westphalen, V P D; Fariniuk, L F; Johann, A C B R

2017-01-01

The study aimed to verify the potential correlation between the detected amount of gram-negative bacteria and the radiographic sizes of the lesions in patients with symptomatic and asymptomatic apical periodontitis. Furthermore, to evaluate whether the expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) and the RANKL/OPG ratio are differentially regulated in both groups. Twenty patients with periapical lesions were divided into two groups: symptomatic (SYM) n=10 and asymptomatic (ASYM) n=10. After periapical surgery, the lesions were collected and processed for histological examination, and immunohistochemistry. The percentage of RANKL- and OPG-immunopositive areas relative to the total area of the microscopic field was calculated. For gram staining, the number of gram-negative cells per microscopic field was assessed. The radiographs of each patient were processed and measured. The Student's t-test and the Pearson correlation coefficient were performed. The SYM group showed a significantly higher number of gram-negative cells (p=0.007) when compared to the ASYM group. A higher number of gram-negative bacteria occurred more frequently in larger periapical lesions and the SYM group (p=0.03). The expression for RANKL and OPG and the RANKL/OPG ratio were not significantly different between the groups. There was a significant positive correlation between the number of bacteria and OPG levels in the SYM group (p=0.01). The number of bacteria seems to influence the symptoms and the radiographic size of a periapical lesion. Gram-negative bacteria may play an important role in OPG activity in the SYM group. Copyright © 2016 Elsevier Ltd. All rights reserved.

New transposon tools tailored for metabolic engineering of Gram-negative microbial cell factories

Directory of Open Access Journals (Sweden)

Esteban eMartínez-García

2014-10-01

Full Text Available Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena, but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5 vectors, termed pBAMDs, for the delivery of gene(s into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic resistance markers (kanamycin, streptomycin, and gentamicin. After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate (PHB synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5 vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the

New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories

Energy Technology Data Exchange (ETDEWEB)

Martínez-García, Esteban; Aparicio, Tomás; Lorenzo, Víctor de; Nikel, Pablo I., E-mail: pablo.nikel@cnb.csic.es [Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), Madrid (Spain)

2014-10-28

Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.

New Transposon Tools Tailored for Metabolic Engineering of Gram-Negative Microbial Cell Factories

International Nuclear Information System (INIS)

Martínez-García, Esteban; Aparicio, Tomás; Lorenzo, Víctor de; Nikel, Pablo I.

2014-01-01

Re-programming microorganisms to modify their existing functions and/or to bestow bacteria with entirely new-to-Nature tasks have largely relied so far on specialized molecular biology tools. Such endeavors are not only relevant in the burgeoning metabolic engineering arena but also instrumental to explore the functioning of complex regulatory networks from a fundamental point of view. À la carte modification of bacterial genomes thus calls for novel tools to make genetic manipulations easier. We propose the use of a series of new broad-host-range mini-Tn5-vectors, termed pBAMDs, for the delivery of gene(s) into the chromosome of Gram-negative bacteria and for generating saturated mutagenesis libraries in gene function studies. These delivery vectors endow the user with the possibility of easy cloning and subsequent insertion of functional cargoes with three different antibiotic-resistance markers (kanamycin, streptomycin, and gentamicin). After validating the pBAMD vectors in the environmental bacterium Pseudomonas putida KT2440, their use was also illustrated by inserting the entire poly(3-hydroxybutyrate) (PHB) synthesis pathway from Cupriavidus necator in the chromosome of a phosphotransacetylase mutant of Escherichia coli. PHB is a completely biodegradable polyester with a number of industrial applications that make it attractive as a potential replacement of oil-based plastics. The non-selective nature of chromosomal insertions of the biosynthetic genes was evidenced by a large landscape of PHB synthesis levels in independent clones. One clone was selected and further characterized as a microbial cell factory for PHB accumulation, and it achieved polymer accumulation levels comparable to those of a plasmid-bearing recombinant. Taken together, our results demonstrate that the new mini-Tn5-vectors can be used to confer interesting phenotypes in Gram-negative bacteria that would be very difficult to engineer through direct manipulation of the structural genes.

Lactococcus lactis TrxD represents a subgroup of thioredoxins prevalent in Gram-positive bacteria containing WCXDC active site motifs

DEFF Research Database (Denmark)

Björnberg, Olof; Efler, Petr; Epie, Denis Ebong

2014-01-01

Three protein disulfide reductases of the thioredoxin superfamily from the industrially important Gram-positive Lactococcus lactis (LlTrxA, LlTrxD and LlNrdH) are compared to the "classical" thioredoxin from Escherichia coil (EcTrx1). LlTrxA resembles EcTrx1 with a WCGPC active site motif and other...... capacity to reduce insulin disulfides and their exposed active site thiol is alkylated at a similar rate at pH 7.0. LlTrxD on the other hand, is alkylated by iodoacetamide at almost 100 fold higher rate and shows no activity towards insulin disulfides. LlTrxA, LlTrxD and L1NrdH are all efficiently reduced...

Prevalence of AmpC β-lactamase among Gram-negative bacteria ...

African Journals Online (AJOL)

Purpose: Infections caused by AmpC-positive bacteria results in high patient morbidity and mortality making their detection clinically important as they cannot be detected in routine susceptibility testing. This study aim to determine the prevalence of AmpC β-lactamase among Gram negative bacteria recovered from clinical ...

Doripenem: an expected arrival in the treatment of infections caused by multidrug-resistant Gram-negative pathogens.

Science.gov (United States)

Poulakou, Garyphallia; Giamarellou, Helen

2008-05-01

Potent new drugs against multidrug-resistant Gram-negative bacteria, namely Pseudomonas aeruginosa and Acinetobacter spp. and pan-drug-resistant Klebsiella pneumoniae, which constitute an increasing medical threat, are almost absent from the future pharmaceutical pipeline. This drug evaluation focuses on the position of doripenem, a novel forthcoming carbapenem. Mechanisms of resistance and new drugs with anti-Gram-negative activity are also briefly reviewed. Literature search was performed for new carbapenems, new antibiotics, doripenem, metallo-beta-lactamase inhibitors, multidrug-resistant pathogens, antipseudomonal antibiotics and multidrug-resistant epidemiology. Doripenem possesses a broad spectrum of activity against Gram-negative bacteria, similar to that of meropenem, while retaining the spectrum of imipenem against Gram-positive pathogens. Against P. aeruginosa, doripenem exhibits rapid bactericidal activity with 2 - 4-fold lower MIC values, compared to meropenem. Exploitation of pharmacokinetic/pharmacodynamic applications could offer a treatment opportunity against strains exhibiting borderline resistance to doripenem. Stability against numerous beta-lactamases, low adverse event potential and more potent in vitro antibacterial activity against P. aeruginosa and A. baumanni compared to the existing carbapenems, are its principal features.

Modulation of Quorum Sensing in a Gram-Positive Pathogen by Linear Molecularly Imprinted Polymers with Anti-infective Properties.

Science.gov (United States)

Motib, Anfal; Guerreiro, Antonio; Al-Bayati, Firas; Piletska, Elena; Manzoor, Irfan; Shafeeq, Sulman; Kadam, Anagha; Kuipers, Oscar; Hiller, Luisa; Cowen, Todd; Piletsky, Sergey; Andrew, Peter W; Yesilkaya, Hasan

2017-12-22

We describe the development, characterization, and biological testing of a new type of linear molecularly imprinted polymer (LMIP) designed to act as an anti-infective by blocking the quorum sensing (QS) mechanism and so abrogating the virulence of the pathogen Streptococcus pneumoniae. The LMIP is prepared (polymerized) in presence of a template molecule, but unlike in traditional molecular imprinting approaches, no cross-linker is used. This results in soluble low-molecular-weight oligomers that can act as a therapeutic agent in vitro and in vivo. The LMIP was characterized by mass spectrometry to determine its monomer composition. Fragments identified were then aligned along the peptide template by computer modeling to predict the possible monomer sequence of the LMIP. These findings provide a proof of principle that LMIPs can be used to block QS, thus setting the stage for the development of LMIPs a novel drug-discovery platform and class of materials to target Gram-positive pathogens. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Retrospective Analysis of Blood Stream Infections and Antibiotic Susceptibility Pattern of Gram Negative Bacteria in a Tertiary Care Cancer Hospital

Directory of Open Access Journals (Sweden)

Radha Rani D

2017-12-01

Full Text Available Background: Bacterial bloodstream infections are important causes of morbidity and mortality globally. The aim of the present study was to determine the bacterial profile of bloodstream infections and their antibiotic susceptibility pattern among the clinically diagnosed cases of sepsis in cancer patients. Methods: In the present study, etiological and antimicrobial susceptibility profile of blood cultures over a period of 1 year at a tertiary cancer care hospital was done. Blood culture positive isolates were identified using standard microbiological methods and by Fully automated BD Phoenix 100. The antibiotic susceptibility pattern of the organisms was performed by Kirby-Bauer disc diffusion method and MIC (Minimum inhibitory concentration was done by Fully automated BD Phoenix 100. Results: There were 1178 blood culture samples, of which 327 (27.7% were identified to be culture positive. Out of 327 positive cultures, 299 (91.4% showed bacterial growth, Gram negative were 161 (53.8% and Gram positive were 138 (46.1%. Candida species were isolated from 13 (3.97% of positive samples and 15 samples showed contamination. The most common Gram-negative isolate was. Escherichia coli (37.80% and Gram-positive isolate was coagulasenegative staphylococci (52.80%. Escherichia coli showed highest sensitivity to amikacin (83.60% and sensitivity to piperacillin+ tazobactum and cefaperazone+sulbactam was 54.09% and 52.45% respectively. High degree of resistance was found to cephalosporins and levofloxacin. Conclusion: The results indicate high level of antimicrobial resistance among Gram negative bacilli in septicemic patients. The results warrant continuous monitoring of antimicrobial pattern so as to build geographical epidemiological data.

Can early MRI distinguish between Kingella kingae and Gram-positive cocci in osteoarticular infections in young children?

Energy Technology Data Exchange (ETDEWEB)

Kanavaki, Aikaterini; Hanquinet, Sylviane; Merlini, Laura [Geneva University Hospital HUG, Unit of Pediatric Radiology, Geneva (Switzerland); Ceroni, Dimitri [Geneva University Hospital, Unit of Pediatric Orthopedics, Geneva (Switzerland); Tchernin, David [Geneva University Hospital, Department of Radiology, Geneva (Switzerland)

2012-01-15

K. kingae is a common causative organism in acute osteoarticular infections (OAIs) in children under 4 years of age. Differentiation between K. kingae and Gram-positive cocci (GPC) is of great interest therapeutically. Our aim was to identify early distinguishing MRI features of OAIs. Thirty-one children younger than 4 years of age with OAI underwent MRI at presentation. Of these, 21 were caused by K. kingae and ten by GPC. Bone and soft tissue reaction, epiphyseal cartilage involvement, bone and subperiosteal abscess formation were compared between the two groups. Interobserver agreement was measured. Bone reaction was less frequent (P=0.0066) and soft tissue reaction less severe (P=0.0087) in the K. kingae group. Epiphysis cartilage abscesses were present only in the K. kingae group (P=0.0118). No difference was found for bone abscess (P=0.1411), subperiosteal abscess (P=1) or joint effusion (P=0.4414). Interobserver agreement was good for all criteria. MRI is useful in differentiating K. kingae from GPC in OAI. Cartilaginous involvement and modest soft tissue and bone reaction suggest K. kingae. (orig.)

Computational design and characterization of a temperature-sensitive plasmid replicon for gram positive thermophiles

Directory of Open Access Journals (Sweden)

Olson Daniel G

2012-05-01

Full Text Available Abstract Background Temperature-sensitive (Ts plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996 for predicting Ts mutants based on the amino-acid sequence of the protein. Results A library of 34 mutant plasmids was designed, synthesized and screened, resulting in 6 mutants which exhibited a Ts phenotype. Of these 6, the one with the most temperature-sensitive phenotype (M166A was compared with the original plasmid. It exhibited lower stability at 48°C and was completely unable to replicate at 55°C. Conclusions The plasmid described in this work could be useful in future efforts to genetically engineer C. thermocellum, and the method used to generate this plasmid may be useful for others trying to make Ts plasmids.

Lipopolysaccharide biogenesis and transport at the outer membrane of Gram-negative bacteria.

Science.gov (United States)

Sperandeo, Paola; Martorana, Alessandra M; Polissi, Alessandra

2017-11-01

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer containing a unique glycolipid, lipopolysaccharide (LPS) in its outer leaflet. LPS molecules confer to the OM peculiar permeability barrier properties enabling Gram-negative bacteria to exclude many toxic compounds, including clinically useful antibiotics, and to survive harsh environments. Transport of LPS poses several problems to the cells due to the amphipatic nature of this molecule. In this review we summarize the current knowledge on the LPS transport machinery, discuss the challenges associated with this process and present the solutions that bacterial cells have evolved to address the problem of LPS transport and assembly at the cell surface. Finally, we discuss how knowledge on LPS biogenesis can be translated for the development of novel antimicrobial therapies. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop. Copyright © 2016. Published by Elsevier B.V.

In vitro activities of the new semisynthetic glycopeptide telavancin (TD-6424), vancomycin, daptomycin, linezolid, and four comparator agents against anaerobic gram-positive species and Corynebacterium spp.

Science.gov (United States)

Goldstein, Ellie J C; Citron, Diane M; Merriam, C Vreni; Warren, Yumi A; Tyrrell, Kerin L; Fernandez, Helen T

2004-06-01

Telavancin is a new semisynthetic glycopeptide anti-infective with multiple mechanisms of action, including inhibition of bacterial membrane phospholipid synthesis and inhibition of bacterial cell wall synthesis. We determined the in vitro activities of telavancin, vancomycin, daptomycin, linezolid, quinupristin-dalfopristin, imipenem, piperacillin-tazobactam, and ampicillin against 268 clinical isolates of anaerobic gram-positive organisms and 31 Corynebacterium strains using agar dilution methods according to National Committee for Clinical Laboratory Standards procedures. Plates with daptomycin were supplemented with Ca(2+) to 50 mg/liter. The MICs at which 90% of isolates tested were inhibited (MIC(90)s) for telavancin and vancomycin were as follows: Actinomyces spp. (n = 45), 0.25 and 1 microg/ml, respectively; Clostridium difficile (n = 14), 0.25 and 1 microg/ml, respectively; Clostridium ramosum (n = 16), 1 and 4 microg/ml, respectively; Clostridium innocuum (n = 15), 4 and 16 microg/ml, respectively; Clostridium clostridioforme (n = 15), 8 and 1 microg/ml, respectively; Eubacterium group (n = 33), 0.25 and 2 microg/ml, respectively; Lactobacillus spp. (n = 26), 0.5 and 4 microg/ml, respectively; Propionibacterium spp. (n = 34), 0.125 and 0.5 microg/ml, respectively; Peptostreptococcus spp. (n = 52), 0.125 and 0.5 microg/ml, respectively; and Corynebacterium spp. (n = 31), 0.03 and 0.5 microg/ml, respectively. The activity of TD-6424 was similar to that of quinupristin-dalfopristin for most strains except C. clostridioforme and Lactobacillus casei, where quinupristin-dalfopristin was three- to fivefold more active. Daptomycin had decreased activity (MIC > 4 microg/ml) against 14 strains of Actinomyces spp. and all C. ramosum, Eubacterium lentum, and Lactobacillus plantarum strains. Linezolid showed decreased activity (MIC > 4 microg/ml) against C. ramosum, two strains of C. difficile, and 15 strains of Lactobacillus spp. Imipenem and piperacillin

Measuring the Level of Agreement Between Cloacal Gram's Stains and Bacterial Cultures in Hispaniolan Amazon Parrots ( Amazona ventralis ).

Science.gov (United States)

Evans, Erika E; Mitchell, Mark A; Whittington, Julia K; Roy, Alma; Tully, Thomas N

2014-12-01

Cloacal or fecal Gram's stains and bacterial cultures are routinely performed during avian physical examinations to assess the microbial flora of the gastrointestinal tract. Although cloacal or fecal Gram's stains and bacterial cultures are considered routine diagnostic procedures, the level of agreement between the individual tests has not been determined. To investigate the level of agreement between results from Gram's stain and bacterial culture when used to assess cloacal or fecal samples from psittacine birds, samples were taken from 21 clinically healthy Hispaniolan Amazon parrots ( Amazona ventralis ) and tested by Gram's stain cytology and bacterial culture. Most bacteria (97.2%) identified by Gram's stain were gram positive. However, gram-negative organisms were identified in 7 of 21 (33.3%; 95% confidence interval: 13.3%-53.3%) birds. Escherichia coli was the only gram-negative organism identified on culture. Agreement between results of Gram's stain and culture was fair (weighted κ = 0.27). The results of this study suggest that Gram's stains and bacterial culture may need to be performed with a parallel testing strategy to limit the likelihood of misclassifying the microbial flora of psittacine patients.

Validation of Aedes aegypti Aag-2 cells as a model for insect immune studies.

Science.gov (United States)

Barletta, Ana Beatriz Ferreira; Silva, Maria Clara L Nascimento; Sorgine, Marcos H Ferreira

2012-07-24

The understanding of mosquito immune responses can provide valuable tools for development of novel mosquito control strategies. Aiming the study at insect innate immunity, continuous insect cell lines have been established and used as research tools due to the fact that they constitute more homogeneous, sensitive, and reproducible systems than the insects from which they originated. More recently, Aag-2, an Aedes aegypti cell lineage, began to be frequently used as a model for studies of mosquito immunity. Nevertheless, to our knowledge, no study has systematically characterized the responses of Aag-2 cell line against different kinds of pathogens and compared its response to those exhibited by whole mosquitoes. For this reason, in this study we characterized gene expression profiles of the Aag-2 cell line in response to different kinds of immune challenges, such as Gram negative and positive bacteria, fungi and viruses, comparing the obtained results with the ones already described in the literature for whole mosquitoes. Aedes aegypti Aag-2 cells were exposed to different immune stimuli (gram-positive and gram negative heat inactivated bacteria, zymosan or Sindbis virus) for 24 hours and the expression of selected marker genes from toll, IMD and Jak/STAT pathways was analyzed by qPCR. Also, cells were incubated with fluorescent latex beads for evaluation of its phagocytosis capacity. Aag-2 cells were stimulated with two concentrations of heat-killed Gram negative (Enterobacter cloacae) or Gram positive (Micrococcus luteus) bacteria, Zymosan or infected with Sindbis virus and the expression of key genes from the main immune related pathways, Toll, IMD and Jak/STAT, were investigated. Our results suggest that Toll and IMD pathways are activated in response to both Gram positive and negative bacteria and Zymosan in Aag-2 cells, displaying an immune profile similar to those described in the literature for whole mosquitoes. The same stimuli were also capable of

Combating multidrug-resistant Gram-negative bacterial infections.

Science.gov (United States)

Xu, Ze-Qi; Flavin, Michael T; Flavin, John

2014-02-01

Multidrug-resistant (MDR) bacterial infections, especially those caused by Gram-negative pathogens, have emerged as one of the world's greatest health threats. The development of novel antibiotics to treat MDR Gram-negative bacteria has, however, stagnated over the last half century. This review provides an overview of recent R&D activities in the search for novel antibiotics against MDR Gram-negatives. It provides emphasis in three key areas. First, the article looks at new analogs of existing antibiotic molecules such as β-lactams, tetracyclines, and aminoglycoside as well as agents against novel bacterial targets such as aminoacyl-tRNA synthetase and peptide deformylase. Second, it also examines alternative strategies to conventional approaches including cationic antimicrobial peptides, siderophores, efflux pump inhibitors, therapeutic antibodies, and renewed interest in abandoned treatments or those with limited indications. Third, the authors aim to provide an update on the current clinical development status for each drug candidate. The traditional analog approach is insufficient to meet the formidable challenge brought forth by MDR superbugs. With the disappointing results of the genomics approach for delivering novel targets and drug candidates, alternative strategies to permeate the bacterial cell membrane, enhance influx, disrupt efflux, and target specific pathogens via therapeutic antibodies are attractive and promising. Coupled with incentivized business models, governmental policies, and a clarified regulatory pathway, it is hoped that the antibiotic pipeline will be filled with an effective armamentarium to safeguard global health.

Plants used in Guatemala for the treatment of respiratory diseases. 1. Screening of 68 plants against gram-positive bacteria.

Science.gov (United States)

Caceres, A; Alvarez, A V; Ovando, A E; Samayoa, B E

1991-02-01

Respiratory ailments are important causes of morbidity and mortality in developing countries. Ethnobotanical surveys and literature reviews conducted in Guatemala during 1986-88 showed that 234 plants from 75 families, most of them of American origin, have been used for the treatment of respiratory ailments. Three Gram-positive bacteria causing respiratory infections (Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes) were used to screen 68 of the most commonly used plants for activity. Twenty-eight of these (41.2%) inhibited the growth of one or more of the bacteria tested. Staphylococcus aureus was inhibited by 18 of the plant extracts, while 7 extracts were effective against Streptococcus pyogenes. Plants of American origin which exhibited antibacterial activity were: Gnaphalium viscosum, Lippia alba, Lippia dulcis, Physalis philadelphica, Satureja brownei, Solanum nigrescens and Tagetes lucida. These preliminary in vitro results provide scientific basis for the use of these plants against bacterial respiratory infections.

Functional validation of putative toxin-antitoxin genes from the Gram-positive pathogen Streptococcus pneumoniae: phd-doc is the fourth bona-fide operon.

Science.gov (United States)

Chan, Wai Ting; Yeo, Chew Chieng; Sadowy, Ewa; Espinosa, Manuel

2014-01-01

Bacterial toxin-antitoxin (TAs) loci usually consist of two genes organized as an operon, where their products are bound together and inert under normal conditions. However, under stressful circumstances the antitoxin, which is more labile, will be degraded more rapidly, thereby unleashing its cognate toxin to act on the cell. This, in turn, causes cell stasis or cell death, depending on the type of TAs and/or time of toxin exposure. Previously based on in silico analyses, we proposed that Streptococcus pneumoniae, a pathogenic Gram-positive bacterium, may harbor between 4 and 10 putative TA loci depending on the strains. Here we have chosen the pneumococcal strain Hungary(19A)-6 which contains all possible 10 TA loci. In addition to the three well-characterized operons, namely relBE2, yefM-yoeB, and pezAT, we show here the functionality of a fourth operon that encodes the pneumococcal equivalent of the phd-doc TA. Transcriptional fusions with gene encoding Green Fluorescent Protein showed that the promoter was slightly repressed by the Phd antitoxin, and exhibited almost background values when both Phd-Doc were expressed together. These findings demonstrate that phd-doc shows the negative self-regulatory features typical for an authentic TA. Further, we also show that the previously proposed TAs XreA-Ant and Bro-XreB, although they exhibit a genetic organization resembling those of typical TAs, did not appear to confer a functional behavior corresponding to bona fide TAs. In addition, we have also discovered new interesting bioinformatics results for the known pneumococcal TAs RelBE2 and PezAT. A global analysis of the four identified toxins-antitoxins in the pneumococcal genomes (PezAT, RelBE2, YefM-YoeB, and Phd-Doc) showed that RelBE2 and Phd-Doc are the most conserved ones. Further, there was good correlation among TA types, clonal complexes and sequence types in the 48 pneumococcal strains analyzed.

Study of bacterial meningitis in children below 5 years with comparative evaluation of gram staining, culture and bacterial antigen detection.

Science.gov (United States)

Yadhav Ml, Kala

2014-04-01

Bacterial meningitis is one of the most serious infections seen in infants and children, which is associated with acute complications and chronic morbidity. Infections of Central Nervous System (CNS) still dominate the scene of childhood neurological disorders in most of the developing tropical countries. To isolate, identify and determine the antibiotic susceptibility patterns of pathogens associated with bacterial meningitis. We also aimed to comparatively evaluate of Gram staining, culture and bacterial antigen detection in cerebrospinal fluid samples. Present comparative study included 100 CSF samples of children below the age of 5 years, who were clinically suspected meningitis cases. The samples were subjected to Gram staining, culture and Latex agglutination test (LAT). The organisms isolated in the study were characterized and antibiotic susceptibility test was done according to standard guidelines. It was done by using Gaussian test. Of the 100 cases, 24 were diagnosed as Acute bacterial meningitis (ABM) cases by. Gram staining, culture and latex agglutination test. 21 (87.5%) cases were culture positive, with 2 cases being positive for polymicrobial isolates. Gram staining was positive in 17 (70.53%) cases and LAT was positive in 18 (33.33%) cases. Streptococcus pneumoniae was the predominant organism which was isolated and it was sensitive to antibiotics. In the present study, male to female ratio was 1.27:1, which showed a male preponderance. With the combination of Gram staining, culture, and LAT, 100% sensitivity and specificity can be achieved (p Gram staining and LAT can detect 85% of cases of ABM. Bacterial meningitis is a medical emergency and making an early diagnosis and providing treatment early are life saving and they reduce chronic morbidity.

[Frequency of intestinal microsporidian infections in HIV-positive patients, as diagnosis by quick hot Gram chromotrope staining and PCR].

Science.gov (United States)

Botero, Jorge H; Montoya, Martha Nelly; Vanegas, Adriana Lucía; Díaz, Abel; Navarro-i-Martínez, Luis; Bornay, Fernando Jorge; Izquierdo, Fernando; del Aguila, Carmen; Agudelo, Sonia del Pilar

2004-12-01

Microsporidia are intracellular obligate parasites, today mainly associated with diarrhea in AIDS patients. Microsporidia prevalence ranges from 8% to 52% in different countries, as evaluated by several diagnostic methods, such as the stain test and PCR. In Medellín, Colombia, its frequency is unknown, and hence, a study was undertaken to determine the frequency of intestinal microsporidiosis in HIV patients, by means of the quick-hot Gram chromotrope test and the PCR. A prospective and descriptive study of an intentional population of all HIV-positive patients was sent to the Grupo Interdisciplinario para el Estudio de las Parasitosis Intestinales laboratory by institutions treating the HIV-positive patients of Medellín between August 2001 and September 2002. The clinical-epidemiological survey included a serial stool test with direct concentration and special stains for coccidiae and intestinal microsporidia. In addition, counts of lymphocytes TCD4+ and viral load were requested. One hundred and three patients with ages ranging from 2-74 years were evaluated. Seventy percent presented with diarrhea--mostly in men (83.5%). The overall frequency of intestinal microsporidiosis was 3.9% and that of other intestinal parasitic infections was 39.8%. Three of the four patients positive for microsporida were infected with Enterocytozoon bieneusi and one with Encephalitozoon intestinalis. The microsporidiosis frequency was relatively low with 3 of the 4 cases associated with protracted diarrhea, counts of LTCD4+ below 100 cel/microl and viral loads up to 100,000 copies.

Multicenter Evaluation of the Vitek MS Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry System for Identification of Gram-Positive Aerobic Bacteria

Science.gov (United States)

Burnham, Carey-Ann D.; Bythrow, Maureen; Garner, Omai B.; Ginocchio, Christine C.; Jennemann, Rebecca; Lewinski, Michael A.; Manji, Ryhana; Mochon, A. Brian; Procop, Gary W.; Richter, Sandra S.; Sercia, Linda; Westblade, Lars F.; Ferraro, Mary Jane; Branda, John A.

2013-01-01

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting. PMID:23658261

Multicenter evaluation of the Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of Gram-positive aerobic bacteria.

Science.gov (United States)

Rychert, Jenna; Burnham, Carey-Ann D; Bythrow, Maureen; Garner, Omai B; Ginocchio, Christine C; Jennemann, Rebecca; Lewinski, Michael A; Manji, Ryhana; Mochon, A Brian; Procop, Gary W; Richter, Sandra S; Sercia, Linda; Westblade, Lars F; Ferraro, Mary Jane; Branda, John A

2013-07-01

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) is gaining momentum as a tool for bacterial identification in the clinical microbiology laboratory. Compared with conventional methods, this technology can more readily and conveniently identify a wide range of organisms. Here, we report the findings from a multicenter study to evaluate the Vitek MS v2.0 system (bioMérieux, Inc.) for the identification of aerobic Gram-positive bacteria. A total of 1,146 unique isolates, representing 13 genera and 42 species, were analyzed, and results were compared to those obtained by nucleic acid sequence-based identification as the reference method. For 1,063 of 1,146 isolates (92.8%), the Vitek MS provided a single identification that was accurate to the species level. For an additional 31 isolates (2.7%), multiple possible identifications were provided, all correct at the genus level. Mixed-genus or single-choice incorrect identifications were provided for 18 isolates (1.6%). Although no identification was obtained for 33 isolates (2.9%), there was no specific bacterial species for which the Vitek MS consistently failed to provide identification. In a subset of 463 isolates representing commonly encountered important pathogens, 95% were accurately identified to the species level and there were no misidentifications. Also, in all but one instance, the Vitek MS correctly differentiated Streptococcus pneumoniae from other viridans group streptococci. The findings demonstrate that the Vitek MS system is highly accurate for the identification of Gram-positive aerobic bacteria in the clinical laboratory setting.

Loss of outer membrane integrity in Gram-negative bacteria by silver ...

Indian Academy of Sciences (India)

plausible mechanism of bacterial cell disintegration ... New generation antimicrobial and smart drugs are the needs of the present era in fighting microbial ... charides (LPSs) in nature where the lipid portion acts ... sive release of LPS molecules and membrane proteins [10]. ... efficacy of antimicrobial action on Gram bacteria.

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Science.gov (United States)

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Cell Size Regulation in Bacteria

Science.gov (United States)

Amir, Ariel

2014-05-01

Various bacteria such as the canonical gram negative Escherichia coli or the well-studied gram positive Bacillus subtilis divide symmetrically after they approximately double their volume. Their size at division is not constant, but is typically distributed over a narrow range. Here, we propose an analytically tractable model for cell size control, and calculate the cell size and interdivision time distributions, as well as the correlations between these variables. We suggest ways of extracting the model parameters from experimental data, and show that existing data for E. coli supports partial size control, and a particular explanation: a cell attempts to add a constant volume from the time of initiation of DNA replication to the next initiation event. This hypothesis accounts for the experimentally observed correlations between mother and daughter cells as well as the exponential dependence of size on growth rate.

Antimicrobial-resistant Gram-negative bacteria in febrile neutropenic patients with cancer: current epidemiology and clinical impact.

Science.gov (United States)

Trecarichi, Enrico M; Tumbarello, Mario

2014-04-01

In the recent years, several studies involving cancer patients have demonstrated a clear trend in the epidemiology of bacterial infections showing a shift in the prevalence from Gram-positive to Gram-negative bacteria and the extensive emergence of antimicrobial-resistant strains among Gram-negatives isolated from the blood. The aim of this systematic review was to examine the recent trends in epidemiology and antimicrobial resistance in Gram-negatives recovered from neutropenic cancer patients, with particular emphasis on the impact of antimicrobial resistance on the clinical outcome of severe infections caused by such microorganisms. Overall, from 2007 to date, the rate of Gram-negative bacteria recovery ranged from 24.7 to 75.8% (mean 51.3%) in cancer patient cohorts. Escherichia coli represented the most common species (mean frequency of isolation 32.1%) among the Gram-negatives, followed by Pseudomonas aeruginosa (mean frequency of isolation 20.1%). An increasing frequency of Acinetobacter spp. and Stenotrophomonas maltophilia was also reported. Increased rates of multidrug-resistant Gram-negative strains have been highlighted among Enterobacteriaceae and nonfermenting Gram-negative rods, despite discontinuation of fluoroquinolone-based antibacterial prophylaxis for neutropenic patients. In addition, antimicrobial resistance and/or the inadequacy of empirical antibiotic treatment have been frequently linked to a worse outcome in cancer patients with bloodstream infections caused by Gram-negative isolates. Sound knowledge of the local distribution of pathogens and their susceptibility patterns and prompt initiation of effective antimicrobial treatment for severe infections caused by Gram-negative bacteria are essential in cancer patients.

Multicenter Assessment of Gram Stain Error Rates.

Science.gov (United States)

Samuel, Linoj P; Balada-Llasat, Joan-Miquel; Harrington, Amanda; Cavagnolo, Robert

2016-06-01

Gram stains remain the cornerstone of diagnostic testing in the microbiology laboratory for the guidance of empirical treatment prior to availability of culture results. Incorrectly interpreted Gram stains may adversely impact patient care, and yet there are no comprehensive studies that have evaluated the reliability of the technique and there are no established standards for performance. In this study, clinical microbiology laboratories at four major tertiary medical care centers evaluated Gram stain error rates across all nonblood specimen types by using standardized criteria. The study focused on several factors that primarily contribute to errors in the process, including poor specimen quality, smear preparation, and interpretation of the smears. The number of specimens during the evaluation period ranged from 976 to 1,864 specimens per site, and there were a total of 6,115 specimens. Gram stain results were discrepant from culture for 5% of all specimens. Fifty-eight percent of discrepant results were specimens with no organisms reported on Gram stain but significant growth on culture, while 42% of discrepant results had reported organisms on Gram stain that were not recovered in culture. Upon review of available slides, 24% (63/263) of discrepant results were due to reader error, which varied significantly based on site (9% to 45%). The Gram stain error rate also varied between sites, ranging from 0.4% to 2.7%. The data demonstrate a significant variability between laboratories in Gram stain performance and affirm the need for ongoing quality assessment by laboratories. Standardized monitoring of Gram stains is an essential quality control tool for laboratories and is necessary for the establishment of a quality benchmark across laboratories. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Embryonic and foetal Islet-1 positive cells in human hearts are also positive to c-Kit

Directory of Open Access Journals (Sweden)

C. Serradifalco

2011-12-01

Full Text Available During embryogenesis, the mammalian heart develops from a primitive heart tube originating from two bilateral primary heart fields located in the lateral plate mesoderm. Cells belongings to the pre-cardiac mesoderm will differentiate into early cardiac progenitors, which express early transcription factors which are also common to the Isl-1 positive cardiac progenitor cells isolated from the developing pharyngeal mesoderm and the foetal and post-natal mice hearts. A second population of cardiac progenitor cells positive to c-Kit has been abundantly isolated from adult hearts. Until now, these two populations have been considered two different sets of progenitor cells present in the heart in different stages of an individual life. In the present study we collected embryonic, foetal and infant hearts, and we tested the hypotheses that c-Kit positive cells, usually isolated from the adult heart, are also present in the intra-uterine life and persist in the adult heart after birth, and that foetal Isl-1 positive cells are also positive to c-Kit. Using immunohistochemistry we studied the temporal distribution of Isl-1 positive and c-Kit/CD105 double positive cells, and by immunofluorescence and confocal analysis we studied the co-localization of c-Kit and Isl-1 positive cells. The results indicated that cardiomyocytes and interstitial cells were positive for c-Kit from the 9th to the 19th gestational week, that cells positive for both c-Kit and CD105 appeared in the interstitium at the 17th gestational week and persisted in the postnatal age, and that the Isl-1 positive cells were a subset of the c-Kit positive population.

Development of rapid phenotypic system for the identification of Gram-negative oxidase-positive bacilli in resource-limited settings.

Science.gov (United States)

Kazmi, Mahmooda; Khan, Adnan; Kazmi, Shahana Urooj

2013-06-01

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology. The diagnosis and surveillance of diseases is dependent, to a great extent, on laboratory services, which cannot function without effective reliable reagents and diagnostics. Despite the advancement in microbiology diagnosis globally, resourcelimited countries still struggle to provide an acceptable diagnosis quality which helps in clinical disease management and improve their mortality and morbidity data. During this study an indigenous product, Quick Test Strip (QTS) NE, was developed for the rapid identification of biochemically slower group of Gram-negative oxidase-positive bacilli that covers 19 different bacterial genera. Some of the members belonging to these groups are well-established human pathogens, e.g. various species of Vibrio, Pseudomonas, Burkholderia, Aeromonas, Achromobacter and Stenotrophomonas. This study also evaluates the performance of QTS-NE by comparing with genotypic characterization methods. A total of 232 clinical and reference bacterial isolates were tested by three different methods. QTSNE provides 100 percent concordant results with other rapid identification and molecular characterization methods and confirms the potential to be used in clinical diagnosis.

Relative uptake of technetium 99m stannous colloid by neutrophils and monocytes is altered by gram-negative infection

International Nuclear Information System (INIS)

Ramsay, Stuart C.; Maggs, Jacqueline A.; Ketheesan, Natkunam; Norton, Robert; LaBrooy, Justin

2005-01-01

Gram-negative infection alters phagocytic cell function; hence, it could affect phagocytic uptake of inorganic colloids by these cells. Neutrophil and monocyte uptake of technetium 99m stannous colloid ( 99m Tc SnC) in whole blood was measured in 10 patients with gram-negative infection (Burkholderia pseudomallei) and 7 controls. Mean uptake per individual neutrophil was reduced in infection. Uptake per monocyte was not significantly different. Blood from six normal individuals was incubated with lysed B. pseudomallei and colloid, which showed reduced neutrophil uptake, but increased monocyte uptake. These results indicate that uptake of 99m Tc SnC stannous colloid can be used to measure alteration in phagocytic cell function. They suggest that infection with B. pseudomallei is associated with reduced phagocytosis by individual neutrophils, possibly through toxic effects of bacterial products. This could have immunopathogenic consequences for this gram-negative infection and may explain why it responds to granulocyte colony-stimulating factor

Comparing Medline citations using modified N-grams

Science.gov (United States)

Nawab, Rao Muhammad Adeel; Stevenson, Mark; Clough, Paul

2014-01-01

Objective We aim to identify duplicate pairs of Medline citations, particularly when the documents are not identical but contain similar information. Materials and methods Duplicate pairs of citations are identified by comparing word n-grams in pairs of documents. N-grams are modified using two approaches which take account of the fact that the document may have been altered. These are: (1) deletion, an item in the n-gram is removed; and (2) substitution, an item in the n-gram is substituted with a similar term obtained from the Unified Medical Language System  Metathesaurus. N-grams are also weighted using a score derived from a language model. Evaluation is carried out using a set of 520 Medline citation pairs, including a set of 260 manually verified duplicate pairs obtained from the Deja Vu database. Results The approach accurately detects duplicate Medline document pairs with an F1 measure score of 0.99. Allowing for word deletions and substitution improves performance. The best results are obtained by combining scores for n-grams of length 1–5 words. Discussion Results show that the detection of duplicate Medline citations can be improved by modifying n-grams and that high performance can also be obtained using only unigrams (F1=0.959), particularly when allowing for substitutions of alternative phrases. PMID:23715801

Gram stain microbiological pattern of upper extremities suppuration at Baptist Medical Centre, Ogbomoso Nigeria: a fifteen month review.

Science.gov (United States)

Oke, A J; Olaolorun, D A; Meier, D E; Tarpley, J L

2011-06-01

Sixty-eight (68) patients with serious upper extremity suppurative infections, presenting within a period of fifteen (15) months, were prospectively studied clinically, Gram stain of aspirates/pus were performed, specimen cultured, planted, and where indicated glucose levels and haemoglobin genotype determined. Half of the patients had hand infections. Staphylococcus aureus was isolated from thirty-nine (39) patients. Gram Negative bacilli, including Salmonella were more isolated from patients with diabetes mellitus or Hgb SS or SC. The Gram stain results correlated with the culture result 90%. When Gram Positive cocci were demonstrated in the primary microscopic examination, cultures were not mandatory. When no organism was demonstrated on primary Gram stain or the patient was diabetic or a sickler, cultures of the specimens were done. The Gram stain, well performed, remains a useful, inexpensive, technologically appropriate laboratory test for abetting decision making in patients with upper extremity suppurative infections. Organisms encountered in this study included: Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhi, Proteus mirabilis, Pseudomonas aeruginosa, and Coliforms.

FOXP3 positive regulatory T-cells in cutaneous and systemic CD30 positive T-cell lymphoproliferations

DEFF Research Database (Denmark)

Gjerdrum, Lise Mette; Woetmann, Anders; Ødum, Niels

2008-01-01

for FOXP3 expression in tumour cells and tumour infiltrating Tregs. Labelling of a majority of the neoplastic cells was seen in one case of C-ALCL. Another three cases (one LyP and two C-ALCL) displayed weak labelling of very occasional atypical T-cells. In the remaining 38 cases the atypical lymphoid...... infiltrate was FOXP3 negative. By contrast, all biopsies contained tumour infiltrating FOXP3-positive Tregs. Significant higher numbers were recorded in ALK negative S-ALCL and LyP than in C-ALCL and S-ALCL positive for ALK. In conclusion, it is shown that FOXP3 expression in cutaneous and systemic CD30...

Electron Transport Chain Is Biochemically Linked to Pilus Assembly Required for Polymicrobial Interactions and Biofilm Formation in the Gram-Positive Actinobacterium Actinomyces oris

Directory of Open Access Journals (Sweden)

Belkys C. Sanchez

2017-06-01

Full Text Available The Gram-positive actinobacteria Actinomyces spp. are key colonizers in the development of oral biofilms due to the inherent ability of Actinomyces to adhere to receptor polysaccharides on the surface of oral streptococci and host cells. This receptor-dependent bacterial interaction, or coaggregation, requires a unique sortase-catalyzed pilus consisting of the pilus shaft FimA and the coaggregation factor CafA forming the pilus tip. While the essential role of the sortase machine SrtC2 in pilus assembly, biofilm formation, and coaggregation has been established, little is known about trans-acting factors contributing to these processes. We report here a large-scale Tn5 transposon screen for mutants defective in Actinomyces oris coaggregation with Streptococcus oralis. We obtained 33 independent clones, 13 of which completely failed to aggregate with S. oralis, and the remainder of which exhibited a range of phenotypes from severely to weakly defective coaggregation. The former had Tn5 insertions in fimA, cafA, or srtC2, as expected; the latter were mapped to genes coding for uncharacterized proteins and various nuo genes encoding the NADH dehydrogenase subunits. Electron microscopy and biochemical analyses of mutants with nonpolar deletions of nuo genes and ubiE, a menaquinone C-methyltransferase-encoding gene downstream of the nuo locus, confirmed the pilus and coaggregation defects. Both nuoA and ubiE mutants were defective in oxidation of MdbA, the major oxidoreductase required for oxidative folding of pilus proteins. Furthermore, supplementation of the ubiE mutant with exogenous menaquinone-4 rescued the cell growth and pilus defects. Altogether, we propose that the A. oris electron transport chain is biochemically linked to pilus assembly via oxidative protein folding.

TmCactin plays an important role in Gram-negative and -positive bacterial infection by regulating expression of 7 AMP genes in Tenebrio molitor

Science.gov (United States)

Jo, Yong Hun; Jung Kim, Yu; Beom Park, Ki; Hwan Seong, Jeong; Gon Kim, Soo; Park, Soyi; Young Noh, Mi; Seok Lee, Yong; Soo Han, Yeon

2017-01-01

Cactin was originally identified as an interactor of the Drosophila IκB factor Cactus and shown to play a role in controlling embryonic polarity and regulating the NF-κB signaling pathway. While subsequent studies have identified the roles for Cactin in the mammalian immune response, the immune function of Cactin in insects has not been described yet. Here, we identified a Cactin gene from the mealworm beetle, Tenebrio molitor (TmCactin) and characterized its functional role in innate immunity. TmCactin was highly expressed in prepupa to last instar stages, and its expression was high in the integument and Malpighian tubules of last instar larvae and adults. TmCactin was induced in larvae after infection with different pathogens and detectable within 3 hours of infection. The highest levels of TmCactin expression were detected at 9 hours post infection. TmCactin RNAi significantly decreased the survival rates of larvae after challenge with Escherichia coli and Staphylococcus aureus, but had no significant effect after challenge with Candida albicans. Furthermore, TmCactin RNAi significantly reduced the expression of seven antimicrobial peptide genes (AMPs) after bacterial challenge. Our results suggest that TmCactin may serve as an important regulator of innate immunity, mediating AMP responses against both Gram-positive and Gram-negative bacteria in T. molitor. PMID:28418029

Hyperspectral microscope imaging methods to classify gram-positive and gram-negative foodborne pathogenic bacteria

Science.gov (United States)

An acousto-optic tunable filter-based hyperspectral microscope imaging method has potential for identification of foodborne pathogenic bacteria from microcolony rapidly with a single cell level. We have successfully developed the method to acquire quality hyperspectral microscopic images from variou...

Classification of gram-positive and gram-negative foodborne pathogenic bacteria with hyperspectral microscope imaging

Science.gov (United States)

Optical method with hyperspectral microscope imaging (HMI) has potential for identification of foodborne pathogenic bacteria from microcolonies rapidly with a cell level. A HMI system that provides both spatial and spectral information could be an effective tool for analyzing spectral characteristic...

Inhibitory effect of beta-pinene, alpha-pinene and eugenol on the growth of potential infectious endocarditis causing Gram-positive bacteria Efeito inibitório de eugenol, beta-pineno e alfa-pineno sobre o crescimento de bactérias Gram-positivas potencialmente causadoras de endocardite infecciosa

Directory of Open Access Journals (Sweden)

Aristides Medeiros Leite

2007-03-01

Full Text Available This study was led with the purpose of evaluating the effectiveness of eugenol, beta-pinene and alpha-pinene in inhibiting the growth of potential infectious endocarditis causing gram-positive bacteria. The phytochemicals Minimum Inhibitory Concentration-MIC was determined by solid medium diffusion procedure, while the interference of the MIC values on the bacterial cell viability was performed by viable cells count. Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae and S. pyogenes strains were used as test microorganisms. The assayed phytochemicals showed effectiveness in inhibiting all assayed bacteria strains presenting MIC values between 2.5 and 40 µL/mL. Eugenol showed the lowest MIC values which were between 2.5 and 5 µL/mL for the most bacteria strains. MIC values found to the phytochemicals were able to inhibit the cell viability of S. aureus providing a total elimination of the bacteria inoculum in a maximum time of 24 hours of exposure. These data showed the interesting antibacterial property of the assayed phytochemicals and support their possible and rational use in the antimicrobial therapy.Este estudo foi conduzido com a proposta de avaliar a efetividade de eugenol, beta-pineno e alfa-pineno em inibir o crescimento de cepas de bactérias Gram-positivas potencialmente causadoras de endocardite infecciosa. A Concentração Inibitória Mínima-CIM dos fitoconstituintes foi determinada através do método de difusão em meio sólido, enquanto a interferência da CIM sobre a viabilidade celular bacteriana foi avaliada através da contagem de células viáveis. Cepas de Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae e S. pyogenes foram utilizadas como microrganismos teste nos ensaios antimicrobianos. Os fitoconstituintes ensaiados mostraram efetividade em inibir todas as cepas bacterianas utilizadas como microrganismos testes apresentando valores de CIM entre 2.5 e 40 µL/mL. Eugenol apresentou os menores

Combinatorial events of insertion sequences and ICE in Gram-negative bacteria.

Science.gov (United States)

Toleman, Mark A; Walsh, Timothy R

2011-09-01

The emergence of antibiotic and antimicrobial resistance in Gram-negative bacteria is incremental and linked to genetic elements that function in a so-called 'one-ended transposition' manner, including ISEcp1, ISCR elements and Tn3-like transposons. The power of these elements lies in their inability to consistently recognize one of their own terminal sequences, while recognizing more genetically distant surrogate sequences. This has the effect of mobilizing the DNA sequence found adjacent to their initial location. In general, resistance in Gram-negatives is closely linked to a few one-off events. These include the capture of the class 1 integron by a Tn5090-like transposon; the formation of the 3' conserved segment (3'-CS); and the fusion of the ISCR1 element to the 3'-CS. The structures formed by these rare events have been massively amplified and disseminated in Gram-negative bacteria, but hitherto, are rarely found in Gram-positives. Such events dominate current resistance gene acquisition and are instrumental in the construction of large resistance gene islands on chromosomes and plasmids. Similar combinatorial events appear to have occurred between conjugative plasmids and phages constructing hybrid elements called integrative and conjugative elements or conjugative transposons. These elements are beginning to be closely linked to some of the more powerful resistance mechanisms such as the extended spectrum β-lactamases, metallo- and AmpC type β-lactamases. Antibiotic resistance in Gram-negative bacteria is dominated by unusual combinatorial mistakes of Insertion sequences and gene fusions which have been selected and amplified by antibiotic pressure enabling the formation of extended resistance islands. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Low-fat papadams from black gram-tapioca blends.

Science.gov (United States)

Annapure, U S; Michael, M; Singhal, R S; Kulkarni, P R

1997-07-01

Papadams, made from black gram (Phaseolus mungo) and largely manufactured on cottage scale are popular in the Indian dietary. Escalating prices of black gram coupled with abundant availability of tapioca flour at almost 1/8th the price of black gram prompted the study of papadam characteristics using blends of black gram and tapioca flour. Tapioca flour up to 25% substitution did not alter the sensory attributes. However, the expansibility of the product decreased on addition of tapioca flour; a fact which could be overcome by using carboxymethyl cellulose (CMC) at 3% level of black gram flour. This decreased the oil content to 19.7% and also increased the expansibility to 5.539%.

Dual beta-lactam therapy for serious Gram-negative infections: is it time to revisit?

Science.gov (United States)

Rahme, Christine; Butterfield, Jill M; Nicasio, Anthony M; Lodise, Thomas P

2014-12-01

We are rapidly approaching a crisis in antibiotic resistance, particularly among Gram-negative pathogens. This, coupled with the slow development of novel antimicrobial agents, underscores the exigency of redeploying existing antimicrobial agents in innovative ways. One therapeutic approach that was heavily studied in the 1980s but abandoned over time is dual beta-lactam therapy. This article reviews the evidence for combination beta-lactam therapy. Overall, in vitro, animal and clinical data are positive and suggest that beta-lactam combinations produce a synergistic effect against Gram-negative pathogens that rivals that of beta-lactam-aminoglycoside or beta-lactam-fluoroquinolone combination therapy. Although the precise mechanism of improved activity is not completely understood, it is likely attributable to an enhanced affinity to the diverse penicillin-binding proteins found among Gram negatives. The collective data indicate that dual beta-lactam therapy should be revisited for serious Gram-negative infections, especially in light of the near availability of potent beta-lactamase inhibitors, which neutralize the effect of problematic beta-lactamases. Copyright © 2014 Elsevier Inc. All rights reserved.

False-Negative Rate of Gram-Stain Microscopy for Diagnosis of Septic Arthritis: Suggestions for Improvement

Directory of Open Access Journals (Sweden)

Paul Stirling

2014-01-01

Full Text Available We quantify the false-negative diagnostic rate of septic arthritis using Gram-stain microscopy of synovial fluid and compare this to values reported in the peer-reviewed literature. We propose a method of improving the diagnostic value of Gram-stain microscopy using Lithium Heparin containers that prevent synovial fluid coagulation. Retrospective study of the Manchester Royal Infirmary microbiology database of patients undergoing synovial fluid Gram-stain and culture between December 2003 and March 2012 was undertaken. The initial cohort of 1896 synovial fluid analyses for suspected septic arthritis was reduced to 143 after exclusion criteria were applied. Analysis of our Gram-stain microscopy yielded 111 false-negative results from a cohort size of 143 positive synovial fluid cultures, giving a false-negative rate of 78%. We report a false-negative rate of Gram-stain microscopy for septic arthritis of 78%. Clinicians should therefore avoid the investigation until a statistically significant data set confirms its efficacy. The investigation's value could be improved by using Lithium Heparin containers to collect homogenous synovial fluid samples. Ongoing research aims to establish how much this could reduce the false-negative rate.

Diagnosis of common bacterial causes of urethritis in men by Gram stain, culture and multiplex PCR.

Science.gov (United States)

Jahan, F; Shamsuzzaman, S M; Akter, S

2014-12-01

Urethritis is one of the most important causes of morbidity and mortality in developing countries. The aim of this study was to detect common bacterial causes of urethritis in men by Gram stain, culture and multiplex PCR.185 male patients who presented at the Skin and venereal clinic of the Dhaka Medical College, Bangladesh with clinical symptoms suggestive of urethritis were enrolled in this study. Urethral discharges were tested for detection of Neisseria gonorrhoeae by Gram stain, culture and PCR. Multiplex PCR assay was done to detect DNA of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma genitalium. Out of 185 participants, 30.27% and 14.6% were infected by Neisseria gonorrhoeae and Chlamydia trachomatis respectively. None of the individuals was found positive for either Ureaplasma urealyticum or Mycoplasma genitalium. Among the Neisseria gonorrhoeae positive patients 27.57% were positive from Gram stain, 26.49% were culture positive, 30.27% were positive by PCR (p<0.001). 32.65% of the Neisseria gonorrhoeae isolates were penicillinase producers and 83.67% were susceptible to ceftriaxone. Considering culture as the gold standard, the sensitivity and specificity of PCR for the detection of Neisseria gonorrhoeae was 100%, and 94.85% respectively with an accuracy of 96.22%. 3.73% of the 134 smear negative and 5.15% of the 136 culture negative samples were positive by PCR. PCR was the most sensitive and rapid method for the diagnosis of urethritis. Multiplex PCR may be a useful approach to laboratory diagnosis of urethritis in men for its high sensitivity and specificity.

Benzyl isothiocyanate, a major component from the roots of Salvadora persica is highly active against Gram-negative bacteria.

Directory of Open Access Journals (Sweden)

Abier Sofrata

Full Text Available Plants produce a number of antimicrobial substances and the roots of the shrub Salvadora persica have been demonstrated to possess antimicrobial activity. Sticks from the roots of S. persica, Miswak sticks, have been used for centuries as a traditional method of cleaning teeth. Diverging reports on the chemical nature and antimicrobial repertoire of the chewing sticks from S. persica led us to explore its antibacterial properties against a panel of pathogenic or commensal bacteria and to identify the antibacterial component/s by methodical chemical characterization. S. persica root essential oil was prepared by steam distillation and solid-phase microextraction was used to sample volatiles released from fresh root. The active compound was identified by gas chromatography-mass spectrometry and antibacterial assays. The antibacterial compound was isolated using medium-pressure liquid chromatography. Transmission electron microscopy was used to visualize the effect on bacterial cells. The main antibacterial component of both S. persica root extracts and volatiles was benzyl isothiocyanate. Root extracts as well as commercial synthetic benzyl isothiocyanate exhibited rapid and strong bactericidal effect against oral pathogens involved in periodontal disease as well as against other Gram-negative bacteria, while Gram-positive bacteria mainly displayed growth inhibition or remained unaffected. The short exposure needed to obtain bactericidal effect implies that the chewing sticks and the essential oil may have a specific role in treatment of periodontal disease in reducing Gram-negative periodontal pathogens. Our results indicate the need for further investigation into the mechanism of the specific killing of Gram-negative bacteria by S. persica root stick extracts and its active component benzyl isothiocyanate.

Anti-ATLA (antibody to adult T-cell leukemia virus-associated antigen), highly positive in OKT4-positive mature T-cell malignancies.

Science.gov (United States)

Tobinai, K; Nagai, M; Setoya, T; Shibata, T; Minato, K; Shimoyama, M

1983-01-01

Serum or plasma specimens from 252 patients with lymphoid malignancies were screened for reactivity with adult T-cell leukemia virus-associated antigen (ATLA), and the relationship between the immunologic phenotype of the tumor cells and ATLA reactivity was determined. Anti-ATLA antibodies were found in 24 (29.3%) of 82 patients with T-cell malignancy. In contrast, the antibodies were found in none of the 106 patients with B-cell malignancy and only rarely in patients with other lymphoid malignancies without blood transfusions. Among the patients with T-cell malignancy, anti-ATLA antibodies were found in 23 (45.1%) of the 51 patients with OKT4-positive mature T-cell (inducer/helper T-cell) malignancy, but in none of the patients with T-cell malignancy of pre-T, thymic T-cell or OKT8-positive mature T-cell (suppressor/cytotoxic T-cell) phenotype. Furthermore, among the OKT4-positive mature T-cell malignancies, the antibodies were found in 16 (84.2%) of 19 patients with ATL and in 5 (27.8%) of 18 patients with mature (peripheral) T-cell lymphoma, in none of four with typical T-chronic lymphocytic leukemia, in one of nine with mycosis fungoides and in the one patient with small-cell variant of Sézary's syndrome. These results suggest that anti-ATLA positive T-cell malignancies with OKT4-positive mature T-cell phenotype must be the same disease, because it is highly possible that they have the same etiology and the same cellular origin. In the atypical cases, it seems necessary to demonstrate monoclonal integration of proviral DNA of ATLV or HTLV into the tumor cells in order to establish the final diagnosis of ATL.

Risk factors for carbapenem resistant bacteraemia and mortality due to gram negative bacteraemia in a developing country

International Nuclear Information System (INIS)

Kalam, K.; Kumar, S.; Ali, S.; Baqi, S.; Qamar, F.

2014-01-01

Objective: To identify the risk factors for carbapenem resistant bacteraemia and mortality due to gram negative bacteraemia in a developing country. Methods: A prospective cohort study was conducted at the Sindh Institute of Urology and Transplantation (SIUT) from June to October 2012. Hospitalized patients > 15 years of age with gram negative bacteraemia were included and followed for a period of 2 weeks for in hospital mortality. Data was collected and analyzed for 243 subjects. Multivariate analysis was used to determine the risk factors for carbapenem resistant bacteraemia and mortality due to gram negative bacteraemia. Crude and adjusted odds ratio and 95% CI are reported. Results: A total of 729 out of 1535 (47.5%) cultures were positive for gram negative isolates. Out of 243 subjects, 117 (48%) had an MDR isolate. Having an MDR isolate on culture (AOR, 2.33; 95% CI, 1.35 -4.0), having multiple positive cultures (AOR, 1.8; 95% CI, 0.94 -3.4) and stay in ICU >48 hours (AOR, 2.0 ; 95% CI, 1.12 -3.78) were identified as significant risk factors for mortality due to gram negative organisms. Risk factors for carbapenem resistant bacteraemia were age >50 years (AOR, 1.83; 95% CI, 1.0-3.5), septic shock on presentation (AOR 2.53; 95% CI, 1.03 -6.2) , ICU stay of >72 hours (AOR 2.40; 95% CI, 1.14-5.0) and receiving immunosuppressant medications (AOR 2.23; 95% CI, 0.74 - 6.7). Conclusion: There is a high burden of MDR and carbapenem resistant gram negative bacteraemia, with a high mortality rate. (author)

Dendritic Cells from Peyer's Patches and Mesenteric Lymph Nodes Differ from Spleen Dendritic Cells in their Response to Commensal Gut Bacteria

DEFF Research Database (Denmark)

Fink, Lisbeth Nielsen; Frøkiær, Hanne

2008-01-01

. Expression of CCR7 and CD103 on the surface of MLN DC, necessary for the induction of gut-homing regulatory T cells, increased with stimulation by Gram-positive commensals. Bacteria-dependent cytokine production (IL-6, IL-10 and TNF-alpha) was similar in spleen and MLN DC, and contaminant cells in these DC...

[Post-marketing surveillance of antibacterial activities of cefozopran against various clinical isolates--I. Gram-positive bacteria].

Science.gov (United States)

Igari, Jun; Oguri, Toyoko; Hiramatsu, Nobuyoshi; Akiyama, Kazumitsu; Koyama, Tsuneo

2003-10-01

As a post-marketing surveillance, the in vitro antibacterial activities of cefozopran (CZOP), an agent of cephems, against various clinical isolates were yearly evaluated and compared with those of other cephems, oxacephems, penicillins, and carbapenems. Changes in the bacterial susceptibility for CZOP were also evaluated with the resistance ratio calculated with breakpoint MIC. Sixteen species (2,363 strains) of Gram-positive bacteria were isolated from the clinical materials annually collected from 1996 to 2001, and consisted of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus epidermidis (MSSE), methicillin-resistant Staphylococcus epidermidis (MRSE), Staphylococcus haemolyticus, Staphylococcus saprophyticus, Enterococcus faecalis, Enterococcus faecium, Enterococcus avium, Streptococcus pyogenes, Streptococcus agalactiae, penicillin-susceptible Streptococcus pneumoniae (PSSP), penicillin-intermediate resistant S. pneumoniae (PISP), penicillin-resistant S. pneumoniae (PRSP), Streptococcus milleri group and Peptostreptococcus spp. The antibacterial activity of CZOP either against MSSA or MSSE was preferable (MIC90: 2 or 0.5 micrograms/mL) and comparable to those of other cephems. CZOP was also effective on MRSE (MIC90: 16 micrograms/mL) but not on MRSA. CZOP and other cephems had low antibacterial activity against S. haemolyticus (MIC90: 64 micrograms/mL). The antibacterial activity of CZOP against S. saprophyticus was comparable to or higher than those of other cephems, but the MIC90 of CZOP in 2001 was higher than those in 1996-2000 (32 vs 1-2 micrograms/mL). The antibacterial activity of CZOP against E. faecalis was comparable to that of cefpirome (CPR; MIC90: 16 micrograms/mL) and higher than those of other cephems. No antibacterial activity of CZOP against E. faecium and E. avium was observed, like other drugs. The antibacterial activity of CZOP against S. pyogenes

Atividade antimicrobiana in vitro de quinupristina/dalfopristina para cocos gram-positivos isolados de cinco centros brasileiros: resultado do estudo de vigilância L-SMART Antimicrobial in vitro activity of quinupristin/dalfopristin against gram-positive cocci isolated from 5 Brazilian centers: results from the local smart (L-SMART surveillance study

Directory of Open Access Journals (Sweden)

Caio Mendes

2002-07-01

/ml, sendo que 45% das cepas testadas foram sensíveis e 55% apresentaram sensibilidade intermediária à associação. Desta forma, pode-se afirmar que a associação Q/D representa uma nova opção para o tratamento endovenoso de infecções causadas por cocos gram-positivos, principalmente para as cepas multirresistentes, sendo também uma alternativa ao uso de glicopeptídeos.A progressive increase of resistance among Gram-positive cocci (GPC towards some antimicrobial agents has been observed for the past few years. This rise of resistance, most often seen in Staphylococcus spp., and Enterococcus spp., has mainly been noticed in hospital environments. Due to these recent patterns of resistance, newly developed antimicrobial agents need to be evaluated for the treatment of infections caused by these multi-resistant microorganisms. Quinupristin/dalfopristin (Q/D, also known as Synercid®, is an antimicrobial agent of intravenous administration, composed of two semi-synthetic derivatives of pristinomycin belonging to the group of streptogramins. The combination of streptogramins B and A at 3:7 ratio has an antimicrobial activity against gram-positive cocci. This combination has potent activity against gram-positive cocci such as Staphylococcus spp., Streptococcus spp. including Streptococcus pneumoniae, and Enterococcus faecium. However, strains of E. faecalis are usually resistant to this compound. The aim of this study was to evaluate the in vitro activity of Q/D and other eight antibiotics against 631 strains of GPC isolated from five Brazilian centers. Additionally, 20 vancomycin-resistant strains of E. faecium provided by a reference center from the United States were also included in this study. Minimal Inhibitory Concentrations (MICs were determined by E-test methodology (AB Biodisk, Solna, Sweden, using standardized and controlled procedures. The evaluated strains were as follows: Staphylococcus aureus (267, coagulase negative Staphylococcus (131, Streptococcus

Diesel degradation and biosurfactant production by Gram-positive ...

African Journals Online (AJOL)

PRECIOUS

2009-11-02

Nov 2, 2009 ... Full Length Research Paper ... Diesel degradation rates and microbial cell number, increased with an increase in glucose ... that are less or non-toxic and represents one of the ... organic compounds (Larkin et al., 2005).

Usefulness of urinal urinalysis and urine gram stain in the diagnosis of rinary tract infection in hospitalized patients Utilidad del citoquímico y la coloración de Gram en muestras de orina en el diagnóstico de las infecciones del tracto urinario en pacientes hospitalizados.

Directory of Open Access Journals (Sweden)

Sergio Jaramillo Velásquez

2005-04-01

Full Text Available Objective: To evaluate the usefulness of urinalysis and of urine Gram stain in the diagnosis of urinary tract infection in hospitalized patients. Design and sample: Urinalysis and Gram stain of non centrifuged urine were carried out according to the requests of physicians, in specimens submitted to the laboratory for urine culture. When this modality of Gram stain had not been requested, it was done with urine sediment. Results were compared with those of the culture. Results: Ten thousand nine hundred and seventeen urine cultures were carried out, as well as 6.762 urinalyses, 2.762 Gram stains of non centrifuged urine, and 8.008 Gram stains of urine sediments. In comparison with the urine culture, urinalyses had 99% sensitivity, 51% specificity, 60% positive predictive value, and 98% negative predictive value. Gram stain of non centrifuged urine had 98% sensitivity, 73% specificity, 82% positive predictive value, and 97% negative predictive value. Gram stain of urine sediment had 95% sensitivity, 78% specificity, 67% positive predictive value, and 97% negative predictive value. Conclusions: Urinalysis, following well defined criteria, and Gram stain have high negative predictive values, that may allow to use them as screening tests to determine the need to request urine culture. Objetivo: evaluar la utilidad del citoquímico de orina y la coloración de Gram en el diagnóstico de la infección del tracto urinario en pacientes hospitalizados. Diseño y muestra : estudio de corte transversal; 10.917 urocultivos. Métodos: se hicieron el citoquímico de orina y la coloración de Gram de la orina sin centrifugar según la solicitud de los médicos tratantes. Cuando no se solicitó este último examen, la coloración de Gram se hizo del sedimento urinario. Se compararon los resultados obtenidos con el resultado del urocultivo. Resultados: se obtuvo el resultado del citoquímico de orina en 6.762 de las muestras. Se solicitaron 2.762 coloraciones de

Infective Endocarditis: Identification of Catalase-Negative, Gram-Positive Cocci from Blood Cultures by Partial 16S rRNA Gene Analysis and by Vitek 2 Examination.

Science.gov (United States)

Abdul-Redha, Rawaa Jalil; Kemp, Michael; Bangsborg, Jette M; Arpi, Magnus; Christensen, Jens Jørgen

2010-01-01

Streptococci, enterococci and Streptococcus-like bacteria are frequent etiologic agents of infective endocarditis and correct species identification can be a laboratory challenge. Viridans streptococci (VS) not seldomly cause contamination of blood cultures. Vitek 2 and partial sequencing of the 16S rRNA gene were applied in order to compare the results of both methods. STRAINS ORIGINATED FROM TWO GROUPS OF PATIENTS: 149 strains from patients with infective endocarditis and 181 strains assessed as blood culture contaminants. Of the 330 strains, based on partial 16S rRNA gene sequencing results, 251 (76%) were VS strains, 10 (3%) were pyogenic streptococcal strains, 54 (16%) were E. faecalis strains and 15 (5%) strains belonged to a group of miscellaneous catalase-negative, Gram-positive cocci. Among VS strains, respectively, 220 (87,6%) and 31 (12,3%) obtained agreeing and non-agreeing identifications with the two methods with respect to allocation to the same VS group. Non-agreeing species identification mostly occurred among strains in the contaminant group, while for endocarditis strains notably fewer disagreeing results were observed.Only 67 of 150 strains in the mitis group strains obtained identical species identifications by the two methods. Most VS strains belonging to the groups of salivarius, anginosus, and mutans obtained agreeing species identifications with the two methods, while this only was the case for 13 of the 21 bovis strains. Pyogenic strains (n=10), Enterococcus faecalis strains (n=54) and a miscellaneous group of catalase-negative, Gram-positive cocci (n=15) seemed well identified by both methods, except that disagreements in identifications in the miscellaneous group of strains occurred for 6 of 15 strains.

Doripenem: position in clinical practice.

Science.gov (United States)

Dedhia, Harakh V; McKnight, Richard

2009-06-01

Doripenem is a novel carbapenem with a broad spectrum of activity against Gram-positive pathogens, anerobes and Gram-negative bacteria, including Pseudomonas aeruginosa. Doripenem exhibits rapid bactericidal activity with two- to fourfold lower MIC values for Gram-negative bacteria, compared with other carbapenems such as imipenem. Doripenem is approved for the treatment of complicated intra-abdominal infection and urinary tract infections. It has been successfully used in the treatment of nosocomial and ventilator-associated pneumonia. It has a potential to be the drug of choice for these conditions. This evaluation focuses on the general review of the drug, including mechanisms of resistance, clinical efficacy and the position of doripenem in clinical practice. Stability against numerous beta-lactamases, low adverse-event potential and more potent in vitro and possibly in vivo activity against P. aeruginosa and Acinetobacter baumanni compared with existing carbapenems are attractive features.

Gram-negative bacteria account for main differences between faecal microbiota from patients with ulcerative colitis and healthy controls

DEFF Research Database (Denmark)

Vigsnæs, Louise Kristine; Brynskov, J.; Steenholdt, C.

2012-01-01

process of the gut mucosa. The aim of this study was to investigate the faecal microbiota in patients either with UC in remission (n=6) or with active disease (n=6), and in healthy controls (n=6). The composition of Gram-negative bacteria and Gram-positive bacteria was examined. Antigenic structures...... of Gram-negative bacteria such as lipopolysaccharides have been related to the inflammatory responses and pathogenesis of inflammatory bowel disease. Dice cluster analysis and principal component analysis of faecal microbiota profiles obtained by denaturing gradient gel electrophoresis and quantitative...... PCR, respectively, revealed that the composition of faecal bacteria from UC patients with active disease differed from the healthy controls and that this difference should be ascribed to Gram-negative bacteria. The analysis did not show any clear grouping of UC patients in remission. Even...

Distribution of CD163-positive cell and MHC class II-positive cell in the normal equine uveal tract.

Science.gov (United States)

Sano, Yuto; Matsuda, Kazuya; Okamoto, Minoru; Takehana, Kazushige; Hirayama, Kazuko; Taniyama, Hiroyuki

2016-02-01

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

CD44-positive cells are candidates for astrocyte precursor cells in developing mouse cerebellum.

Science.gov (United States)

Cai, Na; Kurachi, Masashi; Shibasaki, Koji; Okano-Uchida, Takayuki; Ishizaki, Yasuki

2012-03-01

Neural stem cells are generally considered to be committed to becoming precursor cells before terminally differentiating into either neurons or glial cells during neural development. Neuronal and oligodendrocyte precursor cells have been identified in several areas in the murine central nervous system. The presence of astrocyte precursor cells (APCs) is not so well understood. The present study provides several lines of evidence that CD44-positive cells are APCs in the early postnatal mouse cerebellum. In developing mouse cerebellum, CD44-positive cells, mostly located in the white matter, were positive for the markers of the astrocyte lineage, but negative for the markers of mature astrocytes. CD44-positive cells were purified from postnatal cerebellum by fluorescence-activated cell sorting and characterized in vitro. In the absence of any signaling molecule, many cells died by apoptosis. The surviving cells gradually expressed glial fibrillary acidic protein, a marker for mature astrocytes, indicating that differentiation into mature astrocytes is the default program for these cells. The cells produced no neurospheres nor neurons nor oligodendrocytes under any condition examined, indicating these cells are not neural stem cells. Leukemia inhibitory factor greatly promoted astrocytic differentiation of CD44-positive cells, whereas bone morphogenetic protein 4 (BMP4) did not. Fibroblast growth factor-2 was a potent mitogen for these cells, but was insufficient for survival. BMP4 inhibited activation of caspase-3 and greatly promoted survival, suggesting a novel role for BMP4 in the control of development of astrocytes in cerebellum. We isolated and characterized only CD44 strongly positive large cells and discarded small and/or CD44 weakly positive cells in this study. Further studies are necessary to characterize these cells to help determine whether CD44 is a selective and specific marker for APCs in the developing mouse cerebellum. In conclusion, we succeeded in

Antibiotic-Resistant Gram Negative Bacilli in Meals Delivered at a General Hospital, Italy

Directory of Open Access Journals (Sweden)

Maria Rosa Anna Plano

2009-01-01

Full Text Available This study aimed at detecting the presence of antibiotic-resistant Gram-negatives in samples of meals delivered at the University General Hospital of Palermo, Italy. Antibiotic resistant Gram negatives were isolated in July—September 2007 ffrom cold dishes and food contact surfaces and utensils. Bacterial strains were submitted to susceptibility test and subtyped by random amplification of polymorphic DNA (RAPD. Forty-six of 55 (83.6% food samples and 14 of 17 (82.3% environmental swabs were culture positive for Gram negative bacilli resistant to at least one group of antibacterial drugs. A total of 134 antibiotic resistant strains, 51 fermenters and 83 non-fermenters, were recovered. Fermenters and non-fermenters showed frequencies as high as 97.8% of resistance to two or more groups of antibiotics and non fermenters were 28.9% resistant to more than three groups. Molecular typing detected 34 different profiles among the fermenters and 68 among the non-fermenters. Antibiotic resistance was very common among both fermenters and non-fermenters. However, the wide heterogeneity of RAPD patterns seems to support a prominent role of cross-contamination rather than a clonal expansion of a few resistant isolates. A contribution of commensal Gram negatives colonizing foods to a common bacterial resistance pool should not been overlooked.

Antibiotic-resistant gram negative bacilli in meals delivered at a general hospital, Italy.

Science.gov (United States)

Plano, Maria Rosa Anna; Di Noto, Anna Maria; Firenze, Alberto; Sciortino, Sonia; Mammina, Caterina

2009-01-01

This study aimed at detecting the presence of antibiotic-resistant Gram-negatives in samples of meals delivered at the University General Hospital of Palermo, Italy. Antibiotic resistant Gram negatives were isolated in July-September 2007 ffrom cold dishes and food contact surfaces and utensils. Bacterial strains were submitted to susceptibility test and subtyped by random amplification of polymorphic DNA (RAPD). Forty-six of 55 (83.6%) food samples and 14 of 17 (82.3%) environmental swabs were culture positive for Gram negative bacilli resistant to at least one group of antibacterial drugs. A total of 134 antibiotic resistant strains, 51 fermenters and 83 non-fermenters, were recovered. Fermenters and non-fermenters showed frequencies as high as 97.8% of resistance to two or more groups of antibiotics and non fermenters were 28.9% resistant to more than three groups. Molecular typing detected 34 different profiles among the fermenters and 68 among the non-fermenters. Antibiotic resistance was very common among both fermenters and non-fermenters. However, the wide heterogeneity of RAPD patterns seems to support a prominent role of cross-contamination rather than a clonal expansion of a few resistant isolates. A contribution of commensal Gram negatives colonizing foods to a common bacterial resistance pool should not been overlooked.

Suetônio, dos gramáticos

Directory of Open Access Journals (Sweden)

Marcos Martinho

2014-12-01

Full Text Available O Dos gramáticos de Suetônio divide-se em duas partes: na primeira, o autor narra a introdução, desenvolvimento, florescimento e declínio da gramática em Roma (1-4; na segunda, expõe a biografia de vinte professores de gramática, que ensinaram na Cidade entre o início do séc.  I a.C. e meados do século seguinte (5-24. Quanto à primeira parte, procuro mostrar que Suetônio adota conceitos fisicistas ou naturalistas para narrar a história da gramática em Roma, de modo que aquela se possa comparar à vida de um ser vivo. Assim, a exposição de Suetônio revela-se, na verdade, antes biográfica que historicista. Quanto à segunda parte, procuro mostrar que o Biógrafo se interessa por gramáticos que não só escreveram sobre sua arte no ócio, mas a ensinaram por ofício, isto é, por professores de gramática. Daí, seleciono e comento os elementos narrativos relacionados à docência, a saber: os métodos de ensino, o lugar de ensino, o número e qualidade dos alunos, os preços e honorários dos professores. Por fim, apresento tradução anotada do texto de Suetônio.

Streptomyces sporulation - Genes and regulators involved in bacterial cell differentiation

OpenAIRE

Larsson, Jessica

2010-01-01

Streptomycetes are Gram-positive bacteria with a complex developmental life cycle. They form spores on specialized cells called aerial hyphae, and this sporulation involves alterations in growth, morphogenesis and cell cycle processes like cell division and chromosome segregation. Understanding the developmental mechanisms that streptomycetes have evolved for regulating for example cell division is of general interest in bacterial cell biology. It can also be valuable in the design of new dru...

Gramática Hipertextual

Directory of Open Access Journals (Sweden)

Francilaine Munhoz Moraes

2011-07-01

Full Text Available No meio digital, a construção da notícia se dá pelo hipertexto. Uma linguagem própria ao suporte consolida-se nas duas últimas décadas. Este artigo se dedica a discutir regularidades linguísticas do discurso jornalístico na web. Se há uma linguagem peculiar ao meio, existe também uma gramática correspondente: a gramática hipertextual. Nesse viés, levantamos traços linguísticos da estrutura sintático-semântica, com base na organização da informação em páginas noticiosas eletrônicas. 

Protein classification using modified n-grams and skip-grams.

Science.gov (United States)

Islam, S M Ashiqul; Heil, Benjamin J; Kearney, Christopher Michel; Baker, Erich J

2018-05-01

Classification by supervised machine learning greatly facilitates the annotation of protein characteristics from their primary sequence. However, the feature generation step in this process requires detailed knowledge of attributes used to classify the proteins. Lack of this knowledge risks the selection of irrelevant features, resulting in a faulty model. In this study, we introduce a supervised protein classification method with a novel means of automating the work-intensive feature generation step via a Natural Language Processing (NLP)-dependent model, using a modified combination of n-grams and skip-grams (m-NGSG). A meta-comparison of cross-validation accuracy with twelve training datasets from nine different published studies demonstrates a consistent increase in accuracy of m-NGSG when compared to contemporary classification and feature generation models. We expect this model to accelerate the classification of proteins from primary sequence data and increase the accessibility of protein characteristic prediction to a broader range of scientists. m-NGSG is freely available at Bitbucket: https://bitbucket.org/sm_islam/mngsg/src. A web server is available at watson.ecs.baylor.edu/ngsg. erich_baker@baylor.edu. Supplementary data are available at Bioinformatics online.

Usefulness of gram staining of blood collected from total parenteral nutrition catheter for rapid diagnosis of catheter-related sepsis.

Science.gov (United States)

Moonens, F; el Alami, S; Van Gossum, A; Struelens, M J; Serruys, E

1994-01-01

The accuracy of Gram staining of blood drawn from catheters used to administer total parenteral nutrition was compared with paired quantitative blood cultures for the diagnosis of catheter-related sepsis. Gram staining was positive in 11 of 18 episodes of catheter-related sepsis documented by quantitative culture (sensitivity, 61%) but in none of the 5 episodes of fever unrelated to catheter infection. Thus, this procedure enabled the rapid presumptive diagnosis and guidance of antimicrobial therapy for total parenteral nutrition catheter sepsis, with a positive predictive value of 100% and a negative predictive value of 42%. PMID:7521359

Initial development and structure of biofilms on microbial fuel cell anodes

Directory of Open Access Journals (Sweden)

Keller Jürg

2010-04-01

Full Text Available Abstract Background Microbial fuel cells (MFCs rely on electrochemically active bacteria to capture the chemical energy contained in organics and convert it to electrical energy. Bacteria develop biofilms on the MFC electrodes, allowing considerable conversion capacity and opportunities for extracellular electron transfer (EET. The present knowledge on EET is centred around two Gram-negative models, i.e. Shewanella and Geobacter species, as it is believed that Gram-positives cannot perform EET by themselves as the Gram-negatives can. To understand how bacteria form biofilms within MFCs and how their development, structure and viability affects electron transfer, we performed pure and co-culture experiments. Results Biofilm viability was maintained highest nearer the anode during closed circuit operation (current flowing, in contrast to when the anode was in open circuit (soluble electron acceptor where viability was highest on top of the biofilm, furthest from the anode. Closed circuit anode Pseudomonas aeruginosa biofilms were considerably thinner compared to the open circuit anode (30 ± 3 μm and 42 ± 3 μm respectively, which is likely due to the higher energetic gain of soluble electron acceptors used. The two Gram-positive bacteria used only provided a fraction of current produced by the Gram-negative organisms. Power output of co-cultures Gram-positive Enterococcus faecium and either Gram-negative organisms, increased by 30-70% relative to the single cultures. Over time the co-culture biofilms segregated, in particular, Pseudomonas aeruginosa creating towers piercing through a thin, uniform layer of Enterococcus faecium. P. aeruginosa and E. faecium together generated a current of 1.8 ± 0.4 mA while alone they produced 0.9 ± 0.01 and 0.2 ± 0.05 mA respectively. Conclusion We postulate that this segregation may be an essential difference in strategy for electron transfer and substrate capture between the Gram-negative and the Gram-positive

Humidity-dependent bacterial cells functional morphometry investigations using atomic force microscope.

Science.gov (United States)

Nikiyan, Hike; Vasilchenko, Alexey; Deryabin, Dmitry

2010-01-01

The effect of a relative humidity (RH) in a range of 93-65% on morphological and elastic properties of Bacillus cereus and Escherichia coli cells was evaluated using atomic force microscopy. It is shown that gradual dehumidification of bacteria environment has no significant effect on cell dimensional features and considerably decreases them only at 65% RH. The increasing of the bacteria cell wall roughness and elasticity occurs at the same time. Observed changes indicate that morphological properties of B. cereus are rather stable in wide range of relative humidity, whereas E. coli are more sensitive to drying, significantly increasing roughness and stiffness parameters at RH cell wall structure of gram-positive and gram-negative bacterial cells.

Humidity-Dependent Bacterial Cells Functional Morphometry Investigations Using Atomic Force Microscope

Directory of Open Access Journals (Sweden)

Hike Nikiyan

2010-01-01

Full Text Available The effect of a relative humidity (RH in a range of 93–65% on morphological and elastic properties of Bacillus cereus and Escherichia coli cells was evaluated using atomic force microscopy. It is shown that gradual dehumidification of bacteria environment has no significant effect on cell dimensional features and considerably decreases them only at 65% RH. The increasing of the bacteria cell wall roughness and elasticity occurs at the same time. Observed changes indicate that morphological properties of B. cereus are rather stable in wide range of relative humidity, whereas E. coli are more sensitive to drying, significantly increasing roughness and stiffness parameters at RH ≤ 84% RH. It is discussed the dependence of the response features on differences in cell wall structure of gram-positive and gram-negative bacterial cells.

Septicemia caused by the gram-negative bacterium CDC IV c-2 in an immunocompromised human.

OpenAIRE

Dan, M; Berger, S A; Aderka, D; Levo, Y

1986-01-01

A 37-year-old man with plasma cell leukemia developed nonfatal septicemia caused by the gram-negative bacterium CDC IV c-2. Recovery followed appropriate treatment with antibiotics. The biochemical features of this organism are reviewed.

Detection of Extended Spectrum Beta-Lactamases Among Gram Negative Bacilli Recovered from Cattle Feces In Benin City, Nigeria

Directory of Open Access Journals (Sweden)

Helen Oroboghae OGEFERE

2017-06-01

Full Text Available This study was carried out to determine the prevalence of extended spectrum beta-lactamase (ESBL among Gram negative bacteria isolated from cattle feces in Benin City, Nigeria. A total of 250 Gram negative bacteria isolates were recovered from cattle feces and were processed microbiologically using standard techniques. Emergent colonies were identified and antibacterial susceptibility tests were determined using Kirby-Bauer disk diffusion method. All bacterial isolates were screened for the presence of ESBL using the double-disc synergy method. A total of 37 (14.8% isolates were positive for ESBL, with 33 (13.2% indicated by ceftazidime, while only 4 (1.6% were indicated by both ceftazidime and cefotaxime (P < 0.0001. Of the Gram negative bacterial isolates recovered, Salmonella species was the most prevalent ESBL-producer with 55.0% prevalence (P = 0.0092, while no isolate of Pseudomonas aeruginosa produced ESBL. ESBL-positive isolates showed poor susceptibility to the tested antibacterial agents in comparison with non-ESBL-producers and imipenem was the most active antibiotic. The prevalence of ESBL among Gram negative bacilli recovered from cattle feces was 14.8%. The study advises prudent use of antibiotics in the treatment of cattle and harps on improved hygiene in managing cattle, as they are potential reservoirs of ESBL-producing organisms.

Orbit Classification of Qutrit via the Gram Matrix

International Nuclear Information System (INIS)

Tay, B. A.; Zainuddin, Hishamuddin

2008-01-01

We classify the orbits generated by unitary transformation on the density matrices of the three-state quantum systems (qutrits) via the Gram matrix. The Gram matrix is a real symmetric matrix formed from the Hilbert–Schmidt scalar products of the vectors lying in the tangent space to the orbits. The rank of the Gram matrix determines the dimensions of the orbits, which fall into three classes for qutrits. (general)

Sequence-Based Characterization of Tn5801-Like Genomic Islands in Tetracycline-Resistant Staphylococcus pseudintermedius and Other Gram-positive Bacteria from Humans and Animals

DEFF Research Database (Denmark)

de Vries, Lisbeth Elvira; Hasman, Henrik; Jurado Rabadán, Sonia

2016-01-01

Antibiotic resistance in pathogens is often associated with mobile genetic elements, such as genomic islands (GI) including integrative and conjugative elements (ICEs). These can transfer resistance genes within and between bacteria from humans and/or animals. The aim of this study was to investi......Antibiotic resistance in pathogens is often associated with mobile genetic elements, such as genomic islands (GI) including integrative and conjugative elements (ICEs). These can transfer resistance genes within and between bacteria from humans and/or animals. The aim of this study......-like GIs appear to be relatively common in tetracycline-resistant S. pseudintermedius in Denmark. Almost identical Tn5801-like GIs were identified in different Gram-positive species of pet and human origin, suggesting that horizontal transfer of these elements has occurred between S. pseudintermedius...

Improved methods for binding acma-type protein anchor fusions yo cell-wall material of micro-organisms

NARCIS (Netherlands)

Leenhouts, Cornelis; Ramasamy, R.; Steen, Anton; Kok, Jan; Buist, Girbe; Kuipers, Oscar

2002-01-01

The invention provides a method for improving binding of a proteinaceous substance to cell-wall material of a Gram-positive bacterium, said substance comprising an AcmA cell wall binding domain or homolog or functional derivative thereof, said method comprising treating said cell-wall material with

Drug-resistant gram-negative uropathogens: A review.

Science.gov (United States)

Khoshnood, Saeed; Heidary, Mohsen; Mirnejad, Reza; Bahramian, Aghil; Sedighi, Mansour; Mirzaei, Habibollah

2017-10-01

Urinary tract infection(UTI) caused by Gram-negative bacteria is the second most common infectious presentation in community medical practice. Approximately 150 million people are diagnosed with UTI each year worldwide. Drug resistance in Gram-negative uropathogens is a major global concern which can lead to poor clinical outcomes including treatment failure, development of bacteremia, requirement for intravenous therapy, hospitalization, and extended length of hospital stay. The mechanisms of drug resistance in these bacteria are important due to they are often not identified by routine susceptibility tests and have an exceptional potential for outbreaks. Treatment of UTIs depends on the access to effective drugs, which is now threatened by antibiotic resistant Gram-negative uropathogens. Although several effective antibiotics with activity against highly resistant Gram-negatives are available, there is not a unique antibiotic with activity against the high variety of resistance. Therefore, antimicrobial susceptibility tests, correlation between clinicians and laboratories, development of more rapid diagnostic methods, and continuous monitoring of drug resistance are urgent priorities. In this review, we will discuss about the current global status of drug-resistant Gram-negative uropathogens and their mechanisms of drug resistance to provide new insights into their treatment options. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Isolation of dendritic-cell-like S100β-positive cells in rat anterior pituitary gland.

Science.gov (United States)

Horiguchi, Kotaro; Fujiwara, Ken; Yoshida, Saishu; Higuchi, Masashi; Tsukada, Takehiro; Kanno, Naoko; Yashiro, Takashi; Tateno, Kozue; Osako, Shunji; Kato, Takako; Kato, Yukio

2014-07-01

S100β-protein-positive cells in the anterior pituitary gland appear to possess multifunctional properties. Because of their pleiotropic features, S100β-positive cells are assumed to be of a heterogeneous or even a non-pituitary origin. The observation of various markers has allowed these cells to be classified into populations such as stem/progenitor cells, epithelial cells, astrocytes and dendritic cells. The isolation and characterization of each heterogeneous population is a prerequisite for clarifying the functional character and origin of the cells. We attempt to isolate two of the subpopulations of S100β-positive cells from the anterior lobe. First, from transgenic rats that express green fluorescent protein (GFP) driven by the S100β protein promoter, we fractionate GFP-positive cells with a cell sorter and culture them so that they can interact with laminin, a component of the extracellular matrix. We observe that one morphological type of GFP-positive cells possesses extended cytoplasmic processes and shows high adhesiveness to laminin (process type), whereas the other is round in shape and exhibits low adherence to laminin (round type). We successfully isolate cells of the round type from the cultured GFP-positive cells by taking advantage of their low affinity to laminin and then measure mRNA levels of the two cell types by real-time polymerase chain reaction. The resultant data show that the process type expresses vimentin (mesenchymal cell marker) and glial fibrillary acidic protein (astrocyte marker). The round type expresses dendritic cell markers, CD11b and interleukin-6. Thus, we found a method for isolating dendritic-cell-like S100β-positive cells by means of their property of adhering to laminin.

Rolling-circle plasmids from Bacillus subtilis : complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, pTA1050 and pTA1060, and comparisons with related plasmids from Gram-positive bacteria

NARCIS (Netherlands)

Meijer, W.J.; Schuurs-Wisman, Bea; Terpstra, P; Thorsted, P.; Thomas, C.M.; Holsappel, S; Venema, G; Bron, S

Most small plasmids of Gram-positive bacteria use the rolling-circle mechanism of replication and several of these have been studied in considerable detail at the DNA level and for the function of their genes. Although most of the common laboratory Bacillus subtilis 168 strains do not contain

Stool Gram stain

Science.gov (United States)

... stool sample. The Gram stain method is sometimes used to quickly diagnose bacterial infections. How the Test is Performed You will need to collect a stool sample. There are many ways to collect the sample. You can catch the stool on plastic wrap that is loosely placed over the toilet bowl ...

Staphylococcus aureus ?-Toxin-Dependent Induction of Host Cell Death by Membrane-Derived Vesicles

OpenAIRE

Thay, Bernard; Wai, Sun Nyunt; Oscarsson, Jan

2013-01-01

Staphylococcus aureus causes a wide spectrum of infections in humans, ranging from superficial cutaneous infections, infections in the circum-oral region, to life-threatening bacteremia. It was recently demonstrated that Gram-positive organisms such as S. aureus liberate membrane-derived vesicles (MVs), which analogously to outer membrane vesicles (OMVs) of Gram-negative bacteria can play a role in delivering virulence factors to host cells. In the present study we have shown that cholesterol...

Evaluation of cell lysis procedures and use of a micro fluidic system for an automated DNA-based cell identification in interplanetary missions

Science.gov (United States)

Hall, J. A.; Felnagle, E.; Fries, M.; Spearing, S.; Monaco, L.; Steele, A.

2006-12-01

A Modular Assay System for Solar System Exploration (MASSE) is being developed to include sample handling, pre-treatment, separation and analysis of biological target compounds by both DNA and protein microarrays. To better design sensitive and accurate initial upstream sample handling of the MASSE instrument, experiments investigating the sensitivity and potential extraction bias of commercially available DNA extraction kits between classes of environmentally relevant prokaryotes such as gram-negative bacteria ( Escherichia coli), gram-positive bacteria ( Bacillus megatarium), and Archaea ( Haloarcula marismortui) were performed. For extractions of both planktonic cultures and spiked Mars simulated regolith, FTA ® paper demonstrated the highest sensitivity, with detection as low as ˜1×10 1 cells and ˜3.3×10 2 cells, respectively. In addition to the highest sensitivity, custom modified application of FTA ® paper extraction protocol is the simplest in terms of incorporation into MASSE and displayed little bias in sensitivity with respect to prokaryotic cell type. The implementation of FTA paper for environmental microbiology investigations appears to be a viable and effective option potentially negating the need for other pre-concentration steps such as filtration and negating concerns regarding extraction efficiency of cells. In addition to investigations on useful technology for upstream sample handling in MASSE, we have also evaluated the potential for μTAS to be employed in the MASSE instrument by employing proprietary lab-on-a-chip development technology to investigate the potential for microfluidic cell lysis of different prokaryotic cells employing both chemical and biological lysis agents. Real-time bright-field microscopy and quantitative PMT detection indicated that that gram positive, gram negative and archaeal cells were effectively lyzed in a few seconds using the microfluidic chip protocol developed. This included employing a lysis buffer with

Utility of Gram staining for diagnosis of Malassezia folliculitis.

Science.gov (United States)

Tu, Wei-Ting; Chin, Szu-Ying; Chou, Chia-Lun; Hsu, Che-Yuan; Chen, Yu-Tsung; Liu, Donald; Lee, Woan-Ruoh; Shih, Yi-Hsien

2018-02-01

Malassezia folliculitis (MalF) mimics acne vulgaris and bacterial folliculitis in clinical presentations. The role of Gram staining in rapid diagnosis of MalF has not been well studied. In our study, 32 patients were included to investigate the utility of Gram staining for MalF diagnosis. The final diagnoses of MalF were determined according to clinical presentation, pathological result and treatment response to antifungal agents. Our results show that the sensitivity and specificity of Gram staining are 84.6% and 100%, respectively. In conclusion, Gram staining is a rapid, non-invasive, sensitive and specific method for MalF diagnosis. © 2017 Japanese Dermatological Association.

Trojan Horse Antibiotics-A Novel Way to Circumvent Gram-Negative Bacterial Resistance?

Science.gov (United States)

Tillotson, Glenn S

2016-01-01

Antibiotic resistance has been emerged as a major global health problem. In particular, gram-negative species pose a significant clinical challenge as bacteria develop or acquire more resistance mechanisms. Often, these bacteria possess multiple resistance mechanisms, thus nullifying most of the major classes of drugs. Novel approaches to this issue are urgently required. However, the challenges of developing new agents are immense. Introducing novel agents is fraught with hurdles, thus adapting known antibiotic classes by altering their chemical structure could be a way forward. A chemical addition to existing antibiotics known as a siderophore could be a solution to the gram-negative resistance issue. Siderophore molecules rely on the bacterial innate need for iron ions and thus can utilize a Trojan Horse approach to gain access to the bacterial cell. The current approaches to using this potential method are reviewed.

Antibacterial Activity of Extracellular Protease Isolated From an Algicolous Fungus Xylaria psidii KT30 Against Gram-Positive Bacteria

Directory of Open Access Journals (Sweden)

Taufik Indarmawan

2016-04-01

Full Text Available Infectious diseases became more serious problem for public health in recent years. Although existing antibacterial drugs have been relatively effective, they do not rule out the emergence of resistance to the drug. Therefore, the intensive exploration of new bioactive compounds from natural, especially peptide compounds began in recent decades in order-handling infection. This study aimed to isolate, purify and test the potential application of Xylaria psidii KT30 extracellular protease as antibacterial agent against Gram-positive bacteria. X. psidii KT30, a marine fungus isolated from red seaweed Kappaphycus alvarezii showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Antibacterial compounds of this fungus were predicted as a group of proteases. Extracellular protease exhibited an optimum activity when potato dextrose broth was used as cultivation medium. Furthermore, the highest activity of these proteases was found on fungal extract after day 15 of cultivation with value of 2.33 ± 0.19 U/mL. The partial purification of proteases using G-75 column chromatography resulted in 2 groups of fractions and showed protease activity based on zymogram assay. The extracellular proteases obtained from those fractions have 3 patterns of molecular mass based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis which are 56.62, 89.12, 162.18 kDa.

Clinical significance of coryneform Gram-positive rods from blood identified by MALDI-TOF mass spectrometry and their susceptibility profiles - a retrospective chart review.

Science.gov (United States)

Mushtaq, Ammara; Chen, Derrick J; Strand, Gregory J; Dylla, Brenda L; Cole, Nicolynn C; Mandrekar, Jayawant; Patel, Robin

2016-07-01

With the advent of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), most Gram-positive rods (GPRs) are readily identified; however, their clinical relevance in blood cultures remains unclear. Herein, we assessed the clinical significance of GPRs isolated from blood and identified in the era of MALDI-TOF MS. A retrospective chart review of patients presenting to the Mayo Clinic, Rochester, MN, from January 1, 2013, to October 13, 2015, was performed. Any episode of a positive blood culture for a GPR was included. We assessed the number of bottles positive for a given isolate, time to positivity of blood cultures, patient age, medical history, interpretation of culture results by the healthcare team and whether infectious diseases consultation was obtained. We also evaluated the susceptibility profiles of a larger collection of GPRs tested in the clinical microbiology laboratory of the Mayo Clinic, Rochester, MN from January 1, 2013, to October 31, 2015. There were a total of 246 GPRs isolated from the blood of 181 patients during the study period. 56% (n = 101) were deemed contaminants by the healthcare team and were not treated; 33% (n = 59) were clinically determined to represent true bacteremia and were treated; and 8% (n = 14) were considered of uncertain significance, with patients prescribed treatment regardless. Patient characteristics associated with an isolate being treated on univariate analysis included younger age (P = 0.02), identification to the species level (P = 0.02), higher number of positive blood culture sets (P < 0.0001), lower time to positivity (P < 0.0001), immunosuppression (P = 0.03), and recommendation made by an infectious disease consultant (P = 0.0005). On multivariable analysis, infectious diseases consultation (P = 0.03), higher number of positive blood culture sets (P = 0.0005) and lower time to positivity (P = 0.03) were associated with an isolate being treated. 100, 83, 48 and 34% of GPRs

O-antigen protects gram-negative bacteria from histone killing.

Directory of Open Access Journals (Sweden)

Catherine Chaput

Full Text Available Beyond their traditional role of wrapping DNA, histones display antibacterial activity to Gram-negative and -positive bacteria. To identify bacterial components that allow survival to a histone challenge, we selected resistant bacteria from homologous Escherichia coli libraries that harbor plasmids carrying pieces of the chromosome in different sizes. We identified genes required for exopolysaccharide production and for the synthesis of the polysaccharide domain of the lipopolysaccharide, called O-antigen. Indeed, O-antigen and exopolysaccharide conferred further resistance to histones. Notably, O-antigen also conferred resistance to histones in the pathogens Shigella flexneri and Klebsiella pneumoniae.

Evaluation of the automated system Vitek2 for identification and antimicrobial susceptibility testing of Brazilian Gram-positive cocci strains

Directory of Open Access Journals (Sweden)

Pedro Alves d'Azevedo

Full Text Available Automated instruments offer many advantages for clinical laboratories. Nevertheless, they can have problems identifying and determining susceptibilities of some pathogens. Vitek® 2 (bioMérieux is an automated system that was recently introduced to Brazil. We evaluated the performance of this equipment for Brazilian isolates that had been characterized using reference identification and antimicrobial susceptibility testing methods. Ninety-nine strains of Gram-positive cocci from a local reference center collection were analyzed, consisting of 50 coagulasenegative Staphylococcus (CoNS and 49 Enterococcus and related species. Vitek® 2 correctly identified 79.8% (79/99 of the isolates. Oxacillin resistance was detected in 76% (19/25 of resistant S. epidermidis strains and in 88% (22/25 of other resistant CoNS species strains. Vancomycin resistance was detected in 100% (20/20 of resistant Enterococcus and related species strains. Vitek® 2 performed very well for the identification of S. epidermidis and non-epidermidis staphylococci, and for the detection of vancomycin resistance in Enterococcus and related species. However, the system needs improvement in order to provide reliable results for the characterization of some CoNS species, identification of Enterococcus and related species and for detecting oxacillin resistance in CoNS.

FORMALDEHYDE DISMUTASE ACTIVITIES IN GRAM-POSITIVE BACTERIA OXIDIZING METHANOL

NARCIS (Netherlands)

BYSTRYKH, LV; GOVORUKHINA, NI; VANOPHEM, PW; HEKTOR, HJ; DIJKHUIZEN, L; DUINE, JA; Govorukhina, Natalya; Ophem, Peter W. van; Duine, Johannis A.

Extracts of methanol-grown cells of Amycolatopsis methanolica and Mycobacterium gastri oxidized methanol and ethanol with concomitant reduction of N,N'-dimethyl-4-nitrosoaniline (NDMA). Anion-exchange chromatography revealed the presence of a single enzyme able to catalyse this activity in methanol-

Revisiting the Gram-negative lipoprotein paradigm.

Science.gov (United States)

LoVullo, Eric D; Wright, Lori F; Isabella, Vincent; Huntley, Jason F; Pavelka, Martin S

2015-05-01

The processing of lipoproteins (Lpps) in Gram-negative bacteria is generally considered an essential pathway. Mature lipoproteins in these bacteria are triacylated, with the final fatty acid addition performed by Lnt, an apolipoprotein N-acyltransferase. The mature lipoproteins are then sorted by the Lol system, with most Lpps inserted into the outer membrane (OM). We demonstrate here that the lnt gene is not essential to the Gram-negative pathogen Francisella tularensis subsp. tularensis strain Schu or to the live vaccine strain LVS. An LVS Δlnt mutant has a small-colony phenotype on sucrose medium and increased susceptibility to globomycin and rifampin. We provide data indicating that the OM lipoprotein Tul4A (LpnA) is diacylated but that it, and its paralog Tul4B (LpnB), still sort to the OM in the Δlnt mutant. We present a model in which the Lol sorting pathway of Francisella has a modified ABC transporter system that is capable of recognizing and sorting both triacylated and diacylated lipoproteins, and we show that this modified system is present in many other Gram-negative bacteria. We examined this model using Neisseria gonorrhoeae, which has the same Lol architecture as that of Francisella, and found that the lnt gene is not essential in this organism. This work suggests that Gram-negative bacteria fall into two groups, one in which full lipoprotein processing is essential and one in which the final acylation step is not essential, potentially due to the ability of the Lol sorting pathway in these bacteria to sort immature apolipoproteins to the OM. This paper describes the novel finding that the final stage in lipoprotein processing (normally considered an essential process) is not required by Francisella tularensis or Neisseria gonorrhoeae. The paper provides a potential reason for this and shows that it may be widespread in other Gram-negative bacteria. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Investigating of the antimicrobial effect of total extract of Tribulus terrestris against some gram positive and negative bacteria and candida spp.

Directory of Open Access Journals (Sweden)

Mojdeh Hakemi Vala

2014-08-01

Full Text Available Introduction: In the recent years, due to the wide spread of resistant bacteria on one side and several different reports about the side effects of chemical drugs on the other side, vast researches on the medicinal plants have been started. In this study, antimicrobial effect of total extract of Tribulus terrestris L. and its fraction containing Benzoxazine derivative (Terresoxazine was studied for the first time in Iran.Materials and methods: Total aqueous extract of aerial parts of the plant was prepared and in order to separate the components of aqueous extract, liquid/liquid extraction with Petroleum ether was used. Formation of three layers was the result of this extraction. Layers included water fraction, Petroleum ether fraction and a third layer which was formed at the interface of water and petroleum ether. LC/MS system proved the existence of Benzixazine derivative in the water fraction and the thirds fraction. Antimicrobial effects of total extract, water fraction and the third fraction (which were the layers formed after the extraction process were examined against 10 Gram positive and negative and candida spp by cup plate method and Disk diffusion method. Also, the MIC and MBC were determined by micro dilution method.Results: Of 8 evaluated bacteria and 2 Candida spp, the total extract showed antibacterial effect only against E.coli, P.aeruginosa and B.subtilis. Size of the zone of inhibitation increased with increasing the concentration of the extract. Fraction containing Benzoxazine derivative had no effect against tested microbes. MIC and MBC determination showed that B.subtilis had the least sensitivity to the total extract, comparing to other microorganisms. Besides, comparing the zone of inhibitation of Penicillin 200 mg/ml and the zone of inhibitation of the total aqueous extract shows that the solution of total extract in water with 1000 mg/ml concentration and the solution of total extract in DMSO10% with 750 mg/ml density

PRODIGIOSIN INDUCES AUTOLYSINS IN ACTIVELY GROWN Bacillus subtilis CELLS

Directory of Open Access Journals (Sweden)

Tjasa eDanevcic

2016-01-01

Full Text Available Prodigiosin produced by marine bacterium Vibrio ruber DSM 14379 exhibits a potent antimicrobial activity against a broad range of Gram positive and Gram negative bacteria. The mechanism of prodigiosin antimicrobial action, however, is not known. In this work, the effect of prodigiosin on B. subtilis growth, cell membrane leakage, and induction of autolysins was studied. Treating B. subtilis with prodigiosin resulted in rapid decline of optical density and increased cell membrane leakage measured by β-galactosidase activity. Cell lysis was initiated immediately after treatment with prodigiosin in the middle exponential phase and was completed within two hours. Lytic activity of prodigiosin in mutant strains with impaired autolysin genes lytABCD decreased for 80 % compared to the wild-type strain, while in lytABCDEF mutant strain prodigiosin had no bacteriolytic but only bacteriostatic effect. Fast prodigiosin lytic activity on individual B. subtilis cells was confirmed by a modified comet assay. The results indicate that prodigiosin autolysin induction in B. subtilis is growth phase dependent.

Cell surface engineering of microorganisms towards adsorption of heavy metals.

Science.gov (United States)

Li, Peng-Song; Tao, Hu-Chun

2015-06-01

Heavy metal contamination has become a worldwide environmental concern due to its toxicity, non-degradability and food-chain bioaccumulation. Conventional physical and chemical treatment methods for heavy metal removal have disadvantages such as cost-intensiveness, incomplete removal, secondary pollution and the lack of metal specificity. Microbial biomass-based biosorption is one of the approaches gaining increasing attention because it is effective, cheap, and environmental friendly and can work well at low concentrations. To enhance the adsorption properties of microbial cells to heavy metal ions, the cell surface display of various metal-binding proteins/peptides have been performed using a cell surface engineering approach. The surface engineering of Gram-negative bacteria, Gram-positive bacteria and yeast towards the adsorption of heavy metals are reviewed in this article. The problems and future perspectives of this technology are discussed.

Alkali-treated titanium selectively regulating biological behaviors of bacteria, cancer cells and mesenchymal stem cells.

Science.gov (United States)

Li, Jinhua; Wang, Guifang; Wang, Donghui; Wu, Qianju; Jiang, Xinquan; Liu, Xuanyong

2014-12-15

Many attentions have been paid to the beneficial effect of alkali-treated titanium to bioactivity and osteogenic activity, but few to the other biological effect. In this work, hierarchical micro/nanopore films were prepared on titanium surface by acid etching and alkali treatment and their biological effects on bacteria, cancer cells and mesenchymal stem cells were investigated. Gram-positive Staphylococcus aureus, Gram-negative Escherichia coli, and human cholangiocarcinoma cell line RBE were used to investigate whether alkali-treated titanium can influence behaviors of bacteria and cancer cells. Responses of bone marrow mesenchymal stem cells (BMMSCs) to alkali-treated titanium were also subsequently investigated. The alkali-treated titanium can potently reduce bacterial adhesion, inhibit RBE and BMMSCs proliferation, while can better promote BMMSCs osteogenesis and angiogenesis than acid-etched titanium. The bacteriostatic ability of the alkali-treated titanium is proposed to result from the joint effect of micro/nanotopography and local pH increase at bacterium/material interface due to the hydrolysis of alkali (earth) metal titanate salts. The inhibitory action of cell proliferation is thought to be the effect of local pH increase at cell/material interface which causes the alkalosis of cells. This alkalosis model reported in this work will help to understand the biologic behaviors of various cells on alkali-treated titanium surface and design the intended biomedical applications. Copyright © 2014 Elsevier Inc. All rights reserved.

Effect of Laser Irradiation on Cell Function and Its Implications in Raman Spectroscopy.

Science.gov (United States)

Yuan, Xiaofei; Song, Yanqing; Song, Yizhi; Xu, Jiabao; Wu, Yinhu; Glidle, Andrew; Cusack, Maggie; Ijaz, Umer Z; Cooper, Jonathan M; Huang, Wei E; Yin, Huabing

2018-04-15

Lasers are instrumental in advanced bioimaging and Raman spectroscopy. However, they are also well known for their destructive effects on living organisms, leading to concerns about the adverse effects of laser technologies. To implement Raman spectroscopy for cell analysis and manipulation, such as Raman-activated cell sorting, it is crucial to identify nondestructive conditions for living cells. Here, we evaluated quantitatively the effect of 532-nm laser irradiation on bacterial cell fate and growth at the single-cell level. Using a purpose-built microfluidic platform, we were able to quantify the growth characteristics, i.e., specific growth rates and lag times of individual cells, as well as the survival rate of a population in conjunction with Raman spectroscopy. Representative Gram-negative and Gram-positive species show similar trends in response to a laser irradiation dose. Laser irradiation could compromise the physiological function of cells, and the degree of destruction is both dose and strain dependent, ranging from reduced cell growth to a complete loss of cell metabolic activity and finally to physical disintegration. Gram-positive bacterial cells are more susceptible than Gram-negative bacterial strains to irradiation-induced damage. By directly correlating Raman acquisition with single-cell growth characteristics, we provide evidence of nondestructive characteristics of Raman spectroscopy on individual bacterial cells. However, while strong Raman signals can be obtained without causing cell death, the variety of responses from different strains and from individual cells justifies careful evaluation of Raman acquisition conditions if cell viability is critical. IMPORTANCE In Raman spectroscopy, the use of powerful monochromatic light in laser-based systems facilitates the detection of inherently weak signals. This allows environmentally and clinically relevant microorganisms to be measured at the single-cell level. The significance of being able to

Performance of Gram staining on blood cultures flagged negative by an automated blood culture system.

Science.gov (United States)

Peretz, A; Isakovich, N; Pastukh, N; Koifman, A; Glyatman, T; Brodsky, D

2015-08-01

Blood is one of the most important specimens sent to a microbiology laboratory for culture. Most blood cultures are incubated for 5-7 days, except in cases where there is a suspicion of infection caused by microorganisms that proliferate slowly, or infections expressed by a small number of bacteria in the bloodstream. Therefore, at the end of incubation, misidentification of positive cultures and false-negative results are a real possibility. The aim of this work was to perform a confirmation by Gram staining of the lack of any microorganisms in blood cultures that were identified as negative by the BACTEC™ FX system at the end of incubation. All bottles defined as negative by the BACTEC FX system were Gram-stained using an automatic device and inoculated on solid growth media. In our work, 15 cultures that were defined as negative by the BACTEC FX system at the end of the incubation were found to contain microorganisms when Gram-stained. The main characteristic of most bacteria and fungi growing in the culture bottles that were defined as negative was slow growth. This finding raises a problematic issue concerning the need to perform Gram staining of all blood cultures, which could overload the routine laboratory work, especially laboratories serving large medical centers and receiving a large number of blood cultures.

Embedded Lattice and Properties of Gram Matrix

Directory of Open Access Journals (Sweden)

Futa Yuichi

2017-03-01

Full Text Available In this article, we formalize in Mizar [14] the definition of embedding of lattice and its properties. We formally define an inner product on an embedded module. We also formalize properties of Gram matrix. We formally prove that an inverse of Gram matrix for a rational lattice exists. Lattice of Z-module is necessary for lattice problems, LLL (Lenstra, Lenstra and Lov´asz base reduction algorithm [16] and cryptographic systems with lattice [17].

Synergistic antibacterial effect of silver and ebselen against multidrug-resistant Gram-negative bacterial infections.

Science.gov (United States)

Zou, Lili; Lu, Jun; Wang, Jun; Ren, Xiaoyuan; Zhang, Lanlan; Gao, Yu; Rottenberg, Martin E; Holmgren, Arne

2017-08-01

Multidrug-resistant (MDR) Gram-negative bacteria account for a majority of fatal infections, and development of new antibiotic principles and drugs is therefore of outstanding importance. Here, we report that five most clinically difficult-to-treat MDR Gram-negative bacteria are highly sensitive to a synergistic combination of silver and ebselen. In contrast, silver has no synergistic toxicity with ebselen on mammalian cells. The silver and ebselen combination causes a rapid depletion of glutathione and inhibition of the thioredoxin system in bacteria. Silver ions were identified as strong inhibitors of Escherichia coli thioredoxin and thioredoxin reductase, which are required for ribonucleotide reductase and DNA synthesis and defense against oxidative stress. The bactericidal efficacy of silver and ebselen was further verified in the treatment of mild and acute MDR E. coli peritonitis in mice. These results demonstrate that thiol-dependent redox systems in bacteria can be targeted in the design of new antibacterial drugs. The silver and ebselen combination offers a proof of concept in targeting essential bacterial systems and might be developed for novel efficient treatments against MDR Gram-negative bacterial infections. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Infectious mononucleosis accompanied by clonal proliferation of EBV-infected cells and infection of CD8-positive cells.

Science.gov (United States)

Arai, Ayako; Yamaguchi, Takeshi; Komatsu, Honami; Imadome, Ken-Ichi; Kurata, Morito; Nagata, Kaoru; Miura, Osamu

2014-01-01

A 22-year-old male was admitted for a sustained fever of 2 months, lymphadenopathy, and liver dysfunction. Anti-VCA-IgM antibody was positive, with elevated Epstein-Barr virus (EBV)-DNA load in the peripheral blood. Liver biopsy revealed infiltration of CD8-positive and EBV-positive cells. Most peripheral blood mononuclear cells (PBMCs) were also positive for CD8, and showed detectable levels of EBV-DNA. Monoclonal proliferation of EBV-infected cells was detected in the PBMCs by Southern blotting for EBV-terminal repeat (EBV-TR). Although EBV-positive T-cell lymphoproliferative disease (EBV-T-LPD) was suspected, the symptoms spontaneously resolved within 12 months. Anti-VCA-IgM antibody and the clonal band of EBV-TR were negative 1 year after the onset, while anti-EBNA antibody was positive. The final diagnosis was thus confirmed as infectious mononucleosis (IM). Our results indicate that EBV-infected CD8-positive cells and clonal proliferation of EBV-infected cells may be temporally detected in IM. EBV-T-LPDs should be carefully excluded in such cases.

Five-Year Summary of In Vitro Activity and Resistance Mechanisms of Linezolid against Clinically Important Gram-Positive Cocci in the United States from the LEADER Surveillance Program (2011 to 2015).

Science.gov (United States)

Pfaller, Michael A; Mendes, Rodrigo E; Streit, Jennifer M; Hogan, Patricia A; Flamm, Robert K

2017-07-01

This report describes linezolid susceptibility testing results for 6,741 Gram-positive pathogens from 60 U.S. sites collected during 2015 for the LEADER Program. In addition, the report summarizes linezolid in vitro activity, resistance mechanisms, and molecular typing obtained for 2011 to 2015. During 2015, linezolid showed potent activity in testing against Staphylococcus aureus , inhibiting >99.9% of 3,031 isolates at ≤2 µg/ml. Similarly, linezolid showed coverage against 99.2% of coagulase-negative staphylococci, 99.7% of enterococci, and 100.0% of Streptococcus pneumoniae , virdans group, and beta-hemolytic streptococcus isolates tested. The overall linezolid resistance rate remained a modest linezolid resistance mechanisms. Increased annual trends for the presence of cfr among Staphylococcus aureus isolates were not observed, but 64.3% (9/14) of the isolates with decreased susceptibility (MIC, ≥4 µg/ml) to linezolid carried this transferrable gene (2011 to 2015). The cfr gene was detected in 21.9% (7/32) of linezolid-resistant staphylococci other than S. aureus from 2011 to 2015. The optrA gene was noted in half (2/4) of the population of linezolid-nonsusceptible Enterococcus faecalis isolates from 2011 to 2015, while linezolid-nonsusceptible Enterococcus faecium isolates showed alterations predominantly (16/16) in the 23S rRNA gene (G2576T). This report confirms a long record of linezolid activity against Gram-positive isolates in the United States since regulatory approval in 2000 and reports the oxazolidinones evolving resistance mechanisms. Copyright © 2017 American Society for Microbiology.

Renew or die: The molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram-negative pathogens.

Science.gov (United States)

Domínguez-Gil, Teresa; Molina, Rafael; Alcorlo, Martín; Hermoso, Juan A

2016-09-01

Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway. Copyright © 2016. Published by Elsevier Ltd.

Sorption of lead onto two gram-negative marine bacteria in seawater

Science.gov (United States)

Harvey, Ronald W.; Leckie, James O.

1985-01-01

Laboratory adsorption experiments performed at environmentally significant lead (Pb) and cell concentrations indicate that the marine bacteria examined have significant binding capacities for Pb. However, the behavior governing Pb sorption onto gram-negative bacteria in seawater may be quite complex. The sorption kinetics appear to involve two distinct phases, i.e., a rapid removal of Pb from solution within the first few minutes, followed by a slow but nearly constant removal over many hours. Also, the average binding coefficient, calculated for Pb sorption onto bacteria and a measure of binding intensity, increases with decreasing sorption density (amounts of bacteria-associated Pb per unit bacterial surface) at low cell concentrations (105 cells ml−1), but decreases with decreasing sorption density at higher cell concentrations (107 cells ml−1). The latter effect is apparently due to the production of significant amounts of extra-cellular organics at high cell concentrations that compete directly with bacterial surfaces for available lead. Lead toxicity and active uptake by marine bacteria did not appear significant at the Pb concentrations used.

Probing the Penetration of Antimicrobial Polymyxin Lipopeptides into Gram-Negative Bacteria

Science.gov (United States)

2015-01-01

The dry antibiotic development pipeline coupled with the emergence of multidrug resistant Gram-negative ‘superbugs’ has driven the revival of the polymyxin lipopeptide antibiotics. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections. The lack of molecular imaging probes that possess native polymyxin-like antibacterial activity is a barrier to understanding the resistance mechanisms and the development of a new generation of polymyxin lipopeptides. Here we report the regioselective modification of the polymyxin B core scaffold at the N-terminus with the dansyl fluorophore to generate an active probe that mimics polymyxin B pharmacologically. Time-lapse laser scanning confocal microscopy imaging of the penetration of probe (1) into Gram-negative bacterial cells revealed that the probe initially accumulates in the outer membrane and subsequently penetrates into the inner membrane and finally the cytoplasm. The implementation of this polymyxin-mimetic probe will advance the development of platforms for the discovery of novel polymyxin lipopeptides with efficacy against polymyxin-resistant strains. PMID:24635310

Chromosome and cell wall segregation in Streptococcus faecium ATCC 9790

International Nuclear Information System (INIS)

Higgins, M.L.; Glaser, D.; Dicker, D.T.; Zito, E.T.

1989-01-01

Segregation was studied by measuring the positions of autoradiographic grain clusters in chains formed from single cells containing on average less than one radiolabeled chromosome strand. The degree to which chromosomal and cell wall material cosegregated was quantified by using the methods of S. Cooper and M. Weinberger, dividing the number of chains labeled at the middle. This analysis indicated that in contrast to chromosomal segregation in Escherichia coli and, in some studies, to that in gram-positive rods, chromosomal segregation in Streptococcus faecium was slightly nonrandom and did not vary with growth rate. Results were not significantly affected by strand exchange. In contrast, labeled cell wall segregated predominantly nonrandomly

Increased genetic variability for symbiotic nitrogen fixation in green gram (Vigna radiata L.)

International Nuclear Information System (INIS)

Rosaiah, G.; Kumari, D.S.; Satyanarayana, A.; Seenaiah, P.

1989-01-01

Full text: When green gram is planted after rice in Andhra Pradesh, its nitrogen fixation relies upon local rhizobia that have been able to survive the stress of 5-6 months submergence. No rhizobia strain isolated elsewhere was found superior to native rhizobia. Thus improvement of the host may be the only practicable way to improve nitrogen fixation. 15 mutants obtained from gamma irradiated green gram variety 'LGG 127' were tested along with the parent and the cultivar 'Pant Mung 2'. Nodule no. per plant was higher in the mutants. There was also considerable variation in dry weight of nodules per plant and in seed yield. However the number of nodules per plant showed no correlation with seed yield, nodule size may be more relevant. The N content of the shoots at anthesis was positively correlated with dry weight of nodules, seed protein % and seed yield per plant. (author)

Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta.

Directory of Open Access Journals (Sweden)

Hélène Bierne

Full Text Available Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-λ genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-λ genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-λ genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-λ genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-λ. We also found that IFN-λ genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-λ2/λ3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-λ2. Together, these results suggest that IFN-λ may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.

Screening of antibiotic susceptibility to β-lactam-induced elongation of Gram-negative bacteria based on dielectrophoresis.

Science.gov (United States)

Chung, Cheng-Che; Cheng, I-Fang; Chen, Hung-Mo; Kan, Heng-Chuan; Yang, Wen-Horng; Chang, Hsien-Chang

2012-04-03

We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the β-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA). The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment. Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively. The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose. According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 μm when the bacterial growth is inhibited by the CEZ treatment. In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly. There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria. Thus, the drug resistance of GNB to β-lactam antibiotics can be rapidly assessed. Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method. Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria.

Pulmonary infiltrates during community acquired Gram-negative bacteremia

DEFF Research Database (Denmark)

Fjeldsøe-Nielsen, Hans; Gjeraa, Kirsten; Berthelsen, Birgitte G

2013-01-01

The primary aim of this study was to describe the frequency of pulmonary infiltrates on chest X-ray (CXR) during community acquired Gram-negative bacteremia at a single centre in Denmark.......The primary aim of this study was to describe the frequency of pulmonary infiltrates on chest X-ray (CXR) during community acquired Gram-negative bacteremia at a single centre in Denmark....

Antimicrobial resistance pattern of Gram –negative bacilli isolated of Vali-Asr Hospital wards in Arak

Directory of Open Access Journals (Sweden)

Farshid Didgar

2014-11-01

Full Text Available Background: Infectious diseases are of the most important causes of mortality all around the world particular in developing countries. Recently, the most important thing that has worried medical society is antibiotic resistance. Multi-resistant gram_negative rods are important pathogens in hospitals, causing high rate of mortality.The main goal of this study was to investigate the antimicrobial resistance patterns among common gram-negative bacilli isolated from patients of Vali-Asr Hospital. Material and Methods: This is a cross-sectional descriptive study conducted between the years 2010-2012 in Vali-Asr hospital in Arak. In this study 1120 specimen were examined. Bacterial strains were isolated by conventional methods from various clinical samples of patients including: blood, urine, wound, sputum, CSF, andetc.All isolates were examined for antimicrobial resistance using disc diffusion method. Results: In this study 737 specimen were positive cultures. A total of 332 isolates of Gram-negative bacilli were identified. The most frequent gram negative bacteria were isolated from urine, wound, blood, respiratory secretion and catheter. The most frequent pathogens were E.coli followed by k.pneumonia, entrobacter, p.oaeruginosa, Acinetobacter spp, citrobacter and proteus. High rate of resistance to third generation of cephalospoins & carbapenems observed amang isolates of Acintobacter spp.Prodution of extended spectrum beralactamases (ESBLS was found in 51.4% of all Gram negative bacteria. Conclusion: Antibiotic resistance, particularly multi-drug resistance is frequent among microorganisms of ValiAsr Hospital. Resistance in our country, like other countries have been shown to be increased, so it is highly recommended to prohibit unnecessary prescription of antibiotics.

FGF7 and cell density are required for final differentiation of pancreatic amylase-positive cells from human ES cells.

Science.gov (United States)

Takizawa-Shirasawa, Sakiko; Yoshie, Susumu; Yue, Fengming; Mogi, Akimi; Yokoyama, Tadayuki; Tomotsune, Daihachiro; Sasaki, Katsunori

2013-12-01

The major molecular signals of pancreatic exocrine development are largely unknown. We examine the role of fibroblast growth factor 7 (FGF7) in the final induction of pancreatic amylase-containing exocrine cells from induced-pancreatic progenitor cells derived from human embryonic stem (hES) cells. Our protocol consisted in three steps: Step I, differentiation of definitive endoderm (DE) by activin A treatment of hES cell colonies; Step II, differentiation of pancreatic progenitor cells by re-plating of the cells of Step I onto 24-well plates at high density and stimulation with all-trans retinoic acid; Step III, differentiation of pancreatic exocrine cells with a combination of FGF7, glucagon-like peptide 1 and nicotinamide. The expression levels of pancreatic endodermal markers such as Foxa2, Sox17 and gut tube endoderm marker HNF1β were up-regulated in both Step I and II. Moreover, in Step III, the induced cells expressed pancreatic markers such as amylase, carboxypeptidase A and chymotrypsinogen B, which were similar to those in normal human pancreas. From day 8 in Step III, cells immunohistochemically positive for amylase and for carboxypeptidase A, a pancreatic exocrine cell product, were induced by FGF7. Pancreatic progenitor Pdx1-positive cells were localized in proximity to the amylase-positive cells. In the absence of FGF7, few amylase-positive cells were identified. Thus, our three-step culture protocol for human ES cells effectively induces the differentiation of amylase- and carboxypeptidase-A-containing pancreatic exocrine cells.

Metallo- β-lactamases among Multidrug Resistant (MDR Gram Negative Bacteria Isolated from Clinical Specimens during 2009 in Sanandaj, Kurdistan Province

Directory of Open Access Journals (Sweden)

Himen Salimizand

2012-08-01

Full Text Available Background: Today, there are numerous reports about emerging multi drug resistant gram negative bacteria all around the world, especially in ICUs. Rarely, Metallo-β-lactamase (MBL enzymes are responsible for these cases. Study of MBLs for diagnosing and preventing distribution of the origin of infection are critical issues. In addition, we would like to compare the efficacy of Iranian and foreign- made antibiotic disks. Materials and Methods: During 2009 all entered clinical specimens to the laboratory tested for detecting gram negative bacteria. Isolated bacteria were tested by Kirby-Bauer method to antibiotic susceptibility test by Iranian and foreign (MAST disks. For gram negative carbapenem resistant isolates, PCR technique used to detect VIM, GIM, and SIM variants of MBLs.Results: During one year, 17890 clinical specimens referred Besat laboratory. The most specimen was Urine (8172 followed by blood culture (5190 that in which 1110 gram negative and positives isolated. Out of which, 778 (70% of isolates were gram negatives. MDR gram negatives were 157 (20.2%. Imipenem and meropenem were the most efficient antibiotics (all susceptible and ceftriaxone was the least (19 % susceptible. E. coli was the most prevalent isolate. 79 Gram negative isolates (10.1% were resistant to Iranian-made discs but all susceptible for foreign ones. All 79 isolates were tested by PCR for MBL genes, that, all were negative. Besides, Iranian imipenem and cefepime disks have had distinguishable difference in susceptibility of isolates.Conclusion: Fortunately, none of gram negative isolates were MBL producer, which revealed no colonization of MBL producing bacteria. Iranian-made disks appear efficient except for imipenem and cefepime.

Molecular evolution of the nif gene cluster carrying nifI1 and nifI2 genes in the Gram-positive phototrophic bacterium Heliobacterium chlorum.

Science.gov (United States)

Enkh-Amgalan, Jigjiddorj; Kawasaki, Hiroko; Seki, Tatsuji

2006-01-01

A major nif cluster was detected in the strictly anaerobic, Gram-positive phototrophic bacterium Heliobacterium chlorum. The cluster consisted of 11 genes arranged within a 10 kb region in the order nifI1, nifI2, nifH, nifD, nifK, nifE, nifN, nifX, fdx, nifB and nifV. The phylogenetic position of Hbt. chlorum was the same in the NifH, NifD, NifK, NifE and NifN trees; Hbt. chlorum formed a cluster with Desulfitobacterium hafniense, the closest neighbour of heliobacteria based on the 16S rRNA phylogeny, and two species of the genus Geobacter belonging to the Deltaproteobacteria. Two nifI genes, known to occur in the nif clusters of methanogenic archaea between nifH and nifD, were found upstream of the nifH gene of Hbt. chlorum. The organization of the nif operon and the phylogeny of individual and concatenated gene products showed that the Hbt. chlorum nif operon carrying nifI genes upstream of the nifH gene was an intermediate between the nif operon with nifI downstream of nifH (group II and III of the nitrogenase classification) and the nif operon lacking nifI (group I). Thus, the phylogenetic position of Hbt. chlorum nitrogenase may reflect an evolutionary stage of a divergence of the two nitrogenase groups, with group I consisting of the aerobic diazotrophs and group II consisting of strictly anaerobic prokaryotes.

Molecular studies on bacteriophage endolysins and their potential to control gram-negative bacteria

OpenAIRE

Oliveira, Hugo Alexandre Mendes

2014-01-01

Thesis for PhD degree in Chemical and Biological Engineeering Bacteriophages are viruses that specifically infect bacterial hosts to reproduce. At the end of the infection cycle, progeny virions are confronted with a rigid cell wall that impedes their release into the environment. Consequently, bacteriophages encode hydrolytic enzymes, called endolysins, to digest the peptidoglycan and cause bacteriolysis. In contrast to their extensively studied counterparts, active against Gram-positi...

[GEIPC-SEIMC (Study Group for Infections in the Critically Ill Patient of the Spanish Society for Infectious Diseases and Clinical Microbiology) and GTEI-SEMICYUC ( Working Group on Infectious Diseases of the Spanish Society of Intensive Medicine, Critical Care, and Coronary Units) recommendations for antibiotic treatment of gram-positive cocci infections in the critical patient].

Science.gov (United States)

Astigarraga, P M Olaechea; Montero, J Garnacho; Cerrato, S Grau; Colomo, O Rodríguez; Martínez, M Palomar; Crespo, R Zaragoza; García-Paredes, P Muñoz; Cerdá, E Cerdá; Lerma, F Alvarez

2007-01-01

In recent years, an increment of infections caused by gram-positive cocci has been documented in nosocomial and hospital-acquired-infections. In diverse countries, a rapid development of resistance to common antibiotics against gram-positive cocci has been observed. This situation is exceptional in Spain but our country might be affected in the near future. New antimicrobials active against these multi-drug resistant pathogens are nowadays available. It is essential to improve our current knowledge about pharmacokinetic properties of traditional and new antimicrobials to maximize its effectiveness and to minimize toxicity. These issues are even more important in critically ill patients because inadequate empirical therapy is associated with therapeutic failure and a poor outcome. Experts representing two scientific societies (Grupo de estudio de Infecciones en el Paciente Crítico de la SEIMC and Grupo de trabajo de Enfermedades Infecciosas de la SEMICYUC) have elaborated a consensus document based on the current scientific evidence to summarize recommendations for the treatment of serious infections caused by gram-positive cocci in critically ill patients.

EBV-positive diffuse large B-cell lymphoma of the elderly

NARCIS (Netherlands)

C.Y. Ok (Chi Young); T.G. Papathomas (Thomas); L.J. Medeiros (L. Jeffrey); K.H. Young (Ken)

2013-01-01

textabstractEpstein-Barr virus (EBV) positive diffuse large B-cell lymphoma (DLBCL) of the elderly, initially described in 2003, is a provisional entity in the 2008World Health Organization classification system and is defined as an EBV-positive monoclonal large B-cell proliferation that occurs in

A new rapid method for direct antimicrobial susceptibility testing of bacteria from positive blood cultures.

Science.gov (United States)

Barnini, Simona; Brucculeri, Veronica; Morici, Paola; Ghelardi, Emilia; Florio, Walter; Lupetti, Antonella

2016-08-12

Rapid identification and antimicrobial susceptibility testing (AST) of the causative agent(s) of bloodstream infections can lead to prompt appropriate antimicrobial therapy. To shorten species identification, in this study bacteria were recovered from monomicrobial blood cultures by serum separator tubes and spotted onto the target plate for direct MALDI-TOF MS identification. Proper antibiotics were selected for direct AST based on species identification. In order to obtain rapid AST results, bacteria were recovered from positive blood cultures by two different protocols: by serum separator tubes (further referred to as PR1), or after a short-term subculture in liquid medium (further referred to as PR2). The results were compared with those obtained by the method currently used in our laboratory consisting in identification by MALDI-TOF and AST by Vitek 2 or Sensititre on isolated colonies. The direct MALDI-TOF method concordantly identified with the current method 97.5 % of the Gram-negative bacteria and 96.1 % of the Gram-positive cocci contained in monomicrobial blood cultures. The direct AST by PR1 and PR2 for all isolate/antimicrobial agent combinations was concordant/correct with the current method for 87.8 and 90.5 % of Gram-negative bacteria and for 93.1 and 93.8 % of Gram-positive cocci, respectively. In particular, 100 % categorical agreement was found with levofloxacin for Enterobacteriaceae by both PR1 and PR2, and 99.0 and 100 % categorical agreement was observed with linezolid for Gram-positive cocci by PR1 and PR2, respectively. There was no significant difference in accuracy between PR1 and PR2 for Gram-negative bacteria and Gram-positive cocci. This newly described method seems promising for providing accurate AST results. Most importantly, these results would be available in a few hours from blood culture positivity, which would help clinicians to promptly confirm or streamline an effective antibiotic therapy in patients with bloodstream

Antibacterial effect of protamine assayed by impedimetry

DEFF Research Database (Denmark)

Johansen, Charlotte; Gill, T.; Gram, Lone

1995-01-01

estimating the cell number after protamine treatment, rather than colony counts. Protamine from salmon killed growing Gram- positive bacteria and significantly inhibited growth of Gram- negative bacteria in Tryptone Soy Broth (TSB) at 25 degrees C. In general Gram-positive bacteria were more sensitive...... to protamine than Gram-negative bacteria; the minimum inhibitory concentrations (MIG) determined for Gram-positive strains varied from 20 to 1000 mu g ml(-1) and for Gram-negative strains from 500 mu g ml(-1) to more than 4000 mu g ml(-1). The effect of protamine on non-growing Listeria monocytogenes Scott...... A suspended in buffer was not lethal as was the effect on growing cells; however, protamine (50-500 mu g ml(-1)) killed the Gram-negative fish spoilage bacteria Shewanella putrefaciens when the live cells were suspended in buffer....

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

International Nuclear Information System (INIS)

Fiedler, Tomas; Salamon, Achim; Adam, Stefanie; Herzmann, Nicole; Taubenheim, Jan; Peters, Kirsten

2013-01-01

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC

Impact of bacteria and bacterial components on osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Energy Technology Data Exchange (ETDEWEB)

Fiedler, Tomas, E-mail: tomas.fiedler@med.uni-rostock.de [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Salamon, Achim; Adam, Stefanie; Herzmann, Nicole [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Taubenheim, Jan [Institute for Medical Microbiology, Virology, and Hygiene, Rostock University Medical Center, Schillingallee 70, D-18057 Rostock (Germany); Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany); Peters, Kirsten [Department of Cell Biology, Rostock University Medical Center, Schillingallee 69, D-18057 Rostock (Germany)

2013-11-01

Adult mesenchymal stem cells (MSC) are present in several tissues, e.g. bone marrow, heart muscle, brain and subcutaneous adipose tissue. In invasive infections MSC get in contact with bacteria and bacterial components. Not much is known about how bacterial pathogens interact with MSC and how contact to bacteria influences MSC viability and differentiation potential. In this study we investigated the impact of three different wound infection relevant bacteria, Escherichia coli, Staphylococcus aureus, and Streptococcus pyogenes, and the cell wall components lipopolysaccharide (LPS; Gram-negative bacteria) and lipoteichoic acid (LTA; Gram-positive bacteria) on viability, proliferation, and osteogenic as well as adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells (adMSC). We show that all three tested species were able to attach to and internalize into adMSC. The heat-inactivated Gram-negative E. coli as well as LPS were able to induce proliferation and osteogenic differentiation but reduce adipogenic differentiation of adMSC. Conspicuously, the heat-inactivated Gram-positive species showed the same effects on proliferation and adipogenic differentiation, while its cell wall component LTA exhibited no significant impact on adMSC. Therefore, our data demonstrate that osteogenic and adipogenic differentiation of adMSC is influenced in an oppositional fashion by bacterial antigens and that MSC-governed regeneration is not necessarily reduced under infectious conditions. - Highlights: • Staphylococcus aureus, Streptococcus pyogenes and Escherichia coli bind to and internalize into adMSC. • Heat-inactivated cells of these bacterial species trigger proliferation of adMSC. • Heat-inactivated E. coli and LPS induce osteogenic differentiation of adMSC. • Heat-inactivated E. coli and LPS reduce adipogenic differentiation of adMSC. • LTA does not influence adipogenic or osteogenic differentiation of adMSC.

Sistema de tareas para la enseñanza combinada de la gramática comunicativa y la gramática tradicional de lengua inglesa

Directory of Open Access Journals (Sweden)

Nancy del Carmen Jiménez Figueredo

2015-03-01

Full Text Available Se realizó el diseño de un sistema de tareas para la enseñanza combinada de la gramática comunicativa y la gramática tradicional de lengua inglesa, con metodología cuali-cuantitativa, en la Facultad de Ciencias Médicas “Zoilo Marinello Vidaurreta” de la Universidad de Ciencias Médicas de Las Tunas, durante el período comprendido entre septiembre de 2011 y julio de 2014. El objetivo estuvo direccionado a perfeccionar la corrección lingüística en el proceso de enseñanza-aprendizaje del idioma Inglés. La realización del diseño contó con: una revisión bibliográfica de los principales enfoques utilizados en la enseñanza-aprendizaje del Inglés; la revisión documental, que permitió determinar las insuficiencias lingüísticas y la necesidad de combinar la gramática comunicativa con la gramática tradicional, en los estudiantes del cuarto año de la carrera de Estomatología; el intercambio con profesionales con más de quince años de experiencia, para obtener información acerca del proceso de enseñanza-aprendizaje de la gramática; y la aplicación de una valoración equivalente al juicio de expertos, método de agregados individuales, para valorar la propuesta. Los principales resultados fueron: la caracterización de las insuficiencias provocadas por la absolutización del enfoque comunicativo en la enseñanza de la gramática de la lengua inglesa y el diseño de un sistema de tareas para el proceso de enseñanza-aprendizaje de la gramática inglesa en estos estudiantes

Antibiofilm effect of Nocardiopsis sp. GRG 1 (KT235640) compound against biofilm forming Gram negative bacteria on UTIs.

Science.gov (United States)

Rajivgandhi, Govindan; Vijayan, Ramachandran; Maruthupandy, Muthuchamy; Vaseeharan, Baskaralingam; Manoharan, Natesan

2018-05-01

Urinary tract infections (UTIs) are diverse public health complication and caused by range of pathogens, however mostly Gram negative bacteria cause significant life threatening risks to different populations. The prevalence rate and antimicrobial resistance among the Gram negative uropathogens alarmed significantly heighten the economic burden of these infections. In this study, we investigated the antibiofilm efficiency of Pyrrolo [1,2-a] pyrazine-1,4-dione,hexahydro-3-(2-methylpropyl) extracted from endophytic actinomycetes Nocardiopsis sp. GRG 1 (KT235640) against P. mirabilis and E. coli. The extracted compound was characterized through TLC, HPLC, GC-MS, LC-MS and confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM). The compound, Pyrrolo [1,2-a] pyrazine-1, 4-dione, hexahydro-3-(2-methylpropyl) inhibits both bacterial biofilm formation as well as reduces the viability of preformed biofilms. Furthermore, CLSM image shows cell shrinkage, disorganized cell membrane and loss of viability. The SEM result also confirms the cell wall degradation in treated cells of the bacteria. Hence, the Pyrrolo [1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) is active against P. mirabilis and E. coli. Copyright © 2018 Elsevier Ltd. All rights reserved.

Gram-Scale Synthesis of Highly Active and Durable Octahedral PtNi Nanoparticle Catalysts for Proton Exchange Membrane Fuel Cell

DEFF Research Database (Denmark)

Choi, Juhyuk; Jang, Jue-Hyuk; Roh, Chi-Woo

2018-01-01

for the commercialization of PEMFCs. In this study, we focus on gram-scale synthesis of octahedral PtNi nanoparticles with Pt overlayers (PtNi@Pt) supported on the carbon, resulting in enhanced catalytic activity and durability. Such PtNi@Pt catalysts show high mass activity (1.24 A mgPt−1) at 0.9 V (vs RHE) for the ORR......Proton exchange membrane fuel cells (PEMFC) are regarded as a promising renewable energy source for a future hydrogen energy society. However, highly active and durable catalysts are required for the PEMFCs because of their intrinsic high overpotential at the cathode and operation under the acidic...... condition for oxygen reduction reaction (ORR). Since the discovery of the exceptionally high surface activity of Pt3Ni(111), the octahedral PtNi nanoparticles have been synthesized and tested. Nonetheless, their milligram-scale synthesis method and poor durability make them unsuitable...

Calpain-Mediated positional information directs cell wall orientation to sustain plant stem cell activity, growth and development

Science.gov (United States)

Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental f...

Direct identification of bacteria from positive BacT/ALERT blood culture bottles using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Science.gov (United States)

Mestas, Javier; Felsenstein, Susanna; Bard, Jennifer Dien

2014-11-01

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is a fast and robust method for the identification of bacteria. In this study, we evaluate the performance of a laboratory-developed lysis method (LDT) for the rapid identification of bacteria from positive BacT/ALERT blood culture bottles. Of the 168 positive bottles tested, 159 were monomicrobial, the majority of which were Gram-positive organisms (61.0% versus 39.0%). Using a cut-off score of ≥1.7, 80.4% of the organisms were correctly identified to the species level, and the identification rate of Gram-negative organisms (90.3%) was found to be significantly greater than that of Gram-positive organisms (78.4%). The simplicity and cost-effectiveness of the LDT enable it to be fully integrated into the routine workflow of the clinical microbiology laboratory, allowing for rapid identification of Gram-positive and Gram-negative bacteria within an hour of blood culture positivity. Copyright © 2014 Elsevier Inc. All rights reserved.

White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae.

Science.gov (United States)

de-la-Re-Vega, Enrique; García-Galaz, Alfonso; Díaz-Cinco, Martha E; Sotelo-Mundo, Rogerio R

2006-03-01

C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.

Multiparametric Profiling for Identification of Chemosensitizers against Gram-Negative Bacteria

Directory of Open Access Journals (Sweden)

Vincent Lôme

2018-02-01

Full Text Available Antibiotic resistance is now a worldwide therapeutic problem. Since the beginning of anti-infectious treatment bacteria have rapidly shown an incredible ability to develop and transfer resistance mechanisms. In the last decades, the design variation of pioneer bioactive molecules has strongly improved their activity and the pharmaceutical companies partly won the race against the clock. Since the 1980s, the new classes of antibiotics that emerged were mainly directed to Gram-positive bacteria. Thus, we are now facing to multidrug-resistant Gram-negative bacteria, with no therapeutic options to deal with them. These bacteria are mainly resistant because of their double membrane that conjointly impairs antibiotic accumulation and extrudes these molecules when entered. The main challenge is to allow antibiotics to cross the impermeable envelope and reach their targets. One promising solution would be to associate, in a combination therapy, a usual antibiotic with a non-antibiotic chemosensitizer. Nevertheless, for effective drug discovery, there is a prominent lack of tools required to understand the rules of permeation and accumulation into Gram-negative bacteria. By the use of a multidrug-resistant enterobacteria, we introduce a high-content screening procedure for chemosensitizers discovery by quantitative assessment of drug accumulation, alteration of barriers, and deduction of their activity profile. We assembled and analyzed a control chemicals library to perform the proof of concept. The analysis was based on real-time monitoring of the efflux alteration and measure of the influx increase in the presence of studied compounds in an automatized bio-assay. Then, synergistic activity of compounds with an antibiotic was studied and kinetic data reduction was performed which led to the calculation of a score for each barrier to be altered.

Characterization of nonlymphoid cells in rat spleen, with special reference to strongly Ia-positive branched cells in T-cell areas

International Nuclear Information System (INIS)

Dijkstra, C.D.

1982-01-01

By use of a monoclonal antibody against Ia antigen in an immunoperoxidase method, strongly Ia-positive branched cells are found in the T-cell areas of the splenic white pulp of the rat. In order to further characterize these cells, enzyme histochemical characteristics, phagocytic capacity, and irradiation sensitivity have been studied. Evidence is presented that these strongly Ia-positive branched cells represent interdigitating cells. The influence of whole-body irradiation on interdigitating cells is discussed. Comparison with data from the literature on the in vitro dendritic cell isolated from spleen cell suspensions reveals many similarities between the described interdigitating cell in vivo and the dendritic cell in vitro

Assessing the interactions of a natural antibacterial clay with model Gram-positive and Gram-negative human pathogens

Science.gov (United States)

Londono, S. C.; Williams, L. B.

2013-12-01

The emergence of antibiotic resistant bacteria and increasing accumulations of antibiotics in reclaimed water, drive the quest for new natural antimicrobials. We are studying the antibacterial mechanism(s) of clays that have shown an ability to destroy bacteria or significantly inhibit their growth. One possible mode of action is from soluble transition metal species, particularly reduced Fe, capable of generating deleterious oxygen radical species. Yet another possibility is related to membrane damage as a consequence of physical or electrostatic interaction between clay and bacteria. Both mechanisms could combine to produce cell death. This study addresses a natural antibacterial clay from the NW Amazon basin, South America (AMZ clay). Clay mineralogy is composed of disordered kaolinite (28.9%), halloysite (17.8%) illite (12%) and smectite (16.7%). Mean particle size is 1.6μm and total and specific surface area 278.82 and 51.23 m2/g respectively. The pH of a suspension (200mg/ml) is 4.1 and its Eh is 361mV after 24h of equilibration. The ionic strength of the water in equilibrium with the clay after 24 h. is 6 x10-4M. These conditions, affect the element solubility, speciation, and interactions between clay and bacteria. Standard microbiological methods were used to assess the viability of two model bacteria (Escherichia coli and Bacillus subtilis) after incubation with clay at 37 degC for 24 hrs. A threefold reduction in bacterial viability was observed upon treatment with AMZ clay. We separated the cells from the clay using Nycodenz gradient media and observed the mounts under the TEM and SEM. Results showed several membrane anomalies and structural changes that were not observed in the control cells. Additionally, clay minerals appeared in some places attached to cell walls. Experiments showed that exchanging AMZ clay with KCl caused loss of antibacterial property. Among the exchangeable -and potentially toxic- ions we measured Al+3, Cu+2, Zn+2, Ba+2 and Co+2

Comparative analysis of Gram's stain, PNA-FISH and Sepsityper with MALDI-TOF MS for the identification of yeast direct from positive blood cultures.

Science.gov (United States)

Gorton, Rebecca L; Ramnarain, P; Barker, K; Stone, N; Rattenbury, S; McHugh, T D; Kibbler, C C

2014-10-01

Fungaemia diagnosis could be improved by reducing the time to identification of yeast from blood cultures. This study aimed to evaluate three rapid methods for the identification of yeast direct from blood cultures; Gram's stain analysis, the AdvanDX Peptide Nucleic Acid in Situ Hybridisation Yeast Traffic Light system (PNA-FISH YTL) and Bruker Sepsityper alongside matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Fifty blood cultures spiked with a known single yeast strain were analysed by blinded operators experienced in each method. Identifications were compared with MALDI-TOF MS CHROMagar Candida culture and ITS rRNA sequence-based identifications. On first attempt, success rates of 96% (48/50) and 76% (36/50) were achieved using PNA-FISH YTL and Gram's stain respectively. MALDI-TOF MS demonstrated a success rate of 56% (28/50) when applying manufacturer's species log score thresholds and 76% (38/50) using in-house parameters, including lowering the species log score threshold to >1.5. In conclusion, PNA-FISH YTL demonstrated a high success rate successfully identifying yeast commonly encountered in fungaemia. Sepsityper(™) with MALDI-TOF MS was accurate but increased sensitivity is required. Due to the misidentification of commonly encountered yeast Gram's stain analysis demonstrated limited utility in this setting. © 2014 Blackwell Verlag GmbH.

Expression of chemokine CXCL10 in dendritic-cell-like S100β-positive cells in rat anterior pituitary gland.

Science.gov (United States)

Horiguchi, Kotaro; Fujiwara, Ken; Higuchi, Masashi; Yoshida, Saishu; Tsukada, Takehiro; Ueharu, Hiroki; Chen, Mo; Hasegawa, Rumi; Takigami, Shu; Ohsako, Shunji; Yashiro, Takashi; Kato, Takako; Kato, Yukio

2014-09-01

Chemokines are mostly small secreted polypeptides whose signals are mediated by seven trans-membrane G-protein-coupled receptors. Their functions include the control of leukocytes and the intercellular mediation of cell migration, proliferation, and adhesion in several tissues. We have previously revealed that the CXC chemokine ligand 12 (CXCL12) and its receptor 4 (CXCR4) are expressed in the anterior pituitary gland, and that the CXCL12/CXCR4 axis evokes the migration and interconnection of S100β-protein-positive cells (S100β-positive cells), which do not produce classical anterior pituitary hormones. However, little is known of the cells producing the other CXCLs and CXCRs or of their characteristics in the anterior pituitary. We therefore examined whether CXCLs and CXCRs occurred in the rat anterior pituitary lobe. We used reverse transcription plus the polymerase chain reaction to analyze the expression of Cxcl and Cxcr and identified the cells that expressed Cxcl by in situ hybridization. Transcripts of Cxcl10 and its receptor (Cxcr3 and toll-like receptor 4, Tlr4) were clearly detected: cells expressing Cxcl10 and Tlr4 were identified amongst S100β-positive cells and those expressing Cxcr3 amongst adrenocorticotropic hormone (ACTH)-producing cells. We also investigated Cxcl10 expression in subpopulations of S100β-positive cells. We separated cultured S100β-positive cells into the round-type (dendritic-cell-like) and process-type (astrocyte- or epithelial-cell-like) by their adherent activity to laminin, a component of the extracellular matrix; CXCL10 was expressed only in round-type S100β-positive cells. Thus, CXCL10 produced by a subpopulation of S100β-positive cells probably exerts an autocrine/paracrine effect on S100β-positive cells and ACTH-producing cells in the anterior lobe.

A thermostable Salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids.

Directory of Open Access Journals (Sweden)

Hugo Oliveira

Full Text Available Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68 and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively. Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermostability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.

Disruption and analysis of the clpB, clpC, and clpE genes in Lactococcus lactis: ClpE, a new Clp family in gram-positive bacteria