Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation.

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Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Sarma, Potukuchi Venkata Gurunadha Krishna

2017-03-01

When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and spread the infection.

Novel application for the prevention and treatment of Staphylococcus aureus biofilm formation

Science.gov (United States)

Traba, Christian

Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this dissertation, the application of plasma from two very different facets was studied. In part one, the susceptibility of pre-formed Staphylococcus aureus biofilms on biomaterials to different plasmas was investigated. It was found that the distinct chemical/physical properties of plasmas generated from oxygen, nitrogen, and argon all demonstrated very potent but very different anti-biofilm mechanisms of action. An in depth analysis of these results show: 1) different reactive species produced in each plasma demonstrate specific activity, and 2) the commonly associated etching effect could be manipulated and even controlled, depending on experimental conditions and the discharge gas. These studies provide insights into the anti-biofilm mechanisms of plasma as well as the effects of different reactive species on biofilm inactivation. Under experimental parameters, bacterial cells in Staphylococcus aureus biofilms were killed (>99.9%) by plasmas within minutes of exposure and no bacteria nor biofilm re-growth from discharge gas treated biofilms was observed throughout the life-span of the re-growth experiment. The decontamination ability of plasmas for the treatment of biofilm related infections on biomedical materials was confirmed and novel applications involving the use of low power argon and oxygen for the treatment of biofilm contaminated biomaterials and indwelling devices is proposed. The second facet of this dissertation explores the interaction between biofilm forming Staphylococcus aureus bacteria on different antibacterial/anti-biofilm surfaces. The antibiotic-free anti-fouling surfaces constructed in this study were generated from the plasma-assisted graft polymerization technique. These sophisticated surfaces were stable, biocompatible and capable of preventing biofilm formation on biomaterials and medical devices. Under

Study on biofilm-forming properties of clinical isolates of Staphylococcus aureus.

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Taj, Yasmeen; Essa, Farhan; Aziz, Faisal; Kazmi, Shahana Urooj

2012-05-14

The purpose of this study was to observe the formation of biofilm, an important virulence factor, by isolates of Staphylococcus aureus (S. aureus) in Pakistan by different conventional methods and through electron microscopy. We screened 115 strains of S. aureus isolated from different clinical specimens by tube method (TM), air-liquid interface coverslip assay method, Congo red agar (CRA) method, and scanning electron microscopy (SEM). Out of 115 S. aureus isolates, 63 (54.78%) showed biofilm formation by tube method. Biofilm forming bacteria were further categorized as high producers (n = 23, 20%) and moderate producers (n = 40, 34.78%). TM coordinated well with the coverslip assay for strong biofilm-producing strains in 19 (16.5%) isolates. By coverslip method, weak producers were difficult to differentiate from biofilm negative isolates. Screening on CRA showed biofilm formation only in four (3.47%) strains. Scanning electron micrographs showed the biofilm-forming strains of S. aureus arranged in a matrix on the propylene surface and correlated well with the TM. Biofilm production is a marker of virulence for clinically relevant staphylococcal infections. It can be studied by various methods but screening on CRA is not recommended for investigation of biofilm formation in Staphylococcus aureus. Electron micrograph images correlate well with the biofilm production as observed by TM.

SaeRS Is Responsive to Cellular Respiratory Status and Regulates Fermentative Biofilm Formation in Staphylococcus aureus.

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Mashruwala, Ameya A; Gries, Casey M; Scherr, Tyler D; Kielian, Tammy; Boyd, Jeffrey M

2017-08-01

Biofilms are multicellular communities of microorganisms living as a quorum rather than as individual cells. The bacterial human pathogen Staphylococcus aureus uses oxygen as a terminal electron acceptor during respiration. Infected human tissues are hypoxic or anoxic. We recently reported that impaired respiration elicits a p rogrammed c ell l ysis (PCL) phenomenon in S. aureus leading to the release of cellular polymers that are utilized to form biofilms. PCL is dependent upon the AtlA murein hydrolase and is regulated, in part, by the SrrAB two-component regulatory system (TCRS). In the current study, we report that the SaeRS TCRS also governs fermentative biofilm formation by positively influencing AtlA activity. The SaeRS-modulated factor fibronectin-binding protein A (FnBPA) also contributed to the fermentative biofilm formation phenotype. SaeRS-dependent biofilm formation occurred in response to changes in cellular respiratory status. Genetic evidence presented suggests that a high cellular titer of phosphorylated SaeR is required for biofilm formation. Epistasis analyses found that SaeRS and SrrAB influence biofilm formation independently of one another. Analyses using a mouse model of orthopedic implant-associated biofilm formation found that both SaeRS and SrrAB govern host colonization. Of these two TCRSs, SrrAB was the dominant system driving biofilm formation in vivo We propose a model wherein impaired cellular respiration stimulates SaeRS via an as yet undefined signal molecule(s), resulting in increasing expression of AtlA and FnBPA and biofilm formation. Copyright © 2017 American Society for Microbiology.

A Nonbactericidal Zinc-Complexing Ligand as a Biofilm Inhibitor: Structure-Guided Contrasting Effects on Staphylococcus aureus Biofilm.

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Kapoor, Vidushi; Rai, Rajanikant; Thiyagarajan, Durairaj; Mukherjee, Sandipan; Das, Gopal; Ramesh, Aiyagari

2017-08-04

Zinc-complexing ligands are prospective anti-biofilm agents because of the pivotal role of zinc in the formation of Staphylococcus aureus biofilm. Accordingly, the potential of a thiosemicarbazone (compound C1) and a benzothiazole-based ligand (compound C4) in the prevention of S. aureus biofilm formation was assessed. Compound C1 displayed a bimodal activity, hindering biofilm formation only at low concentrations and promoting biofilm growth at higher concentrations. In the case of C4, a dose-dependent inhibition of S. aureus biofilm growth was observed. Atomic force microscopy analysis suggested that at higher concentrations C1 formed globular aggregates, which perhaps formed a substratum that favored adhesion of cells and biofilm formation. In the case of C4, zinc supplementation experiments validated zinc complexation as a plausible mechanism of inhibition of S. aureus biofilm. Interestingly, C4 was nontoxic to cultured HeLa cells and thus has promise as a therapeutic anti-biofilm agent. The essential understanding of the structure-driven implications of zinc-complexing ligands acquired in this study might assist future screening regimes for identification of potent anti-biofilm agents. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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Khadije Rezaie Keikhaie

2017-02-01

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

Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus.

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Nan, Li; Yang, Ke; Ren, Guogang

2015-06-01

Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu(2+) ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1ppm (2days) to 4.5ppm (7days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu(2+) ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface. Copyright © 2015. Published by Elsevier B.V.

Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus

International Nuclear Information System (INIS)

Nan, Li; Yang, Ke; Ren, Guogang

2015-01-01

Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24 h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7 days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu 2+ ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1 ppm (2 days) to 4.5 ppm (7 days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu 2+ ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface

Anti-biofilm formation of a novel stainless steel against Staphylococcus aureus

Energy Technology Data Exchange (ETDEWEB)

Nan, Li; Yang, Ke [Institute of Metal Research, Chinese Academy of Sciences, Shenyang 110016 (China); Ren, Guogang, E-mail: g.g.ren@herts.ac.uk [University of Hertfordshire, Hatfield AL10 9AB (United Kingdom)

2015-06-01

Staphylococcus aureus (S. aureus) is a bacterium frequently found proliferating on metal surfaces such as stainless steels used in healthcare and food processing facilities. Past research has shown that a novel Cu-bearing 304 type stainless steel (304CuSS) exhibits excellent antibacterial ability (i.e. against S. aureus) in a short time period (24 h.). This work was dedicated to investigate the 304CuSS's inhibition ability towards the S. aureus biofilm formation for an extended period of 7 days after incubation. It was found that the antibacterial rate of the 304CuSS against sessile bacterial cells reached over 99.9% in comparison with the 304SS. The thickness and sizes of the biofilms on the 304SS surfaces increased markedly with period of contact, and thus expected higher risk of bio-contamination, indicated by the changes of surface free energy between biofilm and the steel surfaces. The results demonstrated that the 304CuSS exhibited strong inhibition on the growth and adherence of the biofilms. The surface free energy of the 304CuSS after contact with sessile bacterial cells was much lower than that of the 304SS towards the same culture times. The continuously dissolved Cu{sup 2+} ions well demonstrated the dissolution ability of Cu-rich precipitates after exposure to S. aureus solution, from 3.1 ppm (2 days) to 4.5 ppm (7 days). For this to occur, a hypothesis mechanism might be established for 304CuSS in which the Cu{sup 2+} ions were released from Cu-rich phases that bond with extracellular polymeric substances (EPS) of the microorganisms. And these inhibited the activities of cell protein/enzymes and effectively prevented planktonic bacterial cells attaching to the 304CuSS metal surface.

Comparison of Biofilm Formation between Methicillin-Resistant and Methicillin-Susceptible Isolates of Staphylococcus aureus.

Science.gov (United States)

Ghasemian, Abdolmajid; Najar Peerayeh, Shahin; Bakhshi, Bita; Mirzaee, Mohsen

2016-07-01

The aim of this study was to compare the biofilm formation and the prevalence of biofilm-associated genes between the isolates of methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus. In total, 209 S. aureus isolates were collected. The antibiotic susceptibility test was conducted using nine antibiotics according to the guidelines of Clinical and Laboratory Standards Institute. Phenotypic biofilm formation was performed with microtiter plate assay. The polymerase chain reaction was employed to detect icaA, icaD, icaB, icaC, clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, bbp, mecA, and SCCmec types as well as agr group genes with specific primers. Sixty-four (30.62%) isolates were resistant to methicillin, and 54 (83%) MRSA harbored SCCmec III. Furthermore, 122 (58.3%) isolates belonged to agr group I. Twenty-six (36.1%) MRSA and 42 (28.9%) MSSA isolates were strong biofilm producers (no significant difference). The prevalence of icaA, icaD, icaB, and icaC genes in MSSA isolates was 71, 41, 76, and 72%, respectively. The frequency of clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, and bbp in MSSA was 100, 100, 56, 46, 74, 54, 78, 11, and 1%, respectively. However, in MRSA isolates, the frequency was 97, 97, 64, 51, 76, 56, 79, and 12% with no track of bbp, respectively. Statistical difference between MSSA and MRSA regarding biofilm formation and the frequency of all biofilm-encoding genes was not significant. The majority of the S. aureus isolates harbored clfA, clfB, eno, fib, icaA, and icaD genes.

Iron enhances the peptidyl deformylase activity and biofilm formation in Staphylococcus aureus.

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Swarupa, Vimjam; Chaudhury, Abhijit; Sarma, Potukuchi Venkata Gurunadha Krishna

2018-01-01

Staphylococcus aureus plays a major role in persistent infections and many of these species form structured biofilms on different surfaces which is accompanied by changes in gene expression profiles. Further, iron supplementation plays a critical role in the regulation of several protein(s)/enzyme function, which all aid in the development of active bacterial biofilms. It is well known that for each protein, deformylation is the most crucial step in biosynthesis and is catalyzed by peptidyl deformylase (PDF). Thus, the aim of the current study is to understand the role of iron in biofilm formation and deformylase activity of PDF. Hence, the PDF gene of S. aureus ATCC12600 was PCR amplified using specific primers and sequenced, followed by cloning and expression in Escherichia coli DH5α. The deformylase activity of the purified recombinant PDF was measured in culture supplemented with/without iron where the purified rPDF showed K m of 1.3 mM and V max of 0.035 mM/mg/min, which was close to the native PDF ( K m  = 1.4 mM, V max  = 0.030 mM/mg/min). Interestingly, the K m decreased and PDF activity increased when the culture was supplemented with iron, corroborating with qPCR results showing 100- to 150-fold more expression compared to control in S. aureus and its drug-resistant strains. Further biofilm-forming units (BU) showed an incredible increase (0.42 ± 0.005 to 6.3 ± 0.05 BU), i.e., almost 15-fold elevation in anaerobic conditions, indicating the significance of iron in S. aureus biofilms.

Staphylococcus aureus biofilm formation on different gentamicin-loaded polymethylmethacrylate bone cements

NARCIS (Netherlands)

van de Belt, H; Neut, D; Schenk, W; van Horn, [No Value; van der Mei, HC; Busscher, HJ

In this in vitro study, the formation of a Staphylococcus aureus biofilm on six gentamicin-loaded bone cements (CMW1, CMW3, CMW Endurance, CMW2000, Palacos. and Palamed) was determined in a modified Robbins device over a 3 days time span and related with previously (Van de Belt et al., Biomaterials

Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins

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Sudhir K Shukla

2017-01-01

Methods: Biofilm assay was done in 96-well microtitre plate to evaluate the effect of proteinase K on biofilms of bovine mastitis S. Aureus isolates. Extracellular polymeric substances were extracted and evaluated for their composition (protein, polysaccharides and extracellular DNA, before and after the proteinase K treatment. Results: Biofilm assay showed that 2 μg/ml proteinase K significantly inhibited biofilm development in bap-positive S. aureus V329 as well as other S. aureus isolates (SA7, SA10, SA33, SA352, but not in bap-mutant M556 and SA392 (a weak biofilm-producing strain. Proteinase K treatment on S. aureus planktonic cells showed that there was no inhibition of planktonic growth up to 32 μg/ml of proteinase K. Proteinase K treatment on 24 h old preformed biofilms showed an enhanced dispersion of bap-positive V329 and SA7, SA10, SA33 and SA352 biofilms; however, proteinase K did not affect the bap-mutant S. aureus M556 and SA392 biofilms. Biofilm compositions study before and after proteinase K treatment indicated that Bap might also be involved in eDNA retention in the biofilm matrix that aids in biofilm stability. When proteinase K was used in combination with antibiotics, a synergistic effect in antibiotic efficacy was observed against all biofilm-forming S. aureus isolates. Interpretation & conclusions: Proteinase K inhibited biofilms growth in S. aureus bovine mastitis isolates but did not affect their planktonic growth. An enhanced dispersion of preformed S. aureus biofilms was observed on proteinase K treatment. Proteinase K treatment with antibiotics showed a synergistic effect against S. aureus biofilms. The study suggests that dispersing S. aureus by protease can be of use while devising strategies againstS. aureus biofilms.

Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

DEFF Research Database (Denmark)

Baldry, Mara; Nielsen, Anita; Bojer, Martin S.

2016-01-01

Staphylococcus aureus is a serious human pathogen and antibiotic resistant, community-associated strains, such as the methicillin resistant S. aureus (MRSA) strain USA300, continue to spread. To avoid resistance, anti-virulence therapy has been proposed where toxicity is targeted rather than...... viability. Previously we have shown that norlichexanthone, a small non-reduced tricyclic polyketide produced by fungi and lichens, reduces expression of hla encoding α-hemolysin as well as the regulatory RNAIII of the agr quorum sensing system in S. aureus 8325-4. The aim of the present study was to further...... SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation....

Biofilm formation by Staphylococcus aureus from food contact surfaces in a meat-based broth and sensitivity to sanitizers

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Evandro Leite de Souza

2014-01-01

Full Text Available This study assessed the capacity of adhesion, the detachment kinetic and the biofilm formation by Staphylococcus aureus isolated from food services on stainless steel and polypropylene surfaces (2 x 2 cm when cultivated in a meat-based broth at 28 and 7 ºC. It was also to study the efficacy of the sanitizers sodium hypochlorite (250 mg/L and peracetic acid (30 mg/L in inactivating the bacterial cells in the preformed biofilm. S. aureus strains adhered in high numbers regardless the assayed surface kind and incubation temperature over 72 h. Cells detachment of surfaces revealed high persistence over the incubation period. Number of cells needed for biofilm formation was noted at all experimental systems already after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered on polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacity to adhere and form biofilm on polypropylene and stainless steel surfaces under different growth conditions. Moreover, the cells in biofilm matrix were resistant for total removal when submitted to the exposure to sanitizers.

Reducing Staphylococcus aureus biofilm formation on stainless steel 316L using functionalized self-assembled monolayers.

Science.gov (United States)

Kruszewski, Kristen M; Nistico, Laura; Longwell, Mark J; Hynes, Matthew J; Maurer, Joshua A; Hall-Stoodley, Luanne; Gawalt, Ellen S

2013-05-01

Stainless steel 316L (SS316L) is a common material used in orthopedic implants. Bacterial colonization of the surface and subsequent biofilm development can lead to refractory infection of the implant. Since the greatest risk of infection occurs perioperatively, strategies that reduce bacterial adhesion during this time are important. As a strategy to limit bacterial adhesion and biofilm formation on SS316L, self-assembled monolayers (SAMs) were used to modify the SS316L surface. SAMs with long alkyl chains terminated with hydrophobic (-CH3) or hydrophilic (oligoethylene glycol) tail groups were used to form coatings and in an orthogonal approach, SAMs were used to immobilize gentamicin or vancomycin on SS316L for the first time to form an "active" antimicrobial coating to inhibit early biofilm development. Modified SS316L surfaces were characterized using surface infrared spectroscopy, contact angles, MALDI-TOF mass spectrometry and atomic force microscopy. The ability of SAM-modified SS316L to retard biofilm development by Staphylococcus aureus was functionally tested using confocal scanning laser microscopy with COMSTAT image analysis, scanning electron microscopy and colony forming unit analysis. Neither hydrophobic nor hydrophilic SAMs reduced biofilm development. However, gentamicin-linked and vancomycin-linked SAMs significantly reduced S. aureus biofilm formation for up to 24 and 48 h, respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

Chemical composition of essential oil in Mosla chinensis Maxim cv. Jiangxiangru and its inhibitory effect on Staphylococcus aureus biofilm formation

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Peng Liang

2018-03-01

Full Text Available The essential oil of Mosla chinensis Maxim cv. Jiangxiangru is known for its antibacterial ability. This study aimed to investigate the chemical composition of Jiangxiangru essential oil and its inhibitory effect on Staphylococcus aureus biofilm formation. Gas chromatography/mass spectrometry (GC–MS was used to determine the chemical composition of Jiangxiangru essential oil. Subsequently, the eight major chemical components were quantitatively analyzed using GC– MS, and their minimum inhibitory concentration (MIC values against S. aureus were tested. Biofilm formation was detected by crystal violet semi-quantitative method and silver staining. Of the 59 peaks detected, 29 were identified by GC–MS. Of these peaks, thymol, carvacrol, p-cymene, γ-terpinene, thymol acetate, α-caryophyllene, 3-carene, and carvacryl acetate were present at a relatively higher concentration. The results of the quantitative test showed that thymol, carvacrol, p-cymene, and γ-terpinene were the major components of the essential oil. Among the eight reference substances, only thymol, carvacrol, and thymol acetate had lower MICs compared with the essential oil. Essential oil, carvacrol, carvacryl acetate, α-caryophyllene, and 3-carene showed the better inhibition of S. aureus biofilm formation. When one fourth of the MIC concentrations were used for these substances (0.0625 mg/mL for essential oil, 0.0305 mg/mL for carvacrol, 1.458 mg/mL for carvacryl acetate, 0.1268 mg/mL for α-caryophyllene, and 2.5975 mg/mL for 3-carene, the inhibition rates were over 80%. However, thymol, γ-terpinene, thymol acetate, and p-cymene showed a relatively poor inhibition of S. aureus biofilm formation. When 1× MIC concentrations of these substances were used, the inhibition rates were less than 50%. In conclusion, Jiangxiangru essential oil and its major components, carvacrol, carvacryl acetate, α-caryophyllene, and 3-carene, strongly inhibited biofilm formation in S. aureus.

Reducing Staphylococcus aureus biofilm formation on stainless steel 316L using functionalized self-assembled monolayers

International Nuclear Information System (INIS)

Kruszewski, Kristen M.; Nistico, Laura; Longwell, Mark J.; Hynes, Matthew J.; Maurer, Joshua A.; Hall-Stoodley, Luanne; Gawalt, Ellen S.

2013-01-01

Stainless steel 316L (SS316L) is a common material used in orthopedic implants. Bacterial colonization of the surface and subsequent biofilm development can lead to refractory infection of the implant. Since the greatest risk of infection occurs perioperatively, strategies that reduce bacterial adhesion during this time are important. As a strategy to limit bacterial adhesion and biofilm formation on SS316L, self-assembled monolayers (SAMs) were used to modify the SS316L surface. SAMs with long alkyl chains terminated with hydrophobic (− CH 3 ) or hydrophilic (oligoethylene glycol) tail groups were used to form coatings and in an orthogonal approach, SAMs were used to immobilize gentamicin or vancomycin on SS316L for the first time to form an “active” antimicrobial coating to inhibit early biofilm development. Modified SS316L surfaces were characterized using surface infrared spectroscopy, contact angles, MALDI-TOF mass spectrometry and atomic force microscopy. The ability of SAM-modified SS316L to retard biofilm development by Staphylococcus aureus was functionally tested using confocal scanning laser microscopy with COMSTAT image analysis, scanning electron microscopy and colony forming unit analysis. Neither hydrophobic nor hydrophilic SAMs reduced biofilm development. However, gentamicin-linked and vancomycin-linked SAMs significantly reduced S. aureus biofilm formation for up to 24 and 48 h, respectively. - Highlights: ► SS316L was modified with glycol terminated SAMs in order to reduce biofilm growth. ► Antibiotics gentamicin and vancomycin were immobilized on SS316L via SAMs. ► Only the antibiotic modifications reduced biofilm development on SS316L

Reducing Staphylococcus aureus biofilm formation on stainless steel 316L using functionalized self-assembled monolayers

Energy Technology Data Exchange (ETDEWEB)

Kruszewski, Kristen M., E-mail: kruszewskik@duq.edu [Duquesne University, Department of Chemistry and Biochemistry, 600 Forbes Avenue, Pittsburgh, PA 15282 (United States); Nistico, Laura, E-mail: lnistico@wpahs.org [Allegheny General Hospital, Center for Genomic Sciences, Allegheny-Singer Research Institute, 320 East North Avenue, 11th floor, South Tower, Pittsburgh, PA 15212 (United States); Longwell, Mark J., E-mail: mlongwel@wpahs.org [Allegheny General Hospital, Center for Genomic Sciences, Allegheny-Singer Research Institute, 320 East North Avenue, 11th floor, South Tower, Pittsburgh, PA 15212 (United States); Hynes, Matthew J., E-mail: mjhynes@go.wustl.edu [Washington University in St. Louis, Department of Chemistry, One Brookings Drive, St. Louis, MO 63130 (United States); Maurer, Joshua A., E-mail: maurer@wustl.edu [Washington University in St. Louis, Department of Chemistry, One Brookings Drive, St. Louis, MO 63130 (United States); Hall-Stoodley, Luanne, E-mail: L.Hall-Stoodley@soton.ac.uk [Southampton Wellcome Trust Clinical Research Facility/NIHR Respiratory BRU, University of Southampton Faculty of Medicine, Southampton General Hospital, Tremona Road, Southampton, Hampshire SO16 6YD (United Kingdom); Gawalt, Ellen S., E-mail: gawalte@duq.edu [Duquesne University, Department of Chemistry and Biochemistry, McGowan Institute for Regenerative Medicine, 600 Forbes Avenue, Pittsburgh, PA 15282 (United States)

2013-05-01

Stainless steel 316L (SS316L) is a common material used in orthopedic implants. Bacterial colonization of the surface and subsequent biofilm development can lead to refractory infection of the implant. Since the greatest risk of infection occurs perioperatively, strategies that reduce bacterial adhesion during this time are important. As a strategy to limit bacterial adhesion and biofilm formation on SS316L, self-assembled monolayers (SAMs) were used to modify the SS316L surface. SAMs with long alkyl chains terminated with hydrophobic (− CH{sub 3}) or hydrophilic (oligoethylene glycol) tail groups were used to form coatings and in an orthogonal approach, SAMs were used to immobilize gentamicin or vancomycin on SS316L for the first time to form an “active” antimicrobial coating to inhibit early biofilm development. Modified SS316L surfaces were characterized using surface infrared spectroscopy, contact angles, MALDI-TOF mass spectrometry and atomic force microscopy. The ability of SAM-modified SS316L to retard biofilm development by Staphylococcus aureus was functionally tested using confocal scanning laser microscopy with COMSTAT image analysis, scanning electron microscopy and colony forming unit analysis. Neither hydrophobic nor hydrophilic SAMs reduced biofilm development. However, gentamicin-linked and vancomycin-linked SAMs significantly reduced S. aureus biofilm formation for up to 24 and 48 h, respectively. - Highlights: ► SS316L was modified with glycol terminated SAMs in order to reduce biofilm growth. ► Antibiotics gentamicin and vancomycin were immobilized on SS316L via SAMs. ► Only the antibiotic modifications reduced biofilm development on SS316L.

In vitro biofilm formation by methicillin susceptible and resistant Staphylococcus aureus strains isolated from cystic fibrosis patients

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Antonietta Lambiase

2008-12-01

Full Text Available Staphylococcus aureus is one of the most common pathogens isolated from respiratory tracts of Cystic Fibrosis patients (CF. The infection by this pathogen starts in early infancy, often preceding chronic infections by Pseudomonas aeruginosa. The infection and colonization by methicillin-resistant Staphylococcus aureus (MRSA are, by then, events very frequent among CF patients and this bacterial isolation leads to complications in therapeutic management because of the limited treatment options. Strains of Staphylococcus aureus are able to produce biofilms on natural or synthetic surfaces. Biofilms are sophisticated communities of matrix-encased bacteria and infections by biofilm-producing bacteria are particularly problematic because sessile bacteria can often withstand host immune responses and are generally much more tolerant to antibiotics. The first aim of this work is to evaluate the ability of MRSA strains isolated from respiratory secretions of CF patients to develop biofilms in comparison with methicillin-sensitive Staphylococcus aureus (MSSA strains obtained from respiratory secretions of CF patients.Therefore, our second aim is to evaluate the environmental influence on this ability. To evaluate the development of biofilm on solid matrix and the possible environmental influence,we applied the method described by Christensen et al. We found that a significantly higher number of MRSA strains were biofilm positive compared with MSSA strains (p<0.05.The presence of glucose did not influence the ability to form biofilm in our MRSA strains (p=0.165. MSSA strains are not strong biofilm-producers, but, when grown in TSB added with 0.25% glucose, the number of biofilm-forming strains increases, as expected. These data suggest a possible association between methicillin-resistance and biofilm formation.

Staphylococcus aureus sarA regulates inflammation and colonization during central nervous system biofilm formation.

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Jessica N Snowden

Full Text Available Infection is a frequent and serious complication following the treatment of hydrocephalus with CSF shunts, with limited therapeutic options because of biofilm formation along the catheter surface. Here we evaluated the possibility that the sarA regulatory locus engenders S. aureus more resistant to immune recognition in the central nervous system (CNS based on its reported ability to regulate biofilm formation. We utilized our established model of CNS catheter-associated infection, similar to CSF shunt infections seen in humans, to compare the kinetics of bacterial titers, cytokine production and inflammatory cell influx elicited by wild type S. aureus versus an isogenic sarA mutant. The sarA mutant was more rapidly cleared from infected catheters compared to its isogenic wild type strain. Consistent with this finding, several pro-inflammatory cytokines and chemokines, including IL-17, CXCL1, and IL-1β were significantly increased in the brain following infection with the sarA mutant versus wild type S. aureus, in agreement with the fact that the sarA mutant displayed impaired biofilm growth and favored a planktonic state. Neutrophil influx into the infected hemisphere was also increased in the animals infected with the sarA mutant compared to wild type bacteria. These changes were not attributable to extracellular protease activity, which is increased in the context of SarA mutation, since similar responses were observed between sarA and a sarA/protease mutant. Overall, these results demonstrate that sarA plays an important role in attenuating the inflammatory response during staphylococcal biofilm infection in the CNS via a mechanism that remains to be determined.

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

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

2017-12-01

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

Maggot excretions inhibit biofilm formation on biomaterials.

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Cazander, Gwendolyn; van de Veerdonk, Mariëlle C; Vandenbroucke-Grauls, Christina M J E; Schreurs, Marco W J; Jukema, Gerrolt N

2010-10-01

Biofilm-associated infections in trauma surgery are difficult to treat with conventional therapies. Therefore, it is important to develop new treatment modalities. Maggots in captured bags, which are permeable for larval excretions/secretions, aid in healing severe, infected wounds, suspect for biofilm formation. Therefore we presumed maggot excretions/secretions would reduce biofilm formation. We studied biofilm formation of Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis, and Enterobacter cloacae on polyethylene, titanium, and stainless steel. We compared the quantities of biofilm formation between the bacterial species on the various biomaterials and the quantity of biofilm formation after various incubation times. Maggot excretions/secretions were added to existing biofilms to examine their effect. Comb-like models of the biomaterials, made to fit in a 96-well microtiter plate, were incubated with bacterial suspension. The formed biofilms were stained in crystal violet, which was eluted in ethanol. The optical density (at 595 nm) of the eluate was determined to quantify biofilm formation. Maggot excretions/secretions were pipetted in different concentrations to (nonstained) 7-day-old biofilms, incubated 24 hours, and finally measured. The strongest biofilms were formed by S. aureus and S. epidermidis on polyethylene and the weakest on titanium. The highest quantity of biofilm formation was reached within 7 days for both bacteria. The presence of excretions/secretions reduced biofilm formation on all biomaterials. A maximum of 92% of biofilm reduction was measured. Our observations suggest maggot excretions/secretions decrease biofilm formation and could provide a new treatment for biofilm formation on infected biomaterials.

Effects of Low-Dose Amoxicillin on Staphylococcus aureus USA300 Biofilms.

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Mlynek, Kevin D; Callahan, Mary T; Shimkevitch, Anton V; Farmer, Jackson T; Endres, Jennifer L; Marchand, Mélodie; Bayles, Kenneth W; Horswill, Alexander R; Kaplan, Jeffrey B

2016-05-01

Previous studies showed that sub-MIC levels of β-lactam antibiotics stimulate biofilm formation in most methicillin-resistant Staphylococcus aureus (MRSA) strains. Here, we investigated this process by measuring the effects of sub-MIC amoxicillin on biofilm formation by the epidemic community-associated MRSA strain USA300. We found that sub-MIC amoxicillin increased the ability of USA300 cells to attach to surfaces and form biofilms under both static and flow conditions. We also found that USA300 biofilms cultured in sub-MIC amoxicillin were thicker, contained more pillar and channel structures, and were less porous than biofilms cultured without antibiotic. Biofilm formation in sub-MIC amoxicillin correlated with the production of extracellular DNA (eDNA). However, eDNA released by amoxicillin-induced cell lysis alone was evidently not sufficient to stimulate biofilm. Sub-MIC levels of two other cell wall-active agents with different mechanisms of action-d-cycloserine and fosfomycin-also stimulated eDNA-dependent biofilm, suggesting that biofilm formation may be a mechanistic adaptation to cell wall stress. Screening a USA300 mariner transposon library for mutants deficient in biofilm formation in sub-MIC amoxicillin identified numerous known mediators of S. aureus β-lactam resistance and biofilm formation, as well as novel genes not previously associated with these phenotypes. Our results link cell wall stress and biofilm formation in MRSA and suggest that eDNA-dependent biofilm formation by strain USA300 in low-dose amoxicillin is an inducible phenotype that can be used to identify novel genes impacting MRSA β-lactam resistance and biofilm formation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Susceptibility of Staphylococcus aureus biofilms to reactive discharge gases.

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Traba, Christian; Liang, Jun F

2011-08-01

Formation of bacterial biofilms at solid-liquid interfaces creates numerous problems in both industrial and biomedical sciences. In this study, the susceptibility of Staphylococcus aureus biofilms to discharge gas generated from plasma was tested. It was found that despite distinct chemical/physical properties, discharge gases from oxygen, nitrogen, and argon demonstrated very potent and almost the same anti-biofilm activity. The bacterial cells in S. aureus biofilms were killed (>99.9%) by discharge gas within minutes of exposure. Under optimal experimental conditions, no bacteria and biofilm re-growth from discharge gas treated biofilms was found. Further studies revealed that the anti-biofilm activity of the discharge gas occurred by two distinct mechanisms: (1) killing bacteria in biofilms by causing severe cell membrane damage, and (2) damaging the extracellular polymeric matrix in the architecture of the biofilm to release biofilm from the surface of the solid substratum. Information gathered from this study provides an insight into the anti-biofilm mechanisms of plasma and confirms the applications of discharge gas in the treatment of biofilms and biofilm related bacterial infections.

Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus.

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Shimamura, Yuko; Hirai, Chikako; Sugiyama, Yuka; Shibata, Masaharu; Ozaki, Junya; Murata, Masatsune; Ohashi, Norio; Masuda, Shuichi

2017-12-01

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.

Bioactive Compounds Produced by Hypoxylon fragiforme against Staphylococcus aureus Biofilms

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Kamila Tomoko Yuyama

2017-12-01

Full Text Available Treating infections organized in biofilms is a challenge due to the resistance of the pathogens against antibiotics and host immune cells. Many fungi grow in a wet environment, favorable for the growth of bacterial biofilms, and we speculated that fungi possess some strategies to control these bacterial biofilms. A fungus identified as Hypoxylon fragiforme, was collected in the Harz Mountains, Germany, and its mycelial culture was fermented in different culture media for 67 days to test its biological potential against bacterial biofilms. Sclerin, sclerin diacid and its 3-methyl monoester (methyl 1-(5-hydroxy-6-carboxylic-2,3,4-trimethylphenyl propionate are here described for the first time from this fungus. Sclerin and its diacid interfered with the biofilm formation of the pathogen Staphylococcus aureus, inhibiting 86% and 80% of the biofilm at 256 μg mL−1, respectively, but not killing the bacterium. Interestingly, the monomethylester of sclerin diacid was inactive. Although these compounds did not possess any activity against a pre-formed biofilm, they prevented its formation at subtoxic concentrations. Furthermore, sclerin and its diacid displayed a high specificity against Staphylococcus aureus, indicating a good strategy against pathogenic biofilms when combined with antibiotics.

Investigation of biofilm formation on contact eye lenses caused by methicillin resistant Staphylococcus aureus.

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Khalil, M A; Sonbol, F I

2014-01-01

The objective was to investigate the biofilm-forming capacity of methicillin resistant Staphylococcus aureus (MRSA) isolated from eye lenses of infected patients. A total of 32 MRSA isolated from contact lenses of patients with ocular infections were screened for their biofilm-forming capacity using tube method (TM), Congo red agar (CRA), and microtiter plate (MtP) methods. The effect of some stress factor on the biofilm formation was studied. The biofilm-forming related genes, icaA, icaD and 10 microbial surface components that recognize adhesive matrix molecule (MSCRAMM), of the selected MRSA were also detected using polymerase chain reaction. Of 32 MRSA isolates, 34.37%, 59.37%, and 81.25% showed positive results using CRA, TM or MtP, respectively. Biofilm production was found to be reduced in the presence of ethanol or ethylenediaminetetraacetic acid and at extreme pH values. On the other hand, glucose or heparin leads to a concentration dependent increase of biofilm production by the isolates. The selected biofilm producing MRSA isolate was found to harbor the icaA, icaD and up to nine of 10 tested MSCRAMM genes, whereas the selected non biofilm producing MRSA isolate did not carry any of the tested genes. The MtP method was found to be the most effective phenotypic screening method for detection of biofilm formation by MRSA. Furthermore, the molecular approach should be taken into consideration for the rapid and correct diagnosis of virulent bacteria associated with contact eye lenses.

Evaluation of biofilm formation using milk in a flow cell model and microarray characterization of Staphylococcus aureus strains from bovine mastitis.

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Snel, G G M; Malvisi, M; Pilla, R; Piccinini, R

2014-12-05

It was hypothesized that biofilm could play an important role in the establishment of chronic Staphylococcus aureus bovine mastitis. The in vitro evaluation of biofilm formation can be performed either in closed/static or in flow-based systems. Efforts have been made to characterize the biofilm-forming ability of S. aureus mastitis isolates, however most authors used static systems and matrices other than UHT milk. It is not clear whether such results could be extrapolated to the mammary gland environment. Therefore, the present study aimed to investigate the biofilm-forming ability of S. aureus strains from subclinical bovine mastitis using the static method and a flow-based one. One hundred and twelve strains were tested by the classic tissue culture plate assay (TCP) and 30 out of them were also tested by a dynamic semi-quantitative assay using commercial UHT milk as culture medium (Milk Flow Culture, MFC) or Tryptic Soy Broth as control medium (TS Flow Culture, TSFC). Only 6 (20%) strains formed biofilm in milk under flow conditions, while 36.6% were considered biofilm-producers in TCP, and 93.3% produced biofilm in TSFC. No agreement was found between TCP, MFC and TSFC results. The association between strain genetic profile, determined by microarray, and biofilm-forming ability in milk was evaluated. Biofilm formation in MFC was significantly associated with the presence of those genes commonly found in bovine-associated strains, assigned to clonal complexes typically detected in mastitis. Based on our results, biofilm-forming potential of bovine strains should be critically analysed and tested applying conditions similar to mammary environment. Copyright © 2014 Elsevier B.V. All rights reserved.

Formation of Staphylococcus aureus Biofilm in the Presence of Sublethal Concentrations of Disinfectants Studied via a Transcriptomic Analysis Using Transcriptome Sequencing (RNA-seq)

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Oppelt, J.; Cincarova, L.

2017-01-01

ABSTRACT Staphylococcus aureus is a common biofilm-forming pathogen. Low doses of disinfectants have previously been reported to promote biofilm formation and to increase virulence. The aim of this study was to use transcriptome sequencing (RNA-seq) analysis to investigate global transcriptional changes in S. aureus in response to sublethal concentrations of the commonly used food industry disinfectants ethanol (EtOH) and chloramine T (ChT) and their combination (EtOH_ChT) in order to better understand the effects of these agents on biofilm formation. Treatment with EtOH and EtOH_ChT resulted in more significantly altered expression profiles than treatment with ChT. Our results revealed that EtOH and EtOH_ChT treatments enhanced the expression of genes responsible for regulation of gene expression (sigB), cell surface factors (clfAB), adhesins (sdrDE), and capsular polysaccharides (cap8EFGL), resulting in more intact biofilm. In addition, in this study we were able to identify the pathways involved in the adaptation of S. aureus to the stress of ChT treatment. Further, EtOH suppressed the effect of ChT on gene expression when these agents were used together at sublethal concentrations. These data show that in the presence of sublethal concentrations of tested disinfectants, S. aureus cells trigger protective mechanisms and try to cope with them. IMPORTANCE So far, the effect of disinfectants is not satisfactorily explained. The presented data will allow a better understanding of the mode of disinfectant action with regard to biofilm formation and the ability of bacteria to survive the treatment. Such an understanding could contribute to the effort to eliminate possible sources of bacteria, making disinfectant application as efficient as possible. Biofilm formation plays significant role in the spread and pathogenesis of bacterial species. PMID:29030437

Efficacy of NVC-422 against Staphylococcus aureus biofilms in a sheep biofilm model of sinusitis.

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Singhal, Deepti; Jekle, Andreas; Debabov, Dmitri; Wang, Lu; Khosrovi, Bez; Anderson, Mark; Foreman, Andrew; Wormald, Peter-John

2012-01-01

Bacterial biofilms are a major obstacle in management of recalcitrant chronic rhinosinusitis. NVC-422 is a potent, fast-acting, broad-spectrum, nonantibiotic, antimicrobial with a new mechanism of action effective against biofilm bacteria in in vitro conditions. The aim of this study was to investigate the safety and efficacy of NVC-422 as local antibiofilm treatment in a sheep model of rhinosinusitis. After accessing and occluding frontal sinus ostia in 24 merino sheep via staged endoscopic procedures, S. aureus clinical isolate was instilled in frontal sinuses. Following biofilm formation, ostial obstruction was removed and sinuses irrigated with 0.1% and 0.5% NVC-422 in 5 mM acetate isotonic saline at pH 4.0. Sheep were monitored for adverse effects and euthanized 24 hours after treatment. Frontal sinuses were assessed for infection and changes in mucosa after the treatment. S. aureus biofilms were identified with Baclight-confocal scanning microscopy protocol and the biofilm biomass assayed by applying the COMSTAT2 program to recorded image stacks. After 2 irrigations with 0.1% NVC-422, S. aureus biofilm biomass was reduced when compared to control sinuses (p = 0.0001), though this effect was variable in samples. NVC-422 0.5% solution irrigations reduced biofilm even more significantly and consistently over all samples (p biofilm biomass (p biofilms, with dose-dependent efficacy in this animal model of biofilm-associated sinusitis. Copyright © 2012 American Rhinologic Society-American Academy of Otolaryngic Allergy, LLC.

Effects of Subinhibitory Concentrations of Ceftaroline on Methicillin-Resistant Staphylococcus aureus (MRSA Biofilms.

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María Lázaro-Díez

Full Text Available Ceftaroline (CPT is a novel cephalosporin with in vitro activity against Staphylococcus aureus. Ceftaroline exhibits a level of binding affinity for PBPs in S. aureus including PBP2a of methicillin-resistant S. aureus (MRSA. The aims of this study were to investigate the morphological, physiological and molecular responses of MRSA clinical strains and MRSA biofilms to sub-MICs (1/4 and 1/16 MIC of ceftaroline by using transmission, scanning and confocal microscopy. We have also used quantitative Real-Time PCR to study the effect of sub-MICs of ceftaroline on the expression of the staphylococcal icaA, agrA, sarA and sasF genes in MRSA biofilms. In one set of experiments, ceftaroline was able to inhibit biofilm formation in all strains tested at MIC, however, a strain dependent behavior in presence of sub-MICs of ceftaroline was shown. In a second set of experiments, destruction of preformed biofilms by addition of ceftaroline was evaluated. Ceftaroline was able to inhibit biofilm formation at MIC in all strains tested but not at the sub-MICs. Destruction of preformed biofilms was strain dependent because the biofilm formed by a matrix-producing strain was resistant to a challenge with ceftaroline at MIC, whereas in other strains the biofilm was sensitive. At sub-MICs, the impact of ceftaroline on expression of virulence genes was strain-dependent at 1/4 MIC and no correlation between ceftaroline-enhanced biofilm formation and gene regulation was established at 1/16 MIC. Our findings suggest that sub-MICs of ceftaroline enhance bacterial attachment and biofilm formation by some, but not all, MRSA strains and, therefore, stress the importance of maintaining effective bactericidal concentrations of ceftaroline to fight biofilm-MRSA related infections.

Effects of Fermented Sumach on the Formation of Slime Layer of Staphylococcus aureus

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Sahra Kırmusaoğlu

2012-03-01

Full Text Available Objective: Staphylococcus aureus (S. aureus is one of the most commonly isolated bacterial pathogens in hospitals, and the most frequent cause of nosocomial infections. Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. Implantation of intravenous catheters and surgical implantation of prosthetic implants carry a risk of infection. In order to prevent all these effects of biofilms, a study was designed to observe the possible antibacterial effect of sumach (Rhus coriaria on the biofilm formation of S. aureus. Material and Methods: The influence of varying concentrations of sumach on the formation of biofilms by 13 strains of Staphylococcus aureus was tested by a microelisa assay. Results: The significant differences between varying concentrations of sumach (0.1, 0.2, 0.5 and 1.0 µl/ml were observed in four methicillin resistant Staphylococcus aureus (MRSA and nine methicillin sensitive Staphylococcus aureus (MSSA (p<0.05. In bacteria, a dose-related decrease in the formation of slime, which is a major virulence factor of staphylococcal infections, was observed. Conclusion: In our study, using 0.1, 0.2, 0.5 and 1.0 µl/ml of sumach, thirteen strains lost, 17%, 22%, 28% and 48% respectively of their capacity to produce biofilms. Sumach, which is a herbal product, can decrease the formation of biofilm, which is a major virulence factor in staphylococcal infections.

Melittin and its potential in the destruction and inhibition of the biofilm formation by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa isolated from bovine milk.

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Picoli, Tony; Peter, Cristina Mendes; Zani, João Luíz; Waller, Stefanie Bressan; Lopes, Matheus Gomes; Boesche, Kamilla Neutzling; Vargas, Gilberto D Ávila; Hübner, Silvia de Oliveira; Fischer, Geferson

2017-11-01

Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa stand out in veterinary and human medicine for their role in opportunistic infections and their pathogenic mechanisms, including the biofilms formation. It was investigated the antibacterial activity of melittin and antibiofilm of such bacteria. Twelve strains of these microorganisms isolated from bovine milk were used, as well as the strains S. aureus ATCC 12600, E. coli ATCC 8739 and Pseudomonas aeruginosa ATCC 15442. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution technique. The biofilms were formed in 96-well plates and melittin on these colonies was added at different concentrations and times. Bacteria previously exposed to melittin were evaluated for inhibition of biofilm production. The MIC and MBC were respectively in μg/mL: S. aureus (6-7 and 32-64), E. coli (40-42.5 and 64-128) and P. aeruginosa (65-70 and 64-128). S. aureus biofilms were more sensitive to the action of melittin, since upon exposure to a concentration 10 times lower than the MIC for 4 h, was completely destroyed. In Gram negative bacteria, the pre-formed biofilm was destroyed only when exposed for 4 h under the MIC. With respect to inhibition of biofilm production, S. aureus was the most sensitive again because produced only 37.2% of the biofilm formed by the control (without previous exposure to melittin), when exposed to the MIC, and at a concentration hundred times smaller than MIC, this microorganism produced 75.2% of the biofilm. E. coli was the most resistant bacteria and produced 56.3% of the biofilm, even if previously exposed to melittin MIC. Melittin presents desirable effects in combating microorganisms studied both at your disposal, biofilm destruction and inhibition of the formation, and maybe used in future studies of new strategies to combat infections caused by these pathogens. Copyright © 2017 Elsevier Ltd. All

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

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Xue, Ting; Chen, Xiaolin; Shang, Fei

2014-10-01

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

Combinations of maggot excretions/secretions and antibiotics are effective against Staphylococcus aureus biofilms and the bacteria derived therefrom

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van der Plas, Mariena J A; Dambrot, Cheryl; Dogterom-Ballering, Heleen C M

2010-01-01

OBJECTIVES: Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Previously we reported that maggot excretions/secretions (ES) break down Staphylococcus aureus biofilms but do not kill the bacteria. As many antibiotics are not effective against biofilms we assessed...... the effect of combinations of ES and antibiotics on S. aureus biofilms and on the survival of the bacteria released from the biofilms. METHODS: Effects of ES, antibiotics (vancomycin, daptomycin or clindamycin) and combinations thereof on S. aureus ATCC 29 213 biofilms and bacterial viability were determined...... using microtitre plates and in vitro killing assays. RESULTS: Vancomycin and daptomycin dose-dependently enhanced biofilm formation, whereas clindamycin reduced S. aureus biofilm size. Adding ES to antibiotic incubations caused a complete biofilm breakdown. After a lag time the bacteria derived from...

Cloning, Expression and Characterization of NAD Kinase from Staphylococcus aureus Involved in the Formation of NADP (H: A Key Molecule in the Maintaining of Redox Status and Biofilm Formation

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U Venkateswara Prasad

2017-01-01

Full Text Available Background:Staphylococcus aureus has the ability to form biofilms on any niches, a key pathogenic factor of this organism and this phenomenon is directly related to the concentration of NADPH. The formation of NADP is catalyzed by NAD kinase (NADK and this gene of S. aureus ATCC 12600 was cloned, sequenced, expressed and characterized. Materials and Methods: The NADK gene was polymerase chain reaction amplified from the chromosomal DNA of S. aureus ATCC 12600 and cloned in pQE 30 vector, sequenced and expressed in Escherichia coli DH5α. The pure protein was obtained by passing through nickel metal chelate agarose column. The enzyme kinetics of the enzyme and biofilm assay of the S. aureus was carried out in both aerobic and anaerobic conditions. The kinetics was further confirmed by the ability of the substrates to dock to the NADK structure. Results: The recombinant NADK exhibited single band with a molecular weight of 31kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence (GenBank: JN645814 revealed presence of only one kind of NADK in all S. aureus strains. The enzyme exhibited very high affinity for NAD compared to adenosine triphosphate concurring with the docking results. A root-mean-square deviation value 14.039Š observed when NADK structure was superimposed with its human counterpart suggesting very low homology. In anaerobic conditions, higher biofilm units were found with decreased NADK activity. Conclusion: The results of this study suggest increased NADPH concentration in S. aureus plays a vital role in the biofilm formation and survival of this pathogen in any environmental conditions.

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

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

2016-01-01

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

Bacterial biofilm formation in different surfaces of food industries

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Karine Angélica Dalla Costa

2017-06-01

Full Text Available The term biofilm describes the sessile microbial life form, characterized by microorganism adhesion to any surface and with the production of extracellular polymeric substances. In food industries, the formation of biofilms results in serious problems, since it can be a contamination source of the food product, compromising the final product quality and consumer health. The aim of this study was to verify the adhesion of biofilms (sessile cells of pathogenic and/or deteriorating bacteria against surfaces of the food industry. The bacterial species tested were Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, Listeria monocytogenes ATCC 19117 and Salmonella Typhimurium ATCC 14028. It was used stainless steel and polypropylene coupons as contact surfaces. The results demonstrated that P. aeruginosa and S. Typhimurium showed higher biofilm formation capacity. Statistically, there was no difference in count of P. aeruginosa and S. Typhimurium (p > 0.05 cells. The same occurred between L. monocytogenes and S. aureus. However, the counts of P. aeruginosa and S. Typhimurium cells were statistically higher than S. aureus and L. monocytogenes (p < 0.05. By means of scanning electron microscopy it was also found increased adhesion of P. aeruginosa. The results revealed that P. aeruginosa was the bacterial species with higher biofilm formation capacity among the others.

Antimicrobial activity of zinc and titanium dioxide nanoparticles against biofilm-producing methicillin-resistant Staphylococcus aureus

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Jesline, A.; John, Neetu P.; Narayanan, P. M.; Vani, C.; Murugan, Sevanan

2015-02-01

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens responsible for a wide spectrum of infections and the emergence of bacterial resistance to antibiotics has lead to treatment drawbacks towards large number of drugs. Formation of biofilms is the main contributing factor to antibiotic resistance. The development of reliable processes for the synthesis of zinc oxide nanoparticles is an important aspect of nanotechnology today. Zinc oxide and titanium dioxide nanoparticles comprise well-known inhibitory and bactericidal effects. Emergence of antimicrobial resistance by pathogenic bacteria is a major health problem in recent years. This study was designed to determine the efficacy of zinc and titanium dioxide nanoparticles against biofilm producing methicillin-resistant S. aureus. Biofilm production was detected by tissue culture plate method. Out of 30 MRSA isolates, 22 isolates showed strong biofilm production and 2 showed weak and moderate biofilm formation. Two strong and weak biofilm-producing methicillin-resistant S. aureus isolates were subjected to antimicrobial activity using commercially available zinc and titanium dioxide nanoparticles. Thus, the nanoparticles showed considerably good activity against the isolates, and it can be concluded that they may act as promising, antibacterial agents in the coming years.

Femtosecond laser surface texturing of titanium as a method to reduce the adhesion of Staphylococcus aureus and biofilm formation

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Cunha, Alexandre [Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa (Portugal); Bordeaux University, Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN UMR 5248, CNRS), European Institute of Chemistry and Biology, 2 Rue Robert Escarpit, 33607 Pessac (France); Elie, Anne-Marie [Bordeaux University, CBMN UMR 5248, CNRS, Bordeaux Science Agro, 1 Rue du G. de Gaulle, 33170 Gradignan (France); Plawinski, Laurent [Bordeaux University, Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN UMR 5248, CNRS), European Institute of Chemistry and Biology, 2 Rue Robert Escarpit, 33607 Pessac (France); Serro, Ana Paula [Instituto Superior Técnico, Universidade de Lisboa, CQE-Centro de Química Estrutural, Av. Rovisco Pais 1, 1049-001 Lisbon (Portugal); Botelho do Rego, Ana Maria [Instituto Superior Técnico, Universidade de Lisboa, CQFM-Centro de Química-Física Molecular and Institute of Nanoscience and Nanotechnology - IN, Av. Rovisco Pais 1, 1049-001 Lisbon (Portugal); Almeida, Amélia [Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa (Portugal); Urdaci, Maria C. [Bordeaux University, CBMN UMR 5248, CNRS, Bordeaux Science Agro, 1 Rue du G. de Gaulle, 33170 Gradignan (France); Durrieu, Marie-Christine [Bordeaux University, Institute of Chemistry & Biology of Membranes & Nanoobjects (CBMN UMR 5248, CNRS), European Institute of Chemistry and Biology, 2 Rue Robert Escarpit, 33607 Pessac (France); Vilar, Rui, E-mail: rui.vilar@tecnico.ulisboa.pt [Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais 1, 1049-001 Lisboa (Portugal)

2016-01-01

Graphical abstract: - Highlights: • The short-term adhesion of Staphylococcus aureus onto femtosecond laser textured surfaces of titanium was investigated. • The laser textured surfaces consist of laser-induced periodic surface structures (LIPSS) and nanopillars. • The laser treatment enhances the hydrophilicity and the surface free energy of the material. • The laser treatment reduces significantly the adhesion of S. aureus and biofilm formation. • Femtosecond laser surface texturing of titanium is a simple and promising method for endowing dental and orthopedic implants with antibacterial properties. - Abstract: The aim of the present work was to investigate the possibility of using femtosecond laser surface texturing as a method to reduce the colonization of Grade 2 Titanium alloy surfaces by Staphylococcus aureus and the subsequent formation of biofilm. The laser treatments were carried out with a Yb:KYW chirped-pulse-regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. Two types of surface textures, consisting of laser-induced periodic surface structures (LIPSS) and nanopillars, were produced. The topography, chemical composition and phase constitution of these surfaces were investigated by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, micro-Raman spectroscopy, and X-ray diffraction. Surface wettability was assessed by the sessile drop method using water and diiodomethane as testing liquids. The response of S. aureus put into contact with the laser treated surfaces in controlled conditions was investigated by epifluorescence microscopy and scanning electron microscopy 48 h after cell seeding. The results achieved show that the laser treatment reduces significantly the bacterial adhesion to the surface as well as biofilm formation as compared to a reference polished surfaces and suggest that femtosecond laser texturing is a simple and promising method

The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

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Liu, Qian; Wang, Xing; Qin, Juanxiu; Cheng, Sen; Yeo, Won-Sik; He, Lei; Ma, Xiaowei; Liu, Xiaoyun; Li, Min; Bae, Taeok

2017-01-01

Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA). The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection. PMID:28555174

The ATP-Dependent Protease ClpP Inhibits Biofilm Formation by Regulating Agr and Cell Wall Hydrolase Sle1 in Staphylococcus aureus

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Qian Liu

2017-05-01

Full Text Available Biofilm causes hospital-associated infections on indwelling medical devices. In Staphylococcus aureus, Biofilm formation is controlled by intricately coordinated network of regulating systems, of which the ATP-dependent protease ClpP shows an inhibitory effect. Here, we demonstrate that the inhibitory effect of ClpP on biofilm formation is through Agr and the cell wall hydrolase Sle1. Biofilm formed by clpP mutant consists of proteins and extracellular DNA (eDNA. The increase of the protein was, at least in part, due to the reduced protease activity of the mutant, which was caused by the decreased activity of agr. On the other hand, the increase of eDNA was due to increased cell lysis caused by the higher level of Sle1. Indeed, as compared with wild type, the clpP mutant excreted an increased level of eDNA, and showed higher sensitivity to Triton-induced autolysis. The deletion of sle1 in the clpP mutant decreased the biofilm formation, the level of eDNA, and the Triton-induced autolysis to wild-type levels. Despite the increased biofilm formation capability, however, the clpP mutant showed significantly reduced virulence in a murine model of subcutaneous foreign body infection, indicating that the increased biofilm formation capability cannot compensate for the intrinsic functions of ClpP during infection.

Biofilm formation by Staphylococcus aureus and Salmonella spp. under mono and dual-species conditions and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite

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Maricarmen Iñiguez-Moreno

Full Text Available Abstract The aim of this study was evaluated the biofilm formation by Staphylococcus aureus 4E and Salmonella spp. under mono and dual-species biofilms, onto stainless steel 316 (SS and polypropylene B (PP, and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite. The biofilms were developed by immersion of the surfaces in TSB by 10 d at 37 °C. The results showed that in monospecies biofilms the type of surface not affected the cellular density (p > 0.05. However, in dual-species biofilms on PP the adhesion of Salmonella spp. was favored, 7.61 ± 0.13 Log10 CFU/cm2, compared with monospecies biofilms onto the same surface, 5.91 ± 0.44 Log10 CFU/cm2 (p < 0.05. The mono and dual-species biofilms were subjected to disinfection treatments; and the most effective disinfectant was peracetic acid (3500 ppm, reducing by more than 5 Log10 CFU/cm2, while the least effective was cetrimonium bromide. In addition, S. aureus 4E and Salmonella spp. were more resistant to the disinfectants in mono than in dual-species biofilms (p < 0.05. Therefore, the interspecies interactions between S. aureus 4E and Salmonella spp. had a negative effect on the antimicrobial resistance of each microorganism, compared with the monospecies biofilms.

Bactericidal and Anti-biofilm Effects of Polyhexamethylene Biguanide in Models of Intracellular and Biofilm of Staphylococcus aureus Isolated from Bovine Mastitis

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Nor F. Kamaruzzaman

2017-08-01

Full Text Available Staphylococcus aureus infection is a common cause of mastitis, reducing milk yield, affecting animal welfare and causing huge economic losses within the dairy industry. In addition to the problem of acquired drug resistance, bacterial invasion into udder cells and the formation of surface biofilms are believed to reduce antibiotic efficacy, leading to treatment failure. Here, we investigated the antimicrobial activities of enrofloxacin, an antibiotic that is commonly used in mastitis therapy and polyhexamethylene biguanide (PHMB, an antimicrobial polymer. The antimicrobial activities were tested against intracellular S. aureus in infected Mac-T cells (host cells. Also, fluorescein-tagged PHMB was used to study PHMB uptake and localization with S. aureus within the infected Mac-T cells. Anti-biofilm activities were tested by treating S. aureus biofilms and measuring effects on biofilm mass in vitro. Enrofloxacin and PHMB at 15 mg/L killed between 42 to 92 and 99.9% of intracellular S. aureus, respectively. PHMB-FITC entered and colocalized with the intracellular S. aureus, suggesting direct interaction of the drug with the bacteria inside the host cells. Enrofloxacin and PHMB at 15 mg/L reduced between 10 to 27% and 28 to 37% of biofilms’ mass, respectively. The half-maximal inhibitory concentrations (IC50 obtained from a cytotoxicity assay were 345 ± 91 and 21 ± 2 mg/L for enrofloxacin and PHMB, respectively; therefore, both compounds were tolerated by the host cells at high concentrations. These findings suggest that both antimicrobials are effective against intracellular S. aureus and can disrupt biofilm structures, with PHMB being more potent against intracellular S. aureus, highlighting the potential application of PHMB in mastitis therapy.

Transcriptional and functional analysis of the effects of magnolol: inhibition of autolysis and biofilms in Staphylococcus aureus.

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Wang, Dacheng; Jin, Qi; Xiang, Hua; Wang, Wei; Guo, Na; Zhang, Kaiyu; Tang, Xudong; Meng, Rizeng; Feng, Haihua; Liu, Lihui; Wang, Xiaohong; Liang, Junchao; Shen, Fengge; Xing, Mingxun; Deng, Xuming; Yu, Lu

2011-01-01

The targeting of Staphylococcus aureus biofilm structures are now gaining interest as an alternative strategy for developing new types of antimicrobial agents. Magnolol (MOL) shows inhibitory activity against S. aureus biofilms and Triton X-100-induced autolysis in vitro, although there are no data regarding the molecular mechanisms of MOL action in bacteria. The molecular basis of the markedly reduced autolytic phenotype and biofilm inhibition triggered by MOL were explored using transcriptomic analysis, and the transcription of important genes were verified by real-time RT-PCR. The inhibition of autolysis by MOL was evaluated using quantitative bacteriolytic assays and zymographic analysis, and antibiofilm activity assays and confocal laser scanning microscopy were used to elucidate the inhibition of biofilm formation caused by MOL in 20 clinical isolates or standard strains. The reduction in cidA, atl, sle1, and lytN transcript levels following MOL treatment was consistent with the induced expression of their autolytic repressors lrgA, lrgB, arlR, and sarA. MOL generally inhibited or reversed the expression of most of the genes involved in biofilm production. The growth of S. aureus strain ATCC 25923 in the presence of MOL dose-dependently led to decreases in Triton X-100-induced autolysis, extracellular murein hydrolase activity, and the amount of extracellular DNA (eDNA). MOL may impede biofilm formation by reducing the expression of cidA, a murein hydrolase regulator, to inhibit autolysis and eDNA release, or MOL may directly repress biofilm formation. MOL shows in vitro antimicrobial activity against clinical and standard S. aureus strains grown in planktonic and biofilm cultures, suggesting that the structure of MOL may potentially be used as a basis for the development of drugs targeting biofilms.

Transcriptional and functional analysis of the effects of magnolol: inhibition of autolysis and biofilms in Staphylococcus aureus.

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Dacheng Wang

Full Text Available BACKGROUND: The targeting of Staphylococcus aureus biofilm structures are now gaining interest as an alternative strategy for developing new types of antimicrobial agents. Magnolol (MOL shows inhibitory activity against S. aureus biofilms and Triton X-100-induced autolysis in vitro, although there are no data regarding the molecular mechanisms of MOL action in bacteria. METHODOLOGY/PRINCIPAL FINDINGS: The molecular basis of the markedly reduced autolytic phenotype and biofilm inhibition triggered by MOL were explored using transcriptomic analysis, and the transcription of important genes were verified by real-time RT-PCR. The inhibition of autolysis by MOL was evaluated using quantitative bacteriolytic assays and zymographic analysis, and antibiofilm activity assays and confocal laser scanning microscopy were used to elucidate the inhibition of biofilm formation caused by MOL in 20 clinical isolates or standard strains. The reduction in cidA, atl, sle1, and lytN transcript levels following MOL treatment was consistent with the induced expression of their autolytic repressors lrgA, lrgB, arlR, and sarA. MOL generally inhibited or reversed the expression of most of the genes involved in biofilm production. The growth of S. aureus strain ATCC 25923 in the presence of MOL dose-dependently led to decreases in Triton X-100-induced autolysis, extracellular murein hydrolase activity, and the amount of extracellular DNA (eDNA. MOL may impede biofilm formation by reducing the expression of cidA, a murein hydrolase regulator, to inhibit autolysis and eDNA release, or MOL may directly repress biofilm formation. CONCLUSIONS/SIGNIFICANCE: MOL shows in vitro antimicrobial activity against clinical and standard S. aureus strains grown in planktonic and biofilm cultures, suggesting that the structure of MOL may potentially be used as a basis for the development of drugs targeting biofilms.

Biofilm formation by Staphylococcus aureus and Salmonella spp. under mono and dual-species conditions and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite.

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Iñiguez-Moreno, Maricarmen; Gutiérrez-Lomelí, Melesio; Guerrero-Medina, Pedro Javier; Avila-Novoa, María Guadalupe

The aim of this study was evaluated the biofilm formation by Staphylococcus aureus 4E and Salmonella spp. under mono and dual-species biofilms, onto stainless steel 316 (SS) and polypropylene B (PP), and their sensitivity to cetrimonium bromide, peracetic acid and sodium hypochlorite. The biofilms were developed by immersion of the surfaces in TSB by 10 d at 37°C. The results showed that in monospecies biofilms the type of surface not affected the cellular density (p>0.05). However, in dual-species biofilms on PP the adhesion of Salmonella spp. was favored, 7.61±0.13Log 10 CFU/cm 2 , compared with monospecies biofilms onto the same surface, 5.91±0.44Log 10 CFU/cm 2 (pdisinfection treatments; and the most effective disinfectant was peracetic acid (3500ppm), reducing by more than 5Log 10 CFU/cm 2 , while the least effective was cetrimonium bromide. In addition, S. aureus 4E and Salmonella spp. were more resistant to the disinfectants in mono than in dual-species biofilms (p<0.05). Therefore, the interspecies interactions between S. aureus 4E and Salmonella spp. had a negative effect on the antimicrobial resistance of each microorganism, compared with the monospecies biofilms. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

Silver-Containing Hydroxyapatite Coating Reduces Biofilm Formation by Methicillin-Resistant Staphylococcus aureus In Vitro and In Vivo

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Masaya Ueno

2016-01-01

Full Text Available Biofilm-producing bacteria are the principal causes of infections associated with orthopaedic implants. We previously reported that silver-containing hydroxyapatite (Ag-HA coatings exhibit high antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA. In the present study, we evaluated the effects of Ag-HA coating of implant surfaces on biofilm formation. Titanium disks (14-mm diameter, 1-mm thickness, one surface of which was coated with HA or 0.5%–3.0% Ag-HA with a thermal spraying technique, were used. In vitro, the disks were inoculated with an MRSA suspension containing 4×105 CFU and incubated for 1-2 weeks. In vivo, MRSA-inoculated HA and 3% Ag-HA disks (8.8–10.0 × 108 CFU were implanted subcutaneously on the back of rats for 1–7 days. All disks were subsequently stained with a biofilm dye and observed under a fluorescence microscope, and biofilm coverage rates (BCRs were calculated. The BCRs on the Ag-HA coating were significantly lower than those on the HA coating at all time points in vitro (p<0.05. Similar results were observed in vivo (p<0.001 without argyria. Ag-HA coating reduced biofilm formation by MRSA in vitro and in vivo; therefore, Ag-HA coating might be effective for reducing implant-associated infections.

[Formation of microbial biofilms in causative agents of acute and chronic pyelonephritis].

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Lagun, L V; Atanasova, Iu V; Tapal'skiĭ, D V

2013-01-01

Study the intensity of formation of microbial biofilms by Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus strains isolated during various forms of pyelonephritis. 150 clinical isolates of microorganisms isolated from urine ofpatientswith acute and chronic pyelonephritiswere included into the study. Determination of intensity of film-formation was carried out by staining of the formed biofilms by crystal violet with consequent extraction of the dye and measurement of its concentration in washout solution. Among causative agents ofpyelonephritis P. aeruginosa isolates had the maximum film-forming ability. The intensity of biofilm formation of these isolates was 2-3 time higher than staphylococcus and enterobacteria strains. Strains isolated from patients with chronic pyelonephritis by ability to form biofilms significantly surpassed strains isolated from acute pyelonephritis patients. A higher ability to form microbial biofilms for microorganisms--causative agents of pyelonephritis progressing against the background ofurolithiasis was noted. The ability to form biofilms is determined by both causative agent species and character of the infectious process in which this microorganism participates. Intensive formation of biofilms by E. coli, P. aeruginosa, K. pneumoniae, S. aureus clinical isolates may be an important factor of chronization of urinary tract infections.

Boeravinone B, A Novel Dual Inhibitor of NorA Bacterial Efflux Pump of Staphylococcus aureus and Human P-Glycoprotein, Reduces the Biofilm Formation and Intracellular Invasion of Bacteria.

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Singh, Samsher; Kalia, Nitin P; Joshi, Prashant; Kumar, Ajay; Sharma, Parduman R; Kumar, Ashok; Bharate, Sandip B; Khan, Inshad A

2017-01-01

This study elucidated the role of boeravinone B, a NorA multidrug efflux pump inhibitor, in biofilm inhibition. The effects of boeravinone B plus ciprofloxacin, a NorA substrate, were evaluated in NorA-overexpressing, wild-type, and knocked-out Staphylococcus aureus (SA-1199B, SA-1199, and SA-K1758, respectively). The mechanism of action was confirmed using the ethidium bromide accumulation and efflux assay. The role of boeravinone B as a human P -glycoprotein ( P -gp) inhibitor was examined in the LS-180 (colon cancer) cell line. Moreover, its role in the inhibition of biofilm formation and intracellular invasion of S. aureus in macrophages was studied. Boeravinone B reduced the minimum inhibitory concentration (MIC) of ciprofloxacin against S. aureus and its methicillin-resistant strains; the effect was stronger in SA-1199B. Furthermore, time-kill kinetics revealed that boeravinone B plus ciprofloxacin, at subinhibitory concentration (0.25 × MIC), is as equipotent as that at the MIC level. This combination also had a reduced mutation prevention concentration. Boeravinone B reduced the efflux of ethidium bromide and increased the accumulation, thus strengthening the role as a NorA inhibitor. Biofilm formation was reduced by four-eightfold of the minimal biofilm inhibitory concentration of ciprofloxacin, effectively preventing bacterial entry into macrophages. Boeravinone B effectively inhibited P -gp with half maximal inhibitory concentration (IC 50 ) of 64.85 μM. The study concluded that boeravinone B not only inhibits the NorA-mediated efflux of fluoroquinolones but also considerably inhibits the biofilm formation of S. aureus. Its P -gp inhibition activity demonstrates its potential as a bioavailability and bioefficacy enhancer.

Biofilm formation by clinical isolates and the implications in chronic infections

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Sanchez Carlos J

2013-01-01

Full Text Available Abstract Background Biofilm formation is a major virulence factor contributing to the chronicity of infections. To date few studies have evaluated biofilm formation in infecting isolates of patients including both Gram-positive and Gram-negative multidrug-resistant (MDR species in the context of numerous types of infectious syndromes. Herein, we investigated the biofilm forming capacity in a large collection of single patient infecting isolates and compared the relationship between biofilm formation to various strain characteristics. Methods The biofilm-forming capacity of 205 randomly sampled clinical isolates from patients, collected from various anatomical sites, admitted for treatment at Brooke Army Medical Center (BAMC from 2004–2011, including methicillin-resistant/methicillin susceptible Staphylococcus aureus (MRSA/MSSA (n=23, Acinetobacter baumannii (n=53, Pseudomonas aeruginosa (n=36, Klebsiella pneumoniae (n=54, and Escherichia coli (n=39, were evaluated for biofilm formation using the high-throughput microtiter plate assay and scanning electron microscopy (SEM. Relationships between biofilm formation to clonal type, site of isolate collection, and MDR phenotype were evaluated. Furthermore, in patients with relapsing infections, serial strains were assessed for their ability to form biofilms in vitro. Results Of the 205 clinical isolates tested, 126 strains (61.4% were observed to form biofilms in vitro at levels greater than or equal to the Staphylococcus epidermidis, positive biofilm producing strain, with P. aeruginosa and S. aureus having the greatest number of biofilm producing strains. Biofilm formation was significantly associated with specific clonal types, the site of isolate collection, and strains positive for biofilm formation were more frequently observed to be MDR. In patients with relapsing infections, the majority of serial isolates recovered from these individuals were observed to be strong biofilm producers in vitro

Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

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Jeffrey L Bose

Full Text Available The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM and a glucosaminidase (GL. Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

Contribution of the Staphylococcus aureus Atl AM and GL murein hydrolase activities in cell division, autolysis, and biofilm formation.

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Bose, Jeffrey L; Lehman, McKenzie K; Fey, Paul D; Bayles, Kenneth W

2012-01-01

The most prominent murein hydrolase of Staphylococcus aureus, AtlA, is a bifunctional enzyme that undergoes proteolytic cleavage to yield two catalytically active proteins, an amidase (AM) and a glucosaminidase (GL). Although the bifunctional nature of AtlA has long been recognized, most studies have focused on the combined functions of this protein in cell wall metabolism and biofilm development. In this study, we generated mutant derivatives of the clinical S. aureus isolate, UAMS-1, in which one or both of the AM and GL domains of AtlA have been deleted. Examination of these strains revealed that each mutant exhibited growth rates comparable to the parental strain, but showed clumping phenotypes and lysis profiles that were distinct from the parental strain and each other, suggesting distinct roles in cell wall metabolism. Given the known function of autolysis in the release of genomic DNA for use as a biofilm matrix molecule, we also tested the mutants in biofilm assays and found both AM and GL necessary for biofilm development. Furthermore, the use of enzymatically inactive point mutations revealed that both AM and GL must be catalytically active for S. aureus to form a biofilm. The results of this study provide insight into the relative contributions of AM and GL in S. aureus and demonstrate the contribution of Atl-mediated lysis in biofilm development.

Biofilm production and beta-lactamic resistance in Brazilian Staphylococcus aureus isolates from bovine mastitis

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Viviane Figueira Marques

Full Text Available Abstract Staphylococcus spp. play an important role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most relevant species due to the production of virulence factors such as slime, which is required for biofilm formation. This study aimed to evaluate biofilm production and its possible relation to beta-lactamic resistance in 20 S. aureus isolates from bovine mastitic milk. The isolates were characterized by pheno-genotypic and MALDI TOF-MS assays and tested for genes such as icaA, icaD, bap, agr RNAIII, agr I, agr II, agr III, and agr IV, which are related to slime production and its regulation. Biofilm production in microplates was evaluated considering the intervals determined along the bacterial growth curve. In addition, to determine the most suitable time interval for biofilm analysis, scanning electron microscopy was performed. Furthermore, genes such as mecA and blaZ that are related to beta-lactamic resistance and oxacillin susceptibility were tested. All the studied isolates were biofilm producers and mostly presented icaA and icaD. The Agr type II genes were significantly prevalent. According to the SEM, gradual changes in the bacterial arrangement were observed during biofilm formation along the growth curve phases, and the peak was reached at the stationary phase. In this study, the penicillin resistance was related to the production of beta-lactamase, and the high minimal bactericidal concentration for cefoxitin was possibly associated with biofilm protection. Therefore, further studies are warranted to better understand biofilm formation, possibly contributing to our knowledge about bacterial resistance in vivo.

Antimicrobial activity of essential oils against Staphylococcus aureus biofilms.

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Vázquez-Sánchez, Daniel; Cabo, Marta L; Rodríguez-Herrera, Juan J

2015-12-01

The present study was aimed to evaluate the potential of essential oils to remove the foodborne pathogen Staphylococcus aureus from food-processing facilities. The effectiveness of 19 essential oils against planktonic cells of S. aureus was firstly assessed by minimal inhibitory concentration. Planktonic cells showed a wide variability in resistance to essential oils, with thyme oil as the most effective, followed by lemongrass oil and then vetiver oil. The eight essential oils most effective against planktonic cells were subsequently tested against 48-h-old biofilms formed on stainless steel. All essential oils reduced significantly (p oils were the most effective, but high concentrations were needed to achieve logarithmic reductions over 4 log CFU/cm(2) after 30 min exposure. Alternatively, the use of sub-lethal doses of thyme oil allowed to slow down biofilm formation and to enhance the efficiency of thyme oil and benzalkonium chloride against biofilms. However, some cellular adaptation to thyme oil was detected. Therefore, essential oil-based treatments should be based on the rotation and combination of different essential oils or with other biocides to prevent the emergence of antimicrobial-resistant strains. © The Author(s) 2014.

Antimicrobial susceptibility, virulence factors and biofilm formation among Staphylococcus aureus isolates from hospital infections in Kerman, Iran

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Mohammad Reza Shakibaie

2014-12-01

Full Text Available Objective: The aims of present study were to determine the antimicrobial susceptibility, virulence factors and biofilm formation among MRSA hospital isolates of Staphylococcus aureus. Methods: Thirty non-repetitive strains of S. aureus isolated from three hospitals in Kerman, Iran. Antimicrobial sus­ceptibility was determined by disk diffusion breakpoints method according to CLSI guideline. The minimum inhibitory concentration (MIC of vancomycin and methicillin were measured by the broth microdilution and E-test procedures. Virulence factors (protease, DNase, lecithinase, capsule and hemolysis associated with the above isolates was studied. Biofilm was quantified by microtiter technique. Results: In total, 14 (46.7% S. aureus were isolated from lower respiratory tract, six (20.0% from urinary tract and re­maining 10 (33.3% were recovered from wounds, blood and orthopedic patients. All of the isolates were susceptible to tigecycline, eight (26.7% were found to be resistant to methicillin (MRSA and 4 (13.3% showed reduced susceptibility to vancomycin. No any vancomycin resistant isolate was detected (p≤0.05. MIC results showed that four of the isolates (13.3% exhibited MIC 4 μg/mL to vancomycin while, five (16.6% demonstrated MIC 32 μg/mL to methicillin. The iso­lates were also resistant to amoxicillin/clavulanic acid, tetracycline and tobramycin. It was found that, six (75 % of MRSA strains produced lecithinase, seven (96.7% demonstrated protease and DNase activities as compared to MSSA isolates. Biofilm analysis revealed that twenty (66.7% isolates formed strong, seven (23.3% formed moderate and three (10.0% had weak biofilm. Conclusion: From the results, it can be concluded that, treatment options available for these infections are limited; therefore, monitoring, and management of infections due to MRSA with reduced susceptibility to vancomycin, must be done in order to control spread of these strains in the hospital environment. J

Methicillin-Resistant Staphylococcus aureus Biofilms and Their Influence on Bacterial Adhesion and Cohesion

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Khulood Hamid Dakheel

2016-01-01

Full Text Available Twenty-five methicillin-resistant Staphylococcus aureus (MRSA isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype was spa type t127 (22 out of 25 isolates; the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested.

Biofilm Formation by Bacteria Isolated from Intravenous Catheters

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Sina Hedayati

2015-10-01

Full Text Available Background: Reports on the association of nosocomial bacterial infections with indwelling medical devices such as intravenous catheters (IVC has increased in recent years. The potential to form biofilm on these devices seems to be the main reason for establishment of such infections. The aim of this study was to measure the potential of biofilm formation by bacterialisolates from IVCs.Methods: Seventy-one IVCs were collected from hospitalized patients in ICU, NICU, hematology and oncology wards at Taleghani Hospital from Jan 2010 to Jan 2011. The bacterial isolates were identified using the standard biochemical tests and the potential to form biofilms was determined by the microtiter plate assay method (MTP and colony morphology using Congo red agar plates (CRA.Results: Overall, 54 (71% IVCs were colonized and 76 bacteria were isolated among which, 64 (84.2% were coagulase negative staphylococci (CoNS, 3 (3.9% S. aureus, 3 (3.9% Enterococcus spp., 2 (2.6% E. coli and 4 (5.3% were miscellaneous isolates not further identified. Among the CoNS, biofilm formation was observed in 68.7% and 82.8% of bacteriausing MTP and CRA methods, respectively. S. aureus and E. coli isolates also were biofilm producers but Enterococcus and other unknown isolates were biofilm negative.Conclusions: Our results confirm that the prevalent biofilm forming bacteria on IVCs were CoNS and that was the reason for high rates of nosocomial infections.

Adhesion and biofilm formation by Staphylococcus aureus from food processing plants as affected by growth medium, surface type and incubation temperature

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Heloísa Maria Ângelo Jerônimo

2012-12-01

Full Text Available This study assessed the effect of different growth media [BHI broth, BHI broth plus glucose (10 g/100 mL and BHI broth plus NaCl (5 g/100 mL] and incubation temperatures (28 or 37 ºC on the adherence, detachment and biofilm formation on polypropylene and stainless steel surfaces (2 x 2 cm coupons for a prolonged period (24-72 h by some strains of Staphylococcus aureus (S3, S28 and S54 from food processing plants. The efficacy of the sanitizers sodium hypochlorite (250 mg/mL and peracetic acid (30 mg/mL in reducing the number of viable bacterial cells in a preformed biofilm was also evaluated. S. aureus strains adhered in highest numbers in BHI broth, regardless of the type of surface or incubation temperature. Cell detachment from surfaces revealed high persistence over the incubation period. The number of cells needed for biofilm formation was noted in all experimental systems after 3 days. Peracetic acid and sodium hypochlorite were not efficient in completely removing the cells of S. aureus adhered onto polypropylene and stainless steel surfaces. From these results, the assayed strains revealed high capacities to adhere and form biofilms on polypropylene and stainless steel surfaces under the different growth conditions, and the cells in biofilm matrixes were resistant to total removal when exposed to the sanitizers sodium hypochlorite and peracetic acid.Este estudo teve como objetivo avaliar o efeito de diferentes meios de crescimento [caldo BHI, caldo BHI adicionado de glucose (10 g/100 mL e caldo BHI adicionado de NaCl (5 g/100 mL] e temperaturas de incubação (28 e 37 ºC sobre a adesão, separação e formação de biofilme sobre superfícies (2 x 2 cm de polipropileno e aço inoxidável durante longo tempo de incubação (24-72 h por parte de cepas de Staphylococcus aureus (S3, S58 e S54 isoladas de plantas de processamento de alimentos. Também foi avaliada a eficácia dos sanitizantes hipoclorito de sódio (250 mg/mL e ácido perac

Biofilm Formation and Its Relationship with the Molecular Characteristics of Food-Related Methicillin-Resistant Staphylococcus aureus (MRSA).

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Vergara, Alberto; Normanno, Giovanni; Di Ciccio, Pierluigi; Pedonese, Francesca; Nuvoloni, Roberta; Parisi, Antonio; Santagada, Gianfranco; Colagiorgi, Angelo; Zanardi, Emanuela; Ghidini, Sergio; Ianieri, Adriana

2017-10-01

The capability to produce biofilm is an important persistence and dissemination mechanism of some foodborne bacteria. This paper investigates the relationship between some molecular characteristics (SCCmec, ST, spa-type, agr-type, cna, sarA, icaA, icaD, clfA, fnbA, fnbB, hla, hlb) of 22 food-related methicillin-resistant Staphylococcus aureus (MRSA) strains and their ability to form biofilm on stainless steel and polystyrene. Five (22.7%, 5/22) strains were able to synthesize biofilm on polystyrene, and one of these (4.5%, 1/22) strains was also able to synthesize biofilm on stainless steel. The largest amount of biofilm was formed on polystyrene by 2 MRSA strains isolated from cows' milk, thus raising concern about the dairy industry. The majority of MRSA biofilm producers carried SCCmec type IVa, suggesting that the presence of SCCmecIVa and/or agr type III could be related to the ability to form biofilm. In conclusion, in order to achieve an acceptable level of food safety, Good Hygiene Practices should be strictly implemented along the food chain to reduce the risk of colonization and dissemination of MRSA biofilm-producing strains in the food industry. In this study, some assayed isolates of food-related MRSA demonstrated the capacity to form biofilm. Biofilm formation differed according to surface characteristics and MRSA strains. A relationship was observed between some molecular characteristics and the ability to form biofilms. Few studies have investigated the ability of MRSA to form biofilms, and the majority of these studies have investigated clinical aspects. This work was performed to investigate whether or not there is a difference between MRSA food isolates and MRSA clinical isolates in their ability to form biofilm. These initial findings could provide information that will contribute to a better understanding of these aspects. © 2017 Institute of Food Technologists®.

Role of bacterial efflux pumps in biofilm formation.

Science.gov (United States)

Alav, Ilyas; Sutton, J Mark; Rahman, Khondaker Miraz

2018-02-28

Efflux pumps are widely implicated in antibiotic resistance because they can extrude the majority of clinically relevant antibiotics from within cells to the extracellular environment. However, there is increasing evidence from many studies to suggest that the pumps also play a role in biofilm formation. These studies have involved investigating the effects of efflux pump gene mutagenesis and efflux pump inhibitors on biofilm formation, and measuring the levels of efflux pump gene expression in biofilms. In particular, several key pathogenic species associated with increasing multidrug resistance, such as Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, have been investigated, whilst other studies have focused on Salmonella enterica serovar Typhimurium as a model organism and problematic pathogen. Studies have shown that efflux pumps, including AcrAB-TolC of E. coli, MexAB-OprM of P. aeruginosa, AdeFGH of A. baumannii and AcrD of S. enterica, play important roles in biofilm formation. The substrates for such pumps, and whether changes in their efflux activity affect biofilm formation directly or indirectly, remain to be determined. By understanding the roles that efflux pumps play in biofilm formation, novel therapeutic strategies can be developed to inhibit their function, to help disrupt biofilms and improve the treatment of infections. This review will discuss and evaluate the evidence for the roles of efflux pumps in biofilm formation and the potential approaches to overcome the increasing problem of biofilm-based infections.

Effects of Fermented Sumach on the Formation of Slime Layer of Staphylococcus aureus

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Kırmusaoğlu, Sahra; Yurdugül, Seyhun; Koçoğlu, Esra

2012-01-01

Objective: Staphylococcus aureus (S. aureus) is one of the most commonly isolated bacterial pathogens in hospitals, and the most frequent cause of nosocomial infections. Nosocomial staphylococcal foreign-body infections related to biofilm formation are a serious threat, demanding new therapeutic and preventive strategies. Implantation of intravenous catheters and surgical implantation of prosthetic implants carry a risk of infection. In order to prevent all these effects of biofilms, a study ...

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates

DEFF Research Database (Denmark)

Oliveira, M; Bexiga, R; Nunes, S F

2006-01-01

of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations...... hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed...

Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

DEFF Research Database (Denmark)

Baldry, Mara; Nielsen, Anita; Bojer, Martin S.

2016-01-01

characterise the mode of action of norlichexanthone and its effect on biofilm formation. We find that norlichexanthone reduces expression of both hla and RNAIII also in strain USA300. Structurally, norlichexanthone resembles ω-hydroxyemodin that recently was shown to bind the agr two component response......-hydroxyemodin however, norlichexanthone reduces staphylococcal biofilm formation. Transcriptomic analysis revealed that genes regulated by the SaeRS two-component system are repressed by norlichexanthone when compared to untreated cells, an effect that was mitigated in strain Newman carrying a partially constitutive...... SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation....

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA isolates of swine origin form robust biofilms.

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Tracy L Nicholson

Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA colonization of livestock animals is common and prevalence rates for pigs have been reported to be as high as 49%. Mechanisms contributing to the persistent carriage and high prevalence rates of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA strains in swine herds and production facilities have not been investigated. One explanation for the high prevalence of MRSA in swine herds is the ability of these organisms to exist as biofilms. In this report, the ability of swine LA-MRSA strains, including ST398, ST9, and ST5, to form biofilms was quantified and compared to several swine and human isolates. The contribution of known biofilm matrix components, polysaccharides, proteins and extracellular DNA (eDNA, was tested in all strains as well. All MRSA swine isolates formed robust biofilms similar to human clinical isolates. The addition of Dispersin B had no inhibitory effect on swine MRSA isolates when added at the initiation of biofilm growth or after pre-established mature biofilms formed. In contrast, the addition of proteinase K inhibited biofilm formation in all strains when added at the initiation of biofilm growth and was able to disperse pre-established mature biofilms. Of the LA-MRSA strains tested, we found ST398 strains to be the most sensitive to both inhibition of biofilm formation and dispersal of pre-formed biofilms by DNaseI. Collectively, these findings provide a critical first step in designing strategies to control or eliminate MRSA in swine herds.

Antimicrobial Resistance Profile of Planktonic and Biofilm Cells of Staphylococcus aureus and Coagulase-Negative Staphylococci

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Adilson de Oliveira

2016-09-01

Full Text Available The objective of the present study was to determine the antimicrobial resistance profile of planktonic and biofilm cells of Staphylococcus aureus and coagulase-negative staphylococci (CoNS. Two hundred Staphylococcus spp. strains were studied, including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis, and 20 S. saprophyticus. Biofilm formation was investigated by adherence to polystyrene plates. Positive strains were submitted to the broth microdilution method to determine the minimum inhibitory concentration (MIC for planktonic and biofilm cells and the minimal bactericidal concentration for biofilm cells (MBCB. Forty-nine Staphylococcus spp. strains (14 S. aureus, 13 S. epidermidis, 13 S. saprophyticus, 3 S. haemolyticus, 1 S. hominis, 3 S. warneri, and 2 S. lugdunensis were biofilm producers. These isolates were evaluated regarding their resistance profile. Determination of planktonic cell MIC identified three (21.4% S. aureus strains that were resistant to oxacillin and six (42.8% that were resistant to erythromycin. Among the CoNS, 31 (88.6% strains were resistant to oxacillin, 14 (40% to erythromycin, 18 (51.4% to gentamicin, and 8 (22.8% to sulfamethoxazole/trimethoprim. None of the planktonic isolates were resistant to vancomycin or linezolid. MICs were 2-, 4-, 8-, and up to 16-fold higher for biofilm cells than for planktonic cells. This observation was more common for vancomycin and erythromycin. The MBCB ranged from 8 to >256 µg/mL for oxacillin, 128 to >128 µg/mL for vancomycin, 256 to >256 µg/mL for erythromycin and gentamicin, >64 µg/mL for linezolid, and 32/608 to >32/608 µg/mL for sulfamethoxazole/trimethoprim. The results showed considerably higher MICs for S. aureus and CoNS biofilm cells compared to planktonic cells. Analysis of MBCM confirmed that even high concentrations of vancomycin were unable to eliminate the biofilms of S. aureus and Co

Formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo Biofilm formation by Aeromonas hydrophila and Staphylococcus aureus on stainless steel using milk and different conditions of cultivation

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Cleube Andrade Boari

2009-12-01

Full Text Available O objetivo desta pesquisa consistiu em avaliar a formação de biofilme em aço inoxidável por Aeromonas hydrophila e Staphylococcus aureus usando leite e diferentes condições de cultivo. As variáveis em estudo consistem no cultivo monoespécie e combinado, dos referidos microrganismos e nas temperaturas de 4, 7 e 18 °C. Recipientes contendo 1000 mL de leite, densidade populacional de 10(5 UFC.mL-1 de cada microrganismo e 10 cupons de aço inoxidável (10 × 20 mm foram lacrados e armazenados, sob agitação de 60 rpm, por um período de 10 dias. As análises ocorreram a cada 48 horas. Células sésseis de A.hydrophila e S. aureus foram enumeradas através do plaqueamento seletivo em ágar m-Aeromonas selective e Baird-Parker, respectivamente. Estudos sobre o tempo de geração, enumeração de células planctônicas e observação dos cupons através da microscopia eletrônica de varredura foram conduzidos. S. aureus, em monocultivo, formou biofilme a 18 °C e a 7 °C. Para 4 °C, foi observado um processo de adesão. A presença de A. hydrophila reduziu o desempenho de S. aureus. Nesta condição de cultivo multiespécie houve formação de biofilme a 18 °C. A. hydrophila, tanto em monocultivo quanto em presença de S. aureus, formou biofilme em todas as condições pesquisadas.The aim of this research was to verify the capability of biofilm formation on stainless steel by Aeromonas hydrophila and Staphylococcus aureus using milk and different conditions of cultivation. The variables consisted in mono and multi-species cultivation of these microorganisms and in the temperatures of 4, 7 and 18 °C. Containers containing 1000 mL of milk, population density of 10(5 CFU.mL-1 of each microorganism, and ten suspended chips of stainless steel AISI 304 (10 × 20 mm were used to seal up and storage, under 60 rpm of agitation for 10 days. The analyses were conducted every 48 hours. Sessile cells of A. hydrophila and S. aureus and were enumerated

Pathology and biofilm formation in a porcine model of staphylococcal osteomyelitis

DEFF Research Database (Denmark)

Johansen, L K; Koch, J; Frees, D

2012-01-01

A porcine model was used to examine the potential of human and porcine Staphylococcus aureus isolates to induce haematogenously spread osteomyelitis. Pigs were inoculated in the right femoral artery with one of the following S. aureus strains: S54F9 (from a porcine lung abscess; n = 3 animals), N...... dependent on the strain of bacteria inoculated and on the formation of a biofilm....... with colonies of S. aureus as demonstrated immunohistochemically. By peptide nucleic acid fluorescence in situ hybridization bacterial aggregates were demonstrated to be embedded in an opaque matrix, indicating that the bacteria had formed a biofilm. Development of experimental osteomyelitis was therefore...

Changes in the Expression of Biofilm-Associated Surface Proteins in Staphylococcus aureus Food-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants

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Lenka Cincarova

2016-01-01

Full Text Available Sublethal concentrations (sub-MICs of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on three Staphylococcus aureus biofilm formers (strong B+++, moderate B++, and weak B+ that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25–2.5% ethanol and 2500 μg/mL chloramine T significantly enhanced S. aureus biofilm formation. To visualize differences in biofilm compactness between S. aureus biofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors.

Colloidal silver: a novel treatment for Staphylococcus aureus biofilms?

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Goggin, Rachel; Jardeleza, Camille; Wormald, Peter-John; Vreugde, Sarah

2014-03-01

Colloidal silver is an alternative medicine consisting of silver particles suspended in water. After using this solution as a nasal spray, the symptoms of a previously recalcitrant Staphylococcus aureus (S. aureus)-infected chronic rhinosinusitis patient were observed to have improved markedly. The aim of this study was to determine whether colloidal silver has any direct bactericidal effects on these biofilms in vitro. S. aureus biofilms were grown from the ATCC 25923 reference strain on Minimum Biofilm Eradication Concentration (MBEC) device pegs, and treated with colloidal silver. Concentrations tested ranged from 10 to 150 μL colloidal silver diluted to 200 μL with sterile water in 50 μL cerebrospinal fluid (CSF) broth. Control pegs were exposed to equivalent volumes of CSF broth and sterile water. The sample size was 4 biomass values per treatment or control group. Confocal scanning laser microscopy and COMSTAT software were used to quantify biofilms 24 hours after treatment. Significant differences from control were found for all concentrations tested bar the lowest of 10 μL colloidal silver in 200 μL. At 20 μL colloidal silver, the reduction in biomass was 98.9% (mean difference between control and treatment = -4.0317 μm(3) /μm(2) , p colloidal silver (mean differences = -4.0681 and -4.0675μm(3) /μm(2) , respectively, p Colloidal silver directly attenuates in vitro S. aureus biofilms. © 2014 ARS-AAOA, LLC.

Antimicrobial potential of Eucalyptus globulus against biofilms of Staphylococcus aureus isolated from bovine mastitis

OpenAIRE

Gomes, F. I.; Martins, Natália; Ferreira, Isabel C. F. R.; Henriques, Mariana

2016-01-01

Staphylococcus aureus are among the most common species isolated from bovine mastitis. The pathogenesis of this bacterium is facilitated by a number of virulence factors, including the ability to adhere to abiotic surfaces and/or host tissues often leading to biofilms' formation. From the clinical perspective, the most important feature of Staphytococcus species' biofilms is their high tolerance to the conventional antimicrobial therapy. So, the increasing number of bovine m...

Anti-biofilm activity of biogenic selenium nanoparticles and selenium dioxide against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis.

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Shakibaie, Mojtaba; Forootanfar, Hamid; Golkari, Yaser; Mohammadi-Khorsand, Tayebe; Shakibaie, Mohammad Reza

2015-01-01

The aim of the present study was to investigate the anti-biofilm activity of biologically synthesized selenium nanoparticles (Se NPs) against the biofilm produced by clinically isolated bacterial strains compared to that of selenium dioxide. Thirty strains of Staphylococcus aureus, Pseudomonas aeruginosa, and Proteus mirabilis were isolated from various specimens of the patients hospitalized in different hospitals (Kerman, Iran). Quantification of the biofilm using microtiter plate assay method introduced 30% of S. aureus, 13% of P. aeruginosa and 17% of P. mirabilis isolates as severely adherent strains. Transmission electron micrograph (TEM) of the purified Se NPs (produced by Bacillus sp. MSh-1) showed individual and spherical nano-structure in the size range of 80-220nm. Obtained results of the biofilm formation revealed that selenium nanoparticles inhibited the biofilm of S. aureus, P. aeruginosa, and P. mirabilis by 42%, 34.3%, and 53.4%, respectively, compared to that of the non-treated samples. Effect of temperature and pH on the biofilm formation in the presence of Se NPs and SeO2 was also evaluated. Copyright © 2014 Elsevier GmbH. All rights reserved.

Femtosecond laser surface texturing of titanium as a method to reduce the adhesion of Staphylococcus aureus and biofilm formation

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Cunha, Alexandre; Elie, Anne-Marie; Plawinski, Laurent; Serro, Ana Paula; Botelho do Rego, Ana Maria; Almeida, Amélia; Urdaci, Maria C.; Durrieu, Marie-Christine; Vilar, Rui

2016-01-01

The aim of the present work was to investigate the possibility of using femtosecond laser surface texturing as a method to reduce the colonization of Grade 2 Titanium alloy surfaces by Staphylococcus aureus and the subsequent formation of biofilm. The laser treatments were carried out with a Yb:KYW chirped-pulse-regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. Two types of surface textures, consisting of laser-induced periodic surface structures (LIPSS) and nanopillars, were produced. The topography, chemical composition and phase constitution of these surfaces were investigated by atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, micro-Raman spectroscopy, and X-ray diffraction. Surface wettability was assessed by the sessile drop method using water and diiodomethane as testing liquids. The response of S. aureus put into contact with the laser treated surfaces in controlled conditions was investigated by epifluorescence microscopy and scanning electron microscopy 48 h after cell seeding. The results achieved show that the laser treatment reduces significantly the bacterial adhesion to the surface as well as biofilm formation as compared to a reference polished surfaces and suggest that femtosecond laser texturing is a simple and promising method for endowing dental and orthopedic titanium implants with antibacterial properties, reducing the risk of implant-associated infections without requiring immobilized antibacterial substances, nanoparticles or coatings.

The role of biofilms in persistent infections and factors involved in ica-independent biofilm development and gene regulation in Staphylococcus aureus.

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Figueiredo, Agnes Marie Sá; Ferreira, Fabienne Antunes; Beltrame, Cristiana Ossaille; Côrtes, Marina Farrel

2017-09-01

Staphylococcus aureus biofilms represent a unique micro-environment that directly contribute to the bacterial fitness within hospital settings. The accumulation of this structure on implanted medical devices has frequently caused the development of persistent and chronic S. aureus-associated infections, which represent an important social and economic burden worldwide. ica-independent biofilms are composed of an assortment of bacterial products and modulated by a multifaceted and overlapping regulatory network; therefore, biofilm composition can vary among S. aureus strains. In the microniches formed by biofilms-produced by a number of bacterial species and composed by different structural components-drug refractory cell subpopulations with distinct physiological characteristics can emerge and result in therapeutic failures in patients with recalcitrant bacterial infections. In this review, we highlight the importance of biofilms in the development of persistence and chronicity in some S. aureus diseases, the main molecules associated with ica-independent biofilm development and the regulatory mechanisms that modulate ica-independent biofilm production, accumulation, and dispersion.

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

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

2014-11-01

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

Streptokinase Treatment Reverses Biofilm-Associated Antibiotic Resistance in Staphylococcus aureus

DEFF Research Database (Denmark)

Jørgensen, Nis Pedersen; Zobek, Natalia; Dreier, Cindy

2016-01-01

by combining antibiotic treatment with a fibrinolytic drug. We quantified S. aureus USA300 biofilms grown on peg-lids in brain heart infusion (BHI) broth with 0%–50% human plasma. Young (2 h) and mature (24 h) biofilms were then treated with streptokinase to determine if this lead to dispersal. Then......, the minimal biofilm eradication concentration (MBEC) of 24 h old biofilms was measured for vancomycin and daptomycin alone or in combination with 10 µg/mL rifampicin in the presence or absence of streptokinase in the antibiotic treatment step. Finally, biofilms were visualized by confocal laser scanning...... or daptomycin, which are commonly used antibiotics for treatment of S. aureus infections. Fibronolytic drugs have been used to treat thromboembolic events for decades, and our findings suggest that their use against biofilm infections has the potential to improve the efficacy of antibiotics in treatment of S...

Formation of biofilms by Staphylococcus aureus on stainless steel and glass surfaces and its resistance to some selected chemical sanitizers Formação de biofilme por Staphylococcus aureus na superfície de aço inoxidável e vidro e sua resistência a alguns sanificantes químicos

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Simone Cristina Marques

2007-09-01

Full Text Available The objectives of this work were to verify the capability of Staphylococcus aureus of forming bio-film on stainless steel and glass surfaces; to evaluate the efficiency of sodium dichloroisocyanurate, hydrogen peroxide and peracetic acid in inactivating Staphylococcus aureus cells adhered onto these surfaces; and to visualize biofilm development by scanning electron microscopy before and after sanitizer treatment. The surfaces studied consisted of 10x20mm chips immersed in Petri dishes containing BHI broth inoculated with S. aureus ATCC 25923. Biofilm formation was observed after 15-day incubation, when the cells were removed using the swab technique, followed by Baird Parker agar plating. Also, the efficiency of the chemical sanitizers on the chip surfaces was tested and the non-removed cells were counted on the Baird-Parker agar. After biofilm formation and use of sanitizers, the chips were respectively observed by scanning electronic microscopy following a pre-existing protocol. The obtained results showed biofilm formation on both surfaces, with bacterial count in the order of 10(7 CFU/cm² on and 10(8 CFU/cm² on stainless steel and glass surfaces, respectively. Peracetic acid was the most efficient in removing adhered cells, presenting 5.26 and 4.5 decimal reduction for adhered cells on stainless steel and glass surfaces, respectively.Os objetivos deste trabalho foram verificar a capacidade de Staphylococcus aureus formar biofilme nas superfícies de aço inoxidável e vidro, avaliar a eficiência do dicloroisocianurato de sódio, peróxido de hidrogênio e ácido peracético na inativação de células de S. aureus aderidas e visualização por microscopia eletrônica de varredura, o desenvolvimento antes e depois do tratamento das superfícies com os sanificantes. As superfícies foram cupons 10x200mm imersos em placas de Petri contendo caldo BHI inoculado com cultura de Staphylococcus aureus ATCC 25923. A formação de biofilme foi

[Investigation of biofilm formation properties of staphylococcus isolates].

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Öcal, Duygu Nilüfer; Dolapçı, İştar; Karahan, Zeynep Ceren; Tekeli, Alper

2017-01-01

Biofilm production is an important virulence factor which allows staphylococci to adhere to medical devices. The principal component of biofilm is a "polysaccharide intercellular adhesin (PIA)" which is composed of a beta-1,6-N-acetylglucosamine polymer synthesized by an enzyme (N-acetylglucosamine transferase) encoded by the ica operon found on the bacterial chromosome. This operon is composed of four genes (A, B, C, and D), and a transposable element IS256. In this study, we aimed to determine the biofilm production characteristics of invasive/non-invasive staphylococcus isolates and different staphylococcus species. Biofilm production of 166 staphylococci was phenotypically investigated on Congo Red Agar (CRA); the presence of icaA, icaD and IS256 genes were investigated by polymerase chain reaction (PCR). 74 of the isolates (44.6%) were identified as methicillin resistant Staphylococcus aureus (MRSA), 25 (15.1%) as methicillin sensitive S.aureus (MSSA), 25 (37.3%) as Staphylococcus hominis, 20 (12%) as S.epidermidis, ten (15%) as Staphylococcus haemolyticus, nine (13.4%) as Staphylococcus capitis, two (3%) Staphylococcus saprophyticus and one (1.5%) as Staphylococcus warnerii. Of the MRSA strains, 52 were isolated from blood and 22 from nose; all MSSA strains were isolated from nose cultures. Coagulase-negative staphylococci (CoNS) strains were composed of invasive and non-invasive strains isolated from nose, catheter tip and blood cultures from patients with catheter. Production with CRA method was found to be statistically significant in invasive isolates (paureus isolates produced biofilm on CRA (paureus when compared with CoNS. Carriage of three genes and biofilm formation capacity of invasive isolates can cause refractory infections and the importance of carriage and hospital infections of these bacteria, it is important to prevent the spread of these isolates. A combination of phenotypic and genotypic tests is recommended for the investigation of biofilm

Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology

DEFF Research Database (Denmark)

Jensen, Louise Kruse; Henriksen, Nicole Lind; Bjarnsholt, Thomas

2018-01-01

Aim: Visualization of Staphylococcus aureus biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC). Methods: The ability of S. aureus S54F9 to form biofilm was tested in vitro. Hereafter, infected bone tissue was collected from two different porcine m...

Extended biofilm susceptibility assay for Staphylococcus aureus bovine mastitis isolates: evidence for association between genetic makeup and biofilm susceptibility.

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Melchior, M B; van Osch, M H J; Lam, T J G M; Vernooij, J C M; Gaastra, W; Fink-Gremmels, J

2011-12-01

Staphylococcus aureus is one of the most prevalent causes of bovine mastitis. The antimicrobial treatment of this disease is currently based on antimicrobial susceptibility tests according to Clinical and Laboratory Standards Institute standards. However, various authors have shown a discrepancy between the results of this standard susceptibility test and the actual cure rate of the applied antimicrobial treatment. Increasing evidence suggests that in vivo biofilm formation by Staph. aureus, which is not assessed in the antimicrobial susceptibility tests, is associated with this problem, resulting in disappointing cure rates, especially for infections of longer duration. Previous data obtained with a limited number of strains showed that the extended biofilm antimicrobial susceptibility (EBS) assay reveals differences between strains, which cannot be derived from a standard susceptibility test or from a 24-h biofilm susceptibility test. The objective of this study was to test a collection of Staph. aureus bovine mastitis strains in the EBS assay and to model the effect of antimicrobial exposure, duration of antimicrobial exposure, and genotype profile of the strains on antimicrobial susceptibility. With the results from a previous study with the same collection of strains, the effect of genotype represented by accessory gene regulator gene (agr-type), the presence of insertional sequence 257 (IS257), intercellular adhesion (ica), and the β-lactamase (blaZ) gene were entered as explanatory factors in a logistic regression model. The agr locus of Staph. aureus controls the expression of most of the virulence factors, represses the transcription of several cell wall-associated proteins, and activates several exoproteins during the post-exponential phase. The IS257 gene has been related to biofilm formation in vitro and was found earlier in 50% of the agr-type 2 strains. The ica gene cluster encodes for the production of an extracellular polysaccharide adhesin, termed

Evaluation of various metallic coatings on steel to mitigate biofilm formation.

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Kanematsu, Hideyuki; Ikigai, Hajime; Yoshitake, Michiko

2009-02-01

In marine environments and water systems, it is easy for many structures to form biofilms on their surfaces and to be deteriorated due to the corrosion caused by biofilm formation by bacteria. The authors have investigated the antibacterial effects of metallic elements in practical steels so far to solve food-related problems, using Escherichia coli and Staphylococcus aureus. However, from the viewpoint of material deterioration caused by bacteria and their antifouling measures, we should consider the biofilm behavior as aggregate rather than individual bacterium. Therefore, we picked up Pseudomonas aeruginosa and Pseudoalteromonas carageenovara in this study, since they easily form biofilms in estuarine and marine environments. We investigated what kind of metallic elements could inhibit the biofilm formation at first and then discussed how the thin films of those inhibitory elements on steels could affect biofilm formation. The information would lead to the establishment of effective antifouling measures against corrosion in estuarine and marine environments.

Evaluation of Various Metallic Coatings on Steel to Mitigate Biofilm Formation

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Hajime Ikigai

2009-02-01

Full Text Available In marine environments and water systems, it is easy for many structures to form biofilms on their surfaces and to be deteriorated due to the corrosion caused by biofilm formation by bacteria. The authors have investigated the antibacterial effects of metallic elements in practical steels so far to solve food-related problems, using Escherichia coli and Staphylococcus aureus. However, from the viewpoint of material deterioration caused by bacteria and their antifouling measures, we should consider the biofilm behavior as aggregate rather than individual bacterium. Therefore, we picked up Pseudomonas aeruginosa and Pseudoalteromonas carageenovara in this study, since they easily form biofilms in estuarine and marine environments. We investigated what kind of metallic elements could inhibit the biofilm formation at first and then discussed how the thin films of those inhibitory elements on steels could affect biofilm formation. The information would lead to the establishment of effective antifouling measures against corrosion in estuarine and marine environments.

Effect of repeated tracheostomy tube reprocessing on biofilm formation.

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Rodney, Jennifer; Ojano-Dirain, Carolyn P; Antonelli, Patrick J; Silva, Rodrigo C

2016-04-01

To determine the effect of repeated reprocessing of pediatric tracheostomy tubes (TTs) on biofilm formation. In vitro microbiological study. Pediatric, uncuffed, polyvinyl chloride (PVC) TTs from two different manufacturers (Tracoe Mini and Shiley) were reprocessed mechanically with household detergent and soaked in sodium hypochlorite (bleach). Two TTs of each brand were reprocessed 0 (control), 10, or 20 times. Twenty 2-mm coupons were then obtained from each TT, immersed in human mucus, and cultured with either Staphylococcus aureus or Pseudomonas aeruginosa. Biofilm formation was evaluated with bacterial counts. Bacterial counts of S. aureus for both brands were significantly higher on the TTs that were reprocessed 20 times compared to those that were not reprocessed (Tracoe: P = .040, Shiley: P  attachment. Further investigation is needed to determine the optimal technique and limits of reprocessing TTs in clinical practice. NA. Laryngoscope published by Wiley on behalf of the American Laryngological, Rhinological and Otological Society, Inc, “The Triological Society” and American Laryngological Association (the “Owner”).

Enhancement of photodynamic inactivation of Staphylococcus aureus biofilms by disruptive strategies.

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Gándara, Lautaro; Mamone, Leandro; Bohm, Gabriela Cervini; Buzzola, Fernanda; Casas, Adriana

2017-11-01

Photodynamic inactivation (PDI) has been used to inactivate microorganisms through the use of photosensitizers and visible light. On the one hand, near-infrared treatment (NIRT) has also bactericidal and dispersal effects on biofilms. In addition, dispersal biological tools such as enzymes have also been employed in antibiotic combination treatments. The aim of this work was to use alternative approaches to increase the PDI efficacy, employing combination therapies aimed at the partial disruption of the biofilms, thus potentially increasing photosensitizer or oxygen penetration and interaction with bacteria. To that end, we applied toluidine blue (TB)-PDI treatment to Staphylococcus aureus biofilms previously treated with NIRT or enzymes and investigated the outcome of the combined therapies. TB employed at 0.5 mM induced per se 2-log drop in S. aureus RN6390 biofilm viability. Each NIRT (980-nm laser) and PDI (635-nm laser) treatment induced a further reduction of 1-log of viable counts. The combination of successive 980- and 635-nm laser treatments on TB-treated biofilms induced additive effects, leading to a 4.5-log viable count decrease. Proteinase K treatment applied to S. aureus of the Newman strain induced an additive effect on PDI mortality, leading to an overall 4-log decrease in S. aureus viability. Confocal scanning laser microscopy after biofilm staining with a fluorescent viability test and scanning electron microscopy observations were correlated with colony counts. The NIRT dose employed (227 J/cm 2 ) led to an increase from 21 to 47 °C in the buffer temperature of the biofilm system, and this NIRT dose also induced 100% keratinocyte death. Further work is needed to establish conditions under which biofilm dispersal occurs at lower NIRT doses.

Brief ultrasonication improves detection of biofilm-formative bacteria around a metal implant.

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Kobayashi, Naomi; Bauer, Thomas W; Tuohy, Marion J; Fujishiro, Takaaki; Procop, Gary W

2007-04-01

Biofilms are complex microenvironments produced by microorganisms on surfaces. Ultrasonication disrupts biofilms and may make the microorganism or its DNA available for detection. We determined whether ultrasonication could affect our ability to detect bacteria adherent to a metal substrate. A biofilm-formative Staphylococcus aureus strain was used for an in vitro implant infection model (biofilm-formative condition). We used quantitative culture and real time-polymerase chain reaction to determine the influence of different durations of ultrasound on bacterial adherence and viability. Sonication for 1 minute increased the yield of bacteria. Sonication longer than 5 minutes led to fewer bacterial colonies by conventional culture but not by polymerase chain reaction. This suggests short periods of sonication help release bacteria from the metal substrate by disrupting the biofilm, but longer periods of sonication lyse bacteria prohibiting their detection in microbiologic cultures. A relatively short duration of sonication may be desirable for maximizing detection of biofilm-formative bacteria around implants by culture or polymerase chain reaction.

Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus

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Vandecandelaere, Ilse; Van Nieuwerburgh, Filip; Deforce, Dieter

2017-01-01

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other’s behavior, but additional studies are required necessary to elucidate the exact

Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model.

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Jully Gogoi-Tiwari

Full Text Available Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model.Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection.Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1β and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (p<0.05 in mice inoculated with strong biofilm forming S. aureus than the weak biofilm forming strain.This finding suggests an important role of TNF-α in mammary gland pathology post-infection with strong biofilm-forming S. aureus in the acute mouse mastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage

[Detection of biofilm formation by selected pathogens relevant to the food industry].

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Šilhová-Hrušková, L; Moťková, P; Šilha, D; Vytřasová, J

2015-09-01

Detection of biofilm formation by microbial pathogens relevant to the food industry and comparison of biofilm formation under different conditions of culture. The following microorganisms were selected for the study: Staphylococcus aureus, Listeria innocua, Listeria ivanovii, Cronobacter sakazakii, Cronobacter muytjensii, Arcobacter butzleri, Arcobacter cryaerophilus, Campylobacter jejuni, and Campylobacter coli. To detect biofilm formation the microtiter plate assay, as described by Christensen and culture on stainless steel coupons were used. The biofilm forming capacity was confirmed in all microorganisms tested, both on the microtiter plates and stainless steel coupons. Biofilm formation was influenced by the culture medium, material used, and culture duration as well as by the test microorganism. It was found that different species and strains of the same genus differ in biofilm formation. Differences were also found between the collection strains and isolates from the environment. Some bacteria tended to form biofilm more readily on the surface of the polyethylene microtiter plates and less readily on stainless steel coupons while others appeared to have an opposite tendency. Some pathogens were able to increase the planktonic cell density in the initial suspension even by three orders of magnitude within 72 hours while producing plenty of biofilm. The study of biofilm formation by high risk pathogens is of utmost importance, not only to the food industry. From the obtained results, it is evident that bacterial biofilms form rapidly (within 24 hours in the present study). Due to their architecture, these biofilms are difficult to eradicate, and therefore, it is crucial to prevent biofilm formation.

Development of a Standard Test to Assess the Resistance of Staphylococcus aureus Biofilm Cells to Disinfectants

NARCIS (Netherlands)

Luppens, S.B.I.; Reij, M.W.; Heijden, van der R.W.; Rombouts, F.M.; Abee, T.

2002-01-01

A standardized disinfectant test for Staphylococcus aureus cells in biofilms was developed. Two disinfectants, the membrane-active compound benzalkonium chloride (BAC) and the oxidizing agent sodium hypochlorite, were used to evaluate the biofilm test. S. aureus formed biofilms on glass, stainless

Staphylococcus aureus Quorum Regulator SarA Targeted Compound, 2-[(Methylaminomethyl]phenol Inhibits Biofilm and Down-Regulates Virulence Genes

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P. Balamurugan

2017-07-01

Full Text Available Staphylococcus aureus is a widely acknowledged Gram-positive pathogen for forming biofilm and virulence gene expressions by quorum sensing (QS, a cell to cell communication process. The quorum regulator SarA of S. aureus up-regulates the expression of many virulence factors including biofilm formation to mediate pathogenesis and evasion of the host immune system in the late phases of growth. Thus, inhibiting the production or blocking SarA protein might influence the down-regulation of biofilm and virulence factors. In this context, here we have synthesized 2-[(Methylaminomethyl]phenol, which was specifically targeted toward the quorum regulator SarA through in silico approach in our previous study. The molecule has been evaluated in vitro to validate its antibiofilm activity against clinical S. aureus strains. In addition, antivirulence properties of the inhibitor were confirmed with the observation of a significant reduction in the expression of representative virulence genes like fnbA, hla and hld that are governed under S. aureus QS. Interestingly, the SarA targeted inhibitor showed negligible antimicrobial activity and markedly reduced the minimum inhibitory concentration of conventional antibiotics when used in combination making it a more attractive lead for further clinical tests.

Bovine mastitis: prevalence and antimicrobial susceptibility profile and detection of genes associated with biofilm formation in Staphylococcus aureus

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Valeska Paula Casanova

2016-06-01

Full Text Available Brazil currently ranks as one of the world leaders in food production and exportation. This scenario encourages the development of animal and plant health programs to ensure the production of safe food, helping the country to become an international provider of food for excellence. However, some health problems in dairy production, such as mastitis, have garnered increasing concern. This study aimed to estimate the prevalence of bovine mastitis in select properties located in the western Santa Catarina region, to assess the susceptibility profile to antimicrobial agents used for treatment and to check for the presence of genes (icaA and icaD associated with biofilm formation in Staphylococcus aureus. In 148 milk samples collected, 72.97% had bacterial growth (n = 108. Among the isolated microorganisms, 21.62% (n = 32 were classified as Staphylococcus aureus, 18.91% (n = 28 as Staphylococcus sp. coagulase negative, 7.43% (n = 11 as Corynebacterium sp., 6.76% (n = 10 as Staphylococcus sp. coagulase positive, 5.41% (n = 8 as Nocardia sp. and 12.83% (n = 19 classified in different bacterial genera. Among the isolates submitted for antimicrobial susceptibility testing, it was observed that 8.95% (n = 6/67 had resistance to amoxicillin, 8.04% (n = 7/87 to ampicillin, 5.88% (n = 5/85 to cephalothin, 3.40% (n = 3/88 to ceftiofur and enrofloxacin, 20.45% (n = 18/88 to streptomycin, 17.04% (n = 15/88 to gentamicin and lincomycin, 31.81% (n = 28/88 to neomycin, 14.94% (n = 13/87 to penicillin and 25% (n = 22/88 to tetracycline. Staphylococcus sp. coagulase negative isolates showed higher multidrug resistance when compared to those of S. aureus and Staphylococcus sp. coagulase positive. Thirty-one strains of S. aureus isolates were genotypically tested by polymerase chain reaction (PCR, yielding a positive result for the icaA gene in 83.87% of the samples, 80.64% positive for icaD and 74.19% of these showed both genes. The results reinforce the importance

The anti-biofilm activity of lemongrass (Cymbopogon flexuosus) and grapefruit (Citrus paradisi) essential oils against five strains of Staphylococcus aureus.

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Adukwu, E C; Allen, S C H; Phillips, C A

2012-11-01

To determine the sensitivity of five strains of Staphylococcus aureus to five essential oils (EOs) and to investigate the anti-biofilm activity of lemongrass and grapefruit EOs. Antimicrobial susceptibility screening was carried out using the disk diffusion method. All of the strains tested were susceptible to lemongrass, grapefruit, bergamot and lime EOs with zones of inhibition varying from 2·85 to 8·60 cm although they were resistant to lemon EO. Lemongrass EO inhibited biofilm formation at 0·125% (v/v) as measured by colorimetric assay and at 0·25% (v/v) no metabolic activity was observed as determined by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction. Grapefruit EO did not show any anti-biofilm activity. Following exposure to lemongrass EO extensive disruption to Staph. aureus biofilms was shown under scanning electron microscopy. In comparison to the other EOs tested, lemongrass exhibited the most effective antimicrobial and anti-biofilm activity. The effect of lemongrass EO highlights its potential against antibiotic resistant Staph. aureus in the healthcare environment. © 2012 The Authors Journal of Applied Microbiology © 2012 The Society for Applied Microbiology.

Combined use of Bacteriophage K and a novel Bacteriophage to reduce Staphylococcus aureus biofilm formation

DEFF Research Database (Denmark)

Alves, D.R.; Gaudion, A.; Bean, J.E.

2014-01-01

Biofilms are major causes of impairment of wound healing and patient morbidity. One of the most common and aggressive wound pathogens is Staphylococcus aureus, displaying a large repertoire of virulence factors and commonly reduced susceptibility to antibiotics, such as the spread of methicillin-...

Hygrocin C from marine-derived Streptomyces sp. SCSGAA 0027 inhibits biofilm formation in Bacillus amyloliquefaciens SCSGAB0082 isolated from South China Sea gorgonian.

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Wang, Jie; Nong, Xu-Hua; Amin, Muhammad; Qi, Shu-Hua

2018-02-01

Several ansamycins have been reported to inhibit bacterial biofilm formation and accelerate the eradication of developed biofilms, but little is known about the effect of hygrocin C, an ansamycin, on bacterial biofilm formation. Here, hygrocin C was isolated from the marine-derived Streptomyces sp. SCSGAA 0027 and reported for the first time to be capable of inhibiting the biofilm formation of Staphylococcus aureus and Bacillus amyloliquefaciens SCSGAB0082 with the production of anti-microbial lipopeptides from South China Sea gorgonian Subergorgia suberosa at concentrations of less than minimum inhibitory concentrations. Moreover, hygrocin C also promoted the eradication of developed biofilms, affected the biofilm architecture, and lowered the extracellular polymeric matrix formation, cell motility, and surface hydrophobicity in B. amyloliquefaciens, which was in accordance with the inhibition of biofilm formation. Furthermore, transcriptome analysis revealed that hygrocin C altered the transcripts of several genes associated with bacterial chemotaxis and flagellar, two-component system and the synthesis of arginine and histidine, which are important for bacterial biofilm formation. In conclusion, hygrocin C could be used as a potential biofilm inhibitor against S. aureus and B. amyloliquefaciens. But further genetic investigations are needed to provide more details for elucidation of the molecular mechanisms responsible for the effects of hygrocin C on B. amyloliquefaciens biofilm formation.

msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus.

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Sahukhal, Gyan S; Batte, Justin L; Elasri, Mohamed O

2015-02-01

Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mammary Gland Pathology Subsequent to Acute Infection with Strong versus Weak Biofilm Forming Staphylococcus aureus Bovine Mastitis Isolates: A Pilot Study Using Non-Invasive Mouse Mastitis Model.

Science.gov (United States)

Gogoi-Tiwari, Jully; Williams, Vincent; Waryah, Charlene Babra; Costantino, Paul; Al-Salami, Hani; Mathavan, Sangeetha; Wells, Kelsi; Tiwari, Harish Kumar; Hegde, Nagendra; Isloor, Shrikrishna; Al-Sallami, Hesham; Mukkur, Trilochan

2017-01-01

Biofilm formation by Staphylococcus aureus is an important virulence attribute because of its potential to induce persistent antibiotic resistance, retard phagocytosis and either attenuate or promote inflammation, depending upon the disease syndrome, in vivo. This study was undertaken to evaluate the potential significance of strength of biofilm formation by clinical bovine mastitis-associated S. aureus in mammary tissue damage by using a mouse mastitis model. Two S. aureus strains of the same capsular phenotype with different biofilm forming strengths were used to non-invasively infect mammary glands of lactating mice. Biofilm forming potential of these strains were determined by tissue culture plate method, ica typing and virulence gene profile per detection by PCR. Delivery of the infectious dose of S. aureus was directly through the teat lactiferous duct without invasive scraping of the teat surface. Both bacteriological and histological methods were used for analysis of mammary gland pathology of mice post-infection. Histopathological analysis of the infected mammary glands revealed that mice inoculated with the strong biofilm forming S. aureus strain produced marked acute mastitic lesions, showing profuse infiltration predominantly with neutrophils, with evidence of necrosis in the affected mammary glands. In contrast, the damage was significantly less severe in mammary glands of mice infected with the weak biofilm-forming S. aureus strain. Although both IL-1β and TNF-α inflammatory biomarkers were produced in infected mice, level of TNF-α produced was significantly higher (pmastitis model, and offers an opportunity for the development of novel strategies for reduction of mammary tissue damage, with or without use of antimicrobials and/or anti-inflammatory compounds for the treatment of bovine mastitis.

Commensal Protection of Staphylococcus aureus against Antimicrobials by Candida albicans Biofilm Matrix

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Kong, Eric F.; Tsui, Christina; Kucharíková, Sona; Andes, David

2016-01-01

ABSTRACT Biofilm-associated polymicrobial infections, particularly those involving fungi and bacteria, are responsible for significant morbidity and mortality and tend to be challenging to treat. Candida albicans and Staphylococcus aureus specifically are considered leading opportunistic fungal and bacterial pathogens, respectively, mainly due to their ability to form biofilms on catheters and indwelling medical devices. However, the impact of mixed-species biofilm growth on therapy remains largely understudied. In this study, we investigated the influence of C. albicans secreted cell wall polysaccharides on the response of S. aureus to antibacterial agents in biofilm. Results demonstrated significantly enhanced tolerance for S. aureus to drugs in the presence of C. albicans or its secreted cell wall polysaccharide material. Fluorescence confocal time-lapse microscopy revealed impairment of drug diffusion through the mixed biofilm matrix. Using C. albicans mutant strains with modulated cell wall polysaccharide expression, exogenous supplementation, and enzymatic degradation, the C. albicans-secreted β-1,3-glucan cell wall component was identified as the key matrix constituent providing the bacteria with enhanced drug tolerance. Further, antibody labeling demonstrated rapid coating of the bacteria by the C. albicans matrix material. Importantly, via its effect on the fungal biofilm matrix, the antifungal caspofungin sensitized the bacteria to the drugs. Understanding such symbiotic interactions with clinical relevance between microbial species in biofilms will greatly aid in overcoming the limitations of current therapies and in defining potential new targets for treating polymicrobial infections. PMID:27729510

Biofilm formation and virulence factor analysis of Staphylococcus aureus isolates collected from ovine mastitis.

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Azara, E; Longheu, C; Sanna, G; Tola, S

2017-08-01

To perform a phenotypic and genotypic characterization of 258 Staphylococcus aureus isolates from clinical ovine mastitis and used for the preparation of inactivated autogenous vaccines. The potential for biofilm production was determined by phenotypic test of Congo Red Agar (CRA) and by PCR for the detection of icaA/D genes. Isolates were also screened by PCR for the presence of enterotoxins (sea, seb, sec, sed and see), toxic shock syndrome toxin (tsst), leukotoxins (lukD-E, lukM and lukPV83), haemolysins (hly-β and hly-γ), autolysin (atlA) genes and encoding microbial surface components recognizing adhesive matrix molecules (MSCRAMMs: clfA, clfB, fnbA, fnbB, bbp, cna, eno, fib, epbs, sdrC, sdrD and SdrE). None of the 258 isolates showed biofilm-forming ability on CRA and harboured icaA/D genes. The most frequent pyrogenic toxin superantigen genes amplified were sec plus tsst-1, which were found strictly in combination with 71·3% of the Staph. aureus isolates tested. None of the isolates harboured the genes encoding sea and see. Of the 258 isolates tested, 159 (61·6%) possessed all lukD-E/lukM/lukPV83 genes, 123 (47·7%) harboured both hly-β/hly-γ genes, whereas almost all (97·3%) were PCR positive for atlA gene. With respect to adhesion determinants, 179 (69·4%) isolates presented simultaneously four genes (fnbA, fib, clfA and clfB) for fibronectin- and fibrinogen-binding proteins. In this search, several putative virulence determinants have been identified in ovine Staph. aureus isolates collected in Sardinia. Some of the putative virulence determinants could be considered as components of a vaccine because of their role in ovine mastitis pathogenesis. © 2017 The Society for Applied Microbiology.

The in vitro effect of xylitol on chronic rhinosinusitis biofilms.

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Jain, R; Lee, T; Hardcastle, T; Biswas, K; Radcliff, F; Douglas, R

2016-12-01

Biofilms have been implicated in chronic rhinosinusitis (CRS) and may explain the limited efficacy of antibiotics. There is a need to find more effective, non-antibiotic based therapies for CRS. This study examines the effects of xylitol on CRS biofilms and planktonic bacteria. Crystal violet assay and spectrophotometry were used to quantify the effects of xylitol (5% and 10% solutions) against Staphylococcus epidermidis, Pseudomonas aeruginosa, and Staphylococcus aureus. The disruption of established biofilms, inhibition of biofilm formation and effects on planktonic bacteria growth were investigated and compared to saline and no treatment. Xylitol 5% and 10% significantly reduced biofilm biomass (S. epidermidis), inhibited biofilm formation (S. aureus and P. aeruginosa) and reduced growth of planktonic bacteria (S. epidermidis, S. aureus, and P. aeruginosa). Xylitol 5% inhibited formation of S. epidermidis biofilms more effectively than xylitol 10%. Xylitol 10% reduced S. epidermidis planktonic bacteria more effectively than xylitol 5%. Saline, xylitol 5% and 10% disrupted established biofilms of S. aureus when compared with no treatment. No solution was effective against established P. aeruginosa biofilm. Xylitol has variable activity against biofilms and planktonic bacteria in vitro and may have therapeutic efficacy in the management of CRS.

5-aminolevulinic acid-mediated photodynamic therapy and its strain-dependent combined effect with antibiotics on Staphylococcus aureus biofilm.

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Qing-Zhao Zhang

Full Text Available Staphylococcus aureus (S. aureus is hard to be eradicated, not only due to the emergence of antibiotic resistant strains but also because of its ability to form biofilm. Antibiotics are the major approach to treating biofilm infections, but their effects are unsatisfactory. One of the potential alternative treatments for controlling biofilm infections is photodynamic therapy (PDT, which requires the administration of photosensitizer, followed by light activation. 5-aminolevulinic acid (ALA, a natural photosensitizer prodrug, presents favorable characteristics, such as easy penetration and rapid clearance. These advantages enable ALA-based PDT (ALA-PDT to be well-tolerated by patients and it can be repeatedly applied without cumulative toxicity or serious side effects. ALA-PDT has been proven to be an effective treatment for multidrug resistant pathogens; however, the study of its effect on S. aureus biofilm is limited. Here, we established our PDT system based on the utilization of ALA and a light-emitting diode, and we tested the effect of ALA-PDT on S. aureus biofilm as well as the combined effect of ALA-PDT and antibiotics on S. aureus biofilm. Our results showed that ALA-PDT has a strong antibacterial effect on S. aureus biofilm, which was confirmed by the confocal laser scanning microscope. We also found that lethal photosensitization occurred predominantly in the upper layer of the biofilm, while the residual live bacteria were located in the lower layer of the biofilm. In addition, the improved bactericidal effect was observed in the combined treatment group but in a strain-dependent manner. Our results suggest that ALA-PDT is a potential alternative approach for future clinical use to treat S. aureus biofilm-associated infections, and some patients may benefit from the combined treatment of ALA-PDT and antibiotics, but drug sensitivity testing should be performed in advance.

Biofilms.

Science.gov (United States)

López, Daniel; Vlamakis, Hera; Kolter, Roberto

2010-07-01

The ability to form biofilms is a universal attribute of bacteria. Biofilms are multicellular communities held together by a self-produced extracellular matrix. The mechanisms that different bacteria employ to form biofilms vary, frequently depending on environmental conditions and specific strain attributes. In this review, we emphasize four well-studied model systems to give an overview of how several organisms form biofilms: Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Using these bacteria as examples, we discuss the key features of biofilms as well as mechanisms by which extracellular signals trigger biofilm formation.

Searching for new strategies against biofilm infections: Colistin-AMP combinations against Pseudomonas aeruginosa and Staphylococcus aureus single- and double-species biofilms.

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Paula Jorge

Full Text Available Antimicrobial research is being pressured to look for more effective therapeutics for the ever-growing antibiotic-resistant infections, and antimicrobial peptides (AMP and antimicrobial combinations are promising solutions. This work evaluates colistin-AMP combinations against two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, encompassing non- and resistant strains. Colistin (CST combined with the AMP temporin A (TEMP-A, citropin 1.1 (CIT-1.1 and tachyplesin I linear analogue (TP-I-L was tested against planktonic, single- and double-species biofilm cultures. Overall synergy for planktonic P. aeruginosa and synergy/additiveness for planktonic S. aureus were observed. Biofilm growth prevention was achieved with synergy and additiveness. Pre-established 24 h-old biofilms were harder to eradicate, especially for S. aureus and double-species biofilms; still, some synergy and addictiveness was observed for higher concentrations, including for the biofilms of resistant strains. Different treatment times and growth media did not greatly influence AMP activity. CST revealed low toxicity compared with the other AMP but its combinations were toxic for high concentrations. Overall, combinations reduced effective AMP concentrations, mainly in prevention scenarios. Improvement of effectiveness and toxicity of therapeutic strategies will be further investigated.

Biofilm inhibitory and eradicating activity of wound care products against Staphylococcus aureus and Staphylococcus epidermidis biofilms in an in vitro chronic wound model.

Science.gov (United States)

Brackman, G; De Meyer, L; Nelis, H J; Coenye, T

2013-06-01

Although several factors contribute to wound healing, bacterial infections and the presence of biofilm can significantly affect healing. Despite that this clearly indicates that therapies should address biofilm in wounds, only few wound care products have been evaluated for their antibiofilm effect. For this reason, we developed a rapid quantification approach to investigate the efficacy of wound care products on wounds infected with Staphylococcus spp. An in vitro chronic wound infection model was used in which a fluorescent Staph. aureus strain was used to allow the rapid quantification of the bacterial burden after treatment. A good correlation was observed between the fluorescence signal and the bacterial counts. When evaluated in this model, several commonly used wound dressings and wound care products inhibited biofilm formation resulting in a decrease between one and seven log CFU per biofilm compared with biofilm formed in the absence of products. In contrast, most dressings only moderately affected mature biofilms. Our model allowed the rapid quantification of the bacterial burden after treatment. However, the efficacy of treatment varied between the different types of dressings and/or wound care products. Our model can be used to compare the efficacy of wound care products to inhibit biofilm formation and/or eradicate mature biofilms. In addition, the results indicate that treatment of infected wounds should be started as soon as possible and that novel products with more potent antibiofilm activity are needed. © 2013 The Society for Applied Microbiology.

The Effect of Cryopreserved Human Placental Tissues on Biofilm Formation of Wound-Associated Pathogens.

Science.gov (United States)

Mao, Yong; Singh-Varma, Anya; Hoffman, Tyler; Dhall, Sandeep; Danilkovitch, Alla; Kohn, Joachim

2018-01-08

Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers) clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM) resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT) on biofilm formation of S. aureus and P. aeruginosa , the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.

The Effect of Cryopreserved Human Placental Tissues on Biofilm Formation of Wound-Associated Pathogens

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Yong Mao

2018-01-01

Full Text Available Biofilm, a community of bacteria, is tolerant to antimicrobial agents and ubiquitous in chronic wounds. In a chronic DFU (Diabetic Foot Ulcers clinical trial, the use of a human cryopreserved viable amniotic membrane (CVAM resulted in a high rate of wound closure and reduction of wound-related infections. Our previous study demonstrated that CVAM possesses intrinsic antimicrobial activity against a spectrum of wound-associated bacteria under planktonic culture conditions. In this study, we evaluated the effect of CVAM and cryopreserved viable umbilical tissue (CVUT on biofilm formation of S. aureus and P. aeruginosa, the two most prominent pathogens associated with chronic wounds. Firstly, we showed that, like CVAM, CVUT released antibacterial activity against multiple bacterial pathogens and the devitalization of CVUT reduced its antibacterial activity. The biofilm formation was then measured using a high throughput method and an ex vivo porcine dermal tissue model. We demonstrate that the formation of biofilm was significantly reduced in the presence of CVAM- or CVUT-derived conditioned media compared to control assay medium. The formation of P. aeruginosa biofilm on CVAM-conditioned medium saturated porcine dermal tissues was reduced 97% compared with the biofilm formation on the control medium saturated dermal tissues. The formation of S. auerus biofilm on CVUT-conditioned medium saturated dermal tissues was reduced 72% compared with the biofilm formation on the control tissues. This study is the first to show that human cryopreserved viable placental tissues release factors that inhibit biofilm formation. Our results provide an explanation for the in vivo observation of their ability to support wound healing.

Sustained prevention of biofilm formation on a novel silicone matrix suitable for medical devices

DEFF Research Database (Denmark)

Steffensen, Søren Langer; Merete H., Vestergaard,; Jensen, Minna Grønning

2015-01-01

Bacterial colonization and biofilm formation on medical devices constitute major challenges in clinical long-term use of e.g. catheters due to the risk of (re)infection of patients, which would result in additional use of antibiotics risking bacterial resistance development. The aim of the present...... in the range of 1–20 mg/mL. Devices containing 25% (w/w) hydrogel and loaded with ciprofloxacin displayed a strong antibacterial effect against Staphylococcus aureus bacterial colonization and subsequent biofilm formation on the device material was inhibited for 29 days. In conclusion, the hydrogel...

Formation and retention of staphylococcal biofilms on DLC and its hybrids compared to metals used as biomaterials.

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Myllymaa, Katja; Levon, Jaakko; Tiainen, Veli-Matti; Myllymaa, Sami; Soininen, Antti; Korhonen, Hannu; Kaivosoja, Emilia; Lappalainen, Reijo; Konttinen, Yrjö Tapio

2013-01-01

Staphylococcus epidermidis and Staphylococcus aureus cause most of the implant-related infections. Antibiotic treatment often fails and cure requires surgical intervention. It was hypothesized that biomaterial coatings resistant to biofilms offer a preventive option. Physical vapour deposited diamond-like carbon (DLC) and its polytetrafluoroethylene (DLC-PTFE-h) and polydimethylsiloxane (DLC-PDMS-h) hybrids were compared to titanium (Ti), tantalum (Ta) and chromium (Cr) thin films on silicon wafers for their resistance against formation and/or retention of biofilms produced by S. epidermidis and S. aureus in vitro. Sample surfaces were characterized for surface topography, contact angle and zeta-potential, because such properties might affect the biofilm. Biofilm was stained using calcofluor white and analysed in fluorescence microscopy using morphometry. Sixteen hour incubation was selected in pilot tests; at this checkpoint Ti, Ta, Cr and DLC-PDMS-h were almost fully covered by biofilm, but DLC and DLC-PTFE-h were only partially biofilm coated by S. epidermidis (88±26%, pDLC and its PTFE hybrid offer a potential biofilm hostile surface coating for implants and medical devices. This ability to resist biofilm formation and attachment could not be explained by only one factor, but it seems to be related to a combination of various properties, with electrokinetic streaming potential and protein coating being particularly important for its outcome. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of combinations of putative anti-biofilm agents and antibiotics to eradicate biofilms of Staphylococcus aureus and Pseudomonas aeruginosa.

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Belfield, Katherine; Bayston, Roger; Hajduk, Nadzieja; Levell, Georgia; Birchall, John P; Daniel, Matija

2017-09-01

To evaluate potential anti-biofilm agents for their ability to enhance the activity of antibiotics for local treatment of localized biofilm infections. Staphylococcus aureus and Pseudomonas aeruginosa in vitro biofilm models were developed. The putative antibiotic enhancers N-acetylcysteine, acetylsalicylic acid, sodium salicylate, recombinant human deoxyribonuclease I, dispersin B, hydrogen peroxide and Johnson's Baby Shampoo (JBS) were tested for their anti-biofilm activity alone and their ability to enhance the activity of antibiotics for 7 or 14 days, against 5 day old biofilms. The antibiotic enhancers were paired with rifampicin and clindamycin against S. aureus and gentamicin and ciprofloxacin against P. aeruginosa. Isolates from biofilms that were not eradicated were tested for antibiotic resistance. Antibiotic levels 10× MIC and 100× MIC significantly reduced biofilm, but did not consistently eradicate it. Antibiotics at 100× MIC with 10% JBS for 14 days was the only treatment to eradicate both staphylococcal and pseudomonal biofilms. Recombinant human deoxyribonuclease I significantly reduced staphylococcal biofilm. Emergence of resistance of surviving isolates was minimal and was often associated with the small colony variant phenotype. JBS enhanced the activity of antibiotics and several other promising anti-biofilm agents were identified. Antibiotics with 10% JBS eradicated biofilms produced by both organisms. Such combinations might be useful in local treatment of localized biofilm infections. © The Author 2017. 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.

Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma

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Watters CM

2016-04-01

Full Text Available Chase M Watters,1,2 Tarea Burton,1 Dickson K Kirui,1 Nancy J Millenbaugh1 1Maxillofacial Injury and Disease Department, Naval Medical Research Unit San Antonio, Joint Base San Antonio-Fort Sam Houston, TX, USA; 2Wound Infections Department, Naval Medical Research Center, Silver Spring, MD, USA Abstract: Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%–50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas a-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve

Streptococcus pneumoniae eradicates preformed Staphylococcus aureus biofilms through a mechanism requiring physical contact

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Faidad Khan

2016-09-01

Full Text Available Staphylococcus aureus (Sau strains are a main cause of disease, including nosocomial infections which have been linked to the production of biofilms and the propagation of antibiotic resistance strains such as methicillin-resistant Staphylococcus aureus (MRSA. A previous study found that Streptococcus pneumoniae (Spn strains kill planktonic cultures of Sau strains. In this work, we have further evaluated in detail the eradication of Sau biofilms and investigated ultrastructural interactions of the biofilmicidal effect. Spn strain D39, which produces the competence stimulating peptide 1 (CSP1, reduced Sau biofilms within 8 h of inoculation, while TIGR4, producing CSP2, eradicated Sau biofilms and planktonic cells within 4 h. Differences were not attributed to pherotypes as other Spn strains producing different pheromones eradicated Sau within 4 h. Experiments using Transwell devices, which physically separated both species growing in the same well, demonstrated that direct contact between Spn and Sau was required to efficiently eradicate Sau biofilms and biofilm-released planktonic cells. Physical contact-mediated killing of Sau was not related to production of hydrogen peroxide as an isogenic TIGR4spxB mutant eradicated Sau bacteria within 4 h. Confocal micrographs confirmed eradication of Sau biofilms by TIGR4 and allowed us to visualize ultrastructural point of contacts between Sau and Spn. A time-course study further demonstrated spatial colocalization of Spn chains and Sau tetrads as early as 30 min post-inoculation (Pearson’s coefficient >0.72. Finally, precolonized biofilms produced by Sau strain Newman, or MRSA strain USA300, were eradicated by mid-log phase cultures of washed TIGR4 bacteria within 2 h post-inoculation. In conclusion, Spn strains rapidly eradicate pre-colonized Sau aureus biofilms, including those formed by MRSA strains, by a mechanism(s requiring bacterium-bacterium contact, but independent from the production of

Meningococcal biofilm formation

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Lappann, M.; Haagensen, Janus Anders Juul; Claus, H.

2006-01-01

We show that in a standardized in vitro flow system unencapsulated variants of genetically diverse lineages of Neisseria meningitidis formed biofilms, that could be maintained for more than 96 h. Biofilm cells were resistant to penicillin, but not to rifampin or ciprofloxacin. For some strains......, microcolony formation within biofilms was observed. Microcolony formation in strain MC58 depended on a functional copy of the pilE gene encoding the pilus subunit pilin, and was associated with twitching of cells. Nevertheless, unpiliated pilE mutants formed biofilms showing that attachment and accumulation......X alleles was identified among genetically diverse meningococcal strains. PilX alleles differed in their propensity to support autoaggregation of cells in suspension, but not in their ability to support microcolony formation within biofilms in the continuous flow system....

Curcumin reduces Streptococcus mutans biofilm formation by inhibiting sortase A activity.

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Hu, Ping; Huang, Ping; Chen, Min Wei

2013-10-01

Sortase A is an enzyme responsible for the covalent attachment of Pac proteins to the cell wall in Streptococcus mutans. It has been shown to play a role in modulating the surface properties and the biofilm formation and influence the cariogenicity of S. mutans. Curcumin, an active ingredient of turmeric, was reported to be an inhibitor for Staphylococcus aureus sortase A. The aim of this study was to investigate the inhibitory ability of curcumin against S. mutans sortase A and the effect of curcumin for biofilm formation. The antimicrobial activity of the curcumin to the S. mutans and inhibitory ability of the curcumin against the purified sortase A in vitro were detected. Western-blot and real-time PCR were used to analysis the sortase A mediated Pac protein changes when the S. mutans was cultured with curcumin. The curcumin on the S. mutans biofilm formation was determined by biofilm formation analysis. Curcumin can inhibit purified S. mutans sortase A with a half-maximal inhibitory concentration (IC50) of (10.2±0.7)μmol/l, which is lower than minimum inhibitory concentration (MIC) of 175μmol/l. Curcumin (15μmol/l) was found to release the Pac protein to the supernatant and reduce S. mutans biofilm formation. These results indicated that curcumin is an S. mutans sortase A inhibitor and has promising anti-caries characteristics through an anti-adhesion-mediated mechanism. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Low Fluid Shear Culture of Staphylococcus Aureus Represses hfq Expression and Induces an Attachment-Independent Biofilm Phenotype

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Ott, C. Mark; Castro, S. L.; Nickerson, C. A.; Nelman-Gonzalez, M.

2011-01-01

Background: The opportunistic pathogen, Staphylococcus aureus, experiences fluctuations in fluid shear during infection and colonization of a human host. Colonization frequently occurs at mucus membrane sites such as in the gastrointestinal tract where the bacterium may experience low levels of fluid shear. The response of S. aureus to low fluid shear remains unclear. Methods: S. aureus was cultured to stationary phase using Rotating-Wall Vessel (RWV) bioreactors which produce a physiologically relevant low fluid shear environment. The bacterial aggregates that developed in the RWV were evaluated by electron microscopy as well as for antibiotic resistance and other virulence-associated stressors. Genetic expression profiles for the low-shear cultured S. aureus were determined by microarray analysis and quantitative real-time PCR. Results: Planktonic S. aureus cultures in the low-shear environment formed aggregates completely encased in high amounts of extracellular polymeric substances. In addition, these aggregates demonstrated increased antibiotic resistance indicating attachment-independent biofilm formation. Carotenoid production in the low-shear cultured S. aureus was significantly decreased, and these cultures displayed an increased susceptibility to oxidative stress and killing by whole blood. The hfq gene, associated with low-shear growth in Gram negative organisms, was also found to be down-regulated in S. aureus. Conclusions: Collectively, this data suggests that S. aureus decreases virulence characteristics in favor of a biofilm-dwelling colonization phenotype in response to a low fluid shear environment. Furthermore, the identification of an Hfq response to low-shear culture in S. aureus, in addition to the previously reported responses in Gram negative organisms, strongly suggests an evolutionarily conserved response to mechanical stimuli among structurally diverse prokaryotes.

Effect of peracetic acid on biofilms formed by Staphylococcus aureus and Listeria monocytogenes isolated from dairy plants.

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Lee, S H I; Cappato, L P; Corassin, C H; Cruz, A G; Oliveira, C A F

2016-03-01

This research investigated the removal of adherent cells of 4 strains of Staphylococcus aureus and 1 Listeria monocytogenes strain (previously isolated from dairy plants) from polystyrene microtiter plates using peracetic acid (PAA, 0.5%) for 15, 30, 60, and 120 s, and the inactivation of biofilms formed by those strains on stainless steel coupons using the same treatment times. In the microtiter plates, PAA removed all S. aureus at 15 s compared with control (no PAA treatment). However, L. monocytogenes biofilm was not affected by any PAA treatment. On the stainless steel surface, epifluorescence microscopy using LIVE/DEAD staining (BacLight, Molecular Probes/Thermo Fisher Scientific, Eugene, OR) showed that all strains were damaged within 15 s, with almost 100% of cells inactivated after 30 s. Results of this trial indicate that, although PAA was able to inactivate both S. aureus and L. monocytogenes monospecies biofilms on stainless steel, it was only able to remove adherent cells of S. aureus from polystyrene microplates. The correct use of PAA is critical for eliminating biofilms formed by S. aureus strains found in dairy plants, although further studies are necessary to determine the optimal PAA treatment for removing biofilms of L. monocytogenes. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

NaOCl effect on biofilm produced by Staphylococcus aureus isolated from the milking environment and mastitis infected cows

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Poliana de Castro Melo

2014-02-01

Full Text Available Biofilms constitute a physical barrier, protecting the encased bacteria from detergents and sanitizers. The objective of this work was to analyze the effectiveness of sodium hypochlorite (NaOCl against strains of Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis and Staphylococcus aureus isolated from the milking environment (blowers and milk conducting tubes. The results revealed that, in the presence of NaOCl (150ppm, the number of adhered cells of the twelve S. aureus strains was significantly reduced. When the same strains were evaluated in biofilm condition, different results were obtained. It was found that, after a contact period of five minutes with NaOCl (150ppm, four strains (two strains from milk , one from the blowers and one from a conductive rubber were still able to grow. Although with the increasing contact time between the bacteria and the NaOCl (150ppm, no growth was detected for any of the strains. Concerning the efficiency of NaOCl on total biofilm biomass formation by each S. aureus strain, a decrease was observed when these strains were in contact with 150 ppm NaOCl for a total period of 10 minutes. This study highlights the importance of a correct sanitation protocol of all the milk processing units which can indeed significantly reduce the presence of microorganisms, leading to a decrease of cow´s mastitis and milk contamination.

Chamaecyparis obtusa Essential Oil Inhibits Methicillin-Resistant Staphylococcus aureus Biofilm Formation and Expression of Virulence Factors.

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Kim, Eun-Sook; Kang, Sun-Young; Kim, Young-Hoi; Lee, Young-Eun; Choi, Na-Young; You, Yong-Ouk; Kim, Kang-Ju

2015-07-01

The emergence of antibiotic-resistant bacteria has caused difficulty in treating infectious diseases. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most commonly recognized antibiotic-resistant bacteria. Novel antibiotics are urgently required to treat these bacteria. Raw materials derived from natural sources can be used for the development of novel antibiotics, such as Chamaecyparis obtusa (C. obtusa), which has been traditionally used in treating asthmatic disease. In this study, the antibacterial activity of the essential oil (EO) extracted from C. obtusa leaves against MRSA was investigated. MRSA growth and acid production from glucose metabolism were inhibited at concentrations greater than 0.1 mg/mL C. obtusa EO. MRSA biofilm formation was observed using scanning electron microscopy and safranin staining. C. obtusa EO inhibited MRSA biofilm formation at concentrations greater than 0.1 mg/mL. Using real-time polymerase chain reaction, mRNA expression of virulence factor genes, sea, agrA, and sarA, was observed. agrA expression was inhibited with C. obtusa EO concentrations greater than 0.2 mg/mL, whereas inhibition of sea and sarA expression was also observed at a concentration of 0.3 mg/mL. C. obtusa EO was analyzed by gas chromatography (GC) and GC coupled for mass spectrometry, which identified 59 constituents, accounting to 98.99% of the total EO. These findings suggest that C. obtusa EO has antibacterial effects against MRSA, which might be associated with the major components of C. obtusa EO, such as sabinene (19.06%), α-terpinyl acetate (16.99%), bornyl acetate (10.48%), limonene (8.54%), elemol (7.47%), myrcene (5.86%), γ-terpinene (4.04%), and hibaene (3.01%).

The Activity of Cotinus coggygria Scop. Leaves on Staphylococcus aureus Strains in Planktonic and Biofilm Growth Forms

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Katarína Rendeková

2015-12-01

Full Text Available The purpose of this study was to detect the effectiveness of Cotinus coggygria Scop. leaves methanol extract against planktonic and biofilm growth forms of Staphylococcus aureus. The antimicrobial activity was determined by the broth microdilution test. Minimal inhibitory concentrations and minimal bactericidal concentrations were detected against two collection and ten clinical S. aureus strains. Anti-biofilm activity of the tested extract was detected using 24 h bacterial biofilm on the surface of microtiter plate wells. The biofilm inhibitory activity was evaluated visually after 24 h interaction of extract with biofilm, and the eradicating activity by a regrowth method. The tested extract showed bactericidal activity against all S. aureus strains (methicillin susceptible or methicillin resistant in concentrations ranging from 0.313 to 0.625 mg·mL−1. Biofilm inhibitory concentrations were 10-times higher and biofilm eradicating concentrations 100-times higher (8 and 32 mg·mL−1, respectively. The phytochemical analysis of C. coggygria leaves 60% methanol extract performed by LC-DAD-MS/MS revealed quercetin rhamnoside, methyl gallate, and methyl trigallate as main constituents. Results of our study indicate that C. coggygria, rich in tannins and flavonoids, seems to be a prospective topical antibacterial agent with anti-biofilm activity.

Evaluation of CAMP-Like Effect, Biofilm Formation, and Discrimination of Candida africana from Vaginal Candida albicans Species

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Keyvan Pakshir

2017-01-01

Full Text Available Candida africana as a species recovered from female genital specimens is highly close to C. albicans. The present study was conducted to discriminate C. africana from presumptive vaginal C. albicans strains by molecular assay and evaluate their hemolysin activity, biofilm formation, and cohemolytic effect (CAMP with vaginal bacterial flora. A total of 110 stock vaginal C. albicans isolates were examined by HWP1 gene amplification. Hemolysin activity and the ability of biofilm formation were evaluated by blood plate assay and visual detection methods, respectively. Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus agalactiae were used to evaluate the CAMP-like effects in Sabouraud blood agar media. Based on the size of the amplicons (941 bp, all isolates were identified as C. albicans. All samples were able to produce beta-hemolysin. Moreover, 69 out of 110 of the isolates (62.7% were biofilm-positive, 54 out of 110 Candida isolates (49% demonstrated cohemolytic effects with S. agalactiae, and 48 out of 110 showed this effect with S. aureus (43.6%. All isolates were CAMP-negative with S. epidermidis. We detected all isolates as Candida albicans and almost half of the isolates were CAMP-positive with S. aureus and S. agalactiae, suggesting that these bacteria increase the pathogenicity of Candida in vaginal candidiasis.

Evaluation of CAMP-Like Effect, Biofilm Formation, and Discrimination of Candida africana from Vaginal Candida albicans Species

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Bordbar, Mahboubeh; Nouraei, Hasti; Khodadadi, Hossein

2017-01-01

Candida africana as a species recovered from female genital specimens is highly close to C. albicans. The present study was conducted to discriminate C. africana from presumptive vaginal C. albicans strains by molecular assay and evaluate their hemolysin activity, biofilm formation, and cohemolytic effect (CAMP) with vaginal bacterial flora. A total of 110 stock vaginal C. albicans isolates were examined by HWP1 gene amplification. Hemolysin activity and the ability of biofilm formation were evaluated by blood plate assay and visual detection methods, respectively. Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus agalactiae were used to evaluate the CAMP-like effects in Sabouraud blood agar media. Based on the size of the amplicons (941 bp), all isolates were identified as C. albicans. All samples were able to produce beta-hemolysin. Moreover, 69 out of 110 of the isolates (62.7%) were biofilm-positive, 54 out of 110 Candida isolates (49%) demonstrated cohemolytic effects with S. agalactiae, and 48 out of 110 showed this effect with S. aureus (43.6%). All isolates were CAMP-negative with S. epidermidis. We detected all isolates as Candida albicans and almost half of the isolates were CAMP-positive with S. aureus and S. agalactiae, suggesting that these bacteria increase the pathogenicity of Candida in vaginal candidiasis. PMID:29318048

Comparative proteomic analysis of extracellular proteins expressed by various clonal types of Staphylococcus aureus and during planktonic growth and biofilm development.

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Atshan, Salman S; Shamsudin, Mariana N; Sekawi, Zamberi; Thian Lung, Leslie T; Barantalab, Fatemeh; Liew, Yun K; Alreshidi, Mateg Ali; Abduljaleel, Salwa A; Hamat, Rukman A

2015-01-01

Staphylococcus aureus is well known for its biofilm formation with rapid emergence of new clones circulating worldwide. The main objectives of the study were (1) to identify possible differences in protein expression among various and closely related clonal types of S. aureus, (2) to establish the differences in protein expression in terms of size of protein spots and its intensities between bacteria which are grown statically (biofilm formation) with that of under aeration and agitation, and (3) to compare the differences in protein expression as a function of time (in hours). In this study, we selected six clinical isolates comprising two similar (MRSA-527 and MRSA-524) and four different (MRSA-139, MSSA-12E, MSSA-22d, and MSSA-10E) types identified by spa typing, MLST and SCCmec typing. We performed 2D gel migration comparison. Also, two MRSA isolates (527 and 139) were selected to determine quantitative changes in the level of extracellular proteins at different biofilm growth time points of 12, 24, and 48 h. The study was done using a strategy that combines 2-DGE and LC-MS/MS analysis for absolute quantification and identification of the extracellular proteins. The 2DGE revealed that the proteomic profiles for the isolates belonging to the similar spa, MLST, and SCCmec types were still quite different. Among the extracellular proteins secreted at different time points of biofilm formation, significant changes in protein expression were observed at 48 h incubation as compared to the exponential growth at 12 h incubation. The main conclusion of the work is that the authors do observe differences among isolates, and growth conditions do influence the protein content at different time points of biofilm formation.

Efficacy of silk fibroin–nano silver against Staphylococcus aureus biofilms in a rabbit model of sinusitis

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Jia M

2017-04-01

Full Text Available Minghui Jia,1,2 Zhongchun Chen,2 Yongwei Guo,2 Xin Chen,3 Xia Zhao2 1Department of Otolaryngology-Head and Neck Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 2Department of Otolaryngology-Head and Neck Surgery, Huashan Hospital, Fudan University, 3State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai, China Background: Staphylococcus aureus biofilms contribute significantly to the recalcitrant nature of chronic rhinosinusitis. In previous studies, it has been shown that silk fibroin–nano silver solution can eliminate S. aureus biofilms in vitro, which suggests a potential role of this novel agent in the treatment of biofilm-associated diseases, such as sinusitis.Objective: The aim of this study was to investigate the efficacy of silk fibroin–nano silver solution as a topical anti-biofilm agent in a rabbit model of sinusitis.Methods: Biofilm-associated sinusitis models were established in 24 New Zealand White rabbits by gelatin sponge placement and S. aureus inoculation through a hole drilled into the anterolateral wall of the right maxillary sinus. After 4 weeks, indwelling catheters were placed into the maxillary sinus. Different concentrations of silk fibroin–nano silver solution or normal saline were irrigated slowly into the maxillary sinus via the indwelling catheters. After 7 days of irrigation, the rabbits were sacrificed. The sinus mucosa was harvested and examined for biofilm biomass as well as morphological integrity of the epithelium by scanning electron microscopy.Results: Silk fibroin–nano silver solution was found to be most effective in reducing the biomass of the S. aureus biofilms at a concentration of 384 mg/L, followed by the concentration of 153.6 mg/L, when compared with saline. After treatment with 384 mg/L silk fibroin–nano silver solution, the biofilms were completely

Effect of a solution containing citrate/Methylene Blue/parabens on Staphylococcus aureus bacteria and biofilm, and comparison with various heparin solutions.

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Sauer, Karin; Steczko, Janusz; Ash, Stephen R

2009-05-01

Some antibiotic solutions increase bacterial resistance and may cause toxic side effects. Heparin, frequently used as an anticoagulant in catheter lock solutions, may cause bleeding and stimulate biofilm formation. The aim of this study was to investigate the effect of a new antibacterial/antithrombotic solution, citrate/Methylene Blue/parabens (C/MB/P), versus various heparin solutions on the viability and the structure of preformed mature biofilms of Staphylococcus aureus bacteria. The degree of eradication of both planktonic and sessile microorganisms was evaluated. The changes in the structure of biofilms after exposure to C/MB/P and several concentrations of heparin were analysed by means of confocal laser scanning microscopy. COMSTAT image analysis was utilized to compare biofilm biomass, average and maximum height, surface coverage and roughness coefficient. Viability studies were performed on both biofilms and supernatant solutions. C/MB/P, in contrast to heparin solutions, significantly reduced biofilm biomass and thickness and reduced viability by 5 log when compared with saline treatment. No viable planktonic bacteria were detected and the few remaining biofilm cells appeared to be lysed. In contrast, most heparin solutions only reduced viability up to 1.0 log and failed to eradicate planktonic bacteria. C/MB/P has a rapid bactericidal effect on the preformed, mature biofilm of S. aureus. The structural changes of biofilms treated with C/MB/P, together with the observed log reduction of viable biofilm cells, confirmed the high potential of this solution to eliminate sessile bacteria. Furthermore, the tested solution entirely eliminated planktonic bacteria detached from the biofilm.

Coral-Associated Bacteria as a Promising Antibiofilm Agent against Methicillin-Resistant and -Susceptible Staphylococcus aureus Biofilms

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Shanmugaraj Gowrishankar

2012-01-01

Full Text Available The current study deals with the evaluation of two coral-associated bacterial (CAB extracts to inhibit the biofilm synthesis in vitro as well as the virulence production like hemolysin and exopolysaccharide (EPS, and also to assess their ability to modify the adhesion properties, that is cell surface hydrophobicity (CSH of methicillin-resistant (MRSA and -susceptible Staphylococcus aureus (MSSA. Out of nine CAB screened, the ethyl acetate extract of CAB-E2 (Bacillus firmus and CAB-E4 (Vibrio parahemolyticus have shown excellent antibiofilm activity against S. aureus. CAB-E2 reduced the production of EPS (57–79% and hemolysin (43–70%, which ultimately resulted in the significant inhibition of biofilms (80–87% formed by both MRSA and MSSA. Similarly, CAB-E4 was also found to decrease the production of EPS (43–57%, hemolysin (43–57% and biofilms (80–85% of test pathogens. CLSM analysis also proved the antibiofilm efficacy of CAB extracts. Furthermore, the CAB extracts strongly decreased the CSH of S. aureus. Additionally, FT-IR analysis of S. aureus treated with CAB extracts evidenced the reduction in cellular components compared to their respective controls. Thus, the present study reports for the first time, B. firmus—a coral-associated bacterium, as a promising source of antibiofilm agent against the recalcitrant biofilms formed by multidrug resistant S. aureus.

Dynamics of biofilm formation and the interaction between Candida albicans and methicillin-susceptible (MSSA) and -resistant Staphylococcus aureus (MRSA).

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Zago, Chaiene Evelin; Silva, Sónia; Sanitá, Paula Volpato; Barbugli, Paula Aboud; Dias, Carla Maria Improta; Lordello, Virgínia Barreto; Vergani, Carlos Eduardo

2015-01-01

Polymicrobial biofilms are an understudied and a clinically relevant problem. This study evaluates the interaction between C. albicans, and methicillin- susceptible (MSSA) and resistant (MRSA) S. aureus growing in single- and dual-species biofilms. Single and dual species adhesion (90 min) and biofilms (12, 24, and 48 h) were evaluated by complementary methods: counting colony-forming units (CFU mL-1), XTT-reduction, and crystal violet staining (CV). The secretion of hydrolytic enzymes by the 48 h biofilms was also evaluated using fluorimetric kits. Scanning electron microscopy (SEM) was used to assess biofilm structure. The results from quantification assays were compared using two-way ANOVAs with Tukey post-hoc tests, while data from enzymatic activities were analyzed by one-way Welch-ANOVA followed by Games-Howell post hoc test (α = 0.05). C. albicans, MSSA and MRSA were able to adhere and to form biofilm in both single or mixed cultures. In general, all microorganisms in both growth conditions showed a gradual increase in the number of cells and metabolic activity over time, reaching peak values between 12 h and 48 h (ρ<0.05). C. albicans single- and dual-biofilms had significantly higher total biomass values (ρ<0.05) than single biofilms of bacteria. Except for single MRSA biofilms, all microorganisms in both growth conditions secreted proteinase and phospholipase-C. SEM images revealed extensive adherence of bacteria to hyphal elements of C. albicans. C. albicans, MSSA, and MRSA can co-exist in biofilms without antagonism and in an apparent synergistic effect, with bacteria cells preferentially associated to C. albicans hyphal forms.

Effect of essential oils of Syzygium aromaticum and Cinnamomum zeylanicum and their major components on biofilm production in Staphylococcus aureus strains isolated from milk of cows with mastitis.

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Budri, P E; Silva, N C C; Bonsaglia, E C R; Fernandes Júnior, A; Araújo Júnior, J P; Doyama, J T; Gonçalves, J L; Santos, M V; Fitzgerald-Hughes, D; Rall, V L M

2015-09-01

Bovine mastitis is an inflammation of the mammary glands of cows and causes significant economic losses in dairy cattle. Staphylococcus aureus is one of the microorganisms most commonly isolated. Novel agents are required in agricultural industries to prevent the development of mastitis. The production of biofilm by Staph. aureus facilitates the adhesion of bacteria to solid surfaces and contributes to the transmission and maintenance of these bacteria. The effect of the essential oils of Syzygium aromaticum (clove; EOSA) and Cinnamomum zeylanicum (cinnamon; EOCZ) and their major components, eugenol and cinnamaldehyde, on Staph. aureus biofilm formation on different surfaces was investigated. The results showed a significant inhibition of biofilm production by EOSA on polystyrene and stainless steel surfaces (69.4 and 63.6%, respectively). However, its major component, eugenol, was less effective on polystyrene and stainless steel (52.8 and 19.6%, respectively). Both EOCZ and its major component, cinnamaldehyde, significantly reduced biofilm formation on polystyrene (74.7 and 69.6%, respectively) and on stainless steel surfaces (45.3 and 44.9%, respectively). These findings suggest that EOSA, EOCZ, and cinnamaldehyde may be considered for applications such as sanitization in the food industry. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Bacteriophage Therapy for Staphylococcus aureus Biofilm-Infected Wounds: A New Approach to Chronic Wound Care

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2013-02-01

lidocaine and 1:100,000 epineph- rine at the planned wound sites. Six full-thickness dermal wounds, 6 mm in diameter, were created on the ventral ear...action were ineffective against S. aureus biofilm, as was seen with P. aeruginosa biofilm.22 Given the durability of biofilm in the face of a harsh

Biofilms of a Bacillus subtilis hospital isolate protect Staphylococcus aureus from biocide action.

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Arnaud Bridier

Full Text Available The development of a biofilm constitutes a survival strategy by providing bacteria a protective environment safe from stresses such as microbicide action and can thus lead to important health-care problems. In this study, biofilm resistance of a Bacillus subtilis strain (called hereafter ND(medical recently isolated from endoscope washer-disinfectors to peracetic acid was investigated and its ability to protect the pathogen Staphylococcus aureus in mixed biofilms was evaluated. Biocide action within Bacillus subtilis biofilms was visualised in real time using a non-invasive 4D confocal imaging method. The resistance of single species and mixed biofilms to peracetic acid was quantified using standard plate counting methods and their architecture was explored using confocal imaging and electronic microscopy. The results showed that the ND(medical strain demonstrates the ability to make very large amount of biofilm together with hyper-resistance to the concentration of PAA used in many formulations (3500 ppm. Evidences strongly suggest that the enhanced resistance of the ND(medical strain was related to the specific three-dimensional structure of the biofilm and the large amount of the extracellular matrix produced which can hinder the penetration of peracetic acid. When grown in mixed biofilm with Staphylococcus aureus, the ND(medical strain demonstrated the ability to protect the pathogen from PAA action, thus enabling its persistence in the environment. This work points out the ability of bacteria to adapt to an extremely hostile environment, and the necessity of considering multi-organism ecosystems instead of single species model to decipher the mechanisms of biofilm resistance to antimicrobials agents.

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

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Aslantaş, Özkan; Demir, Cemil

2016-11-01

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

In vitro production of biofilm in a flow cell system in a strain of Pseudomonas aeruginosa and Staphylococcus aureus and determination of efficiency of ciprofloxacin against them

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Soham Gupta

2011-01-01

Full Text Available Background: Microorganisms develop biofilm on various medical devices. The process is particularly relevant in public health since biofilm associated organisms are much more resistant to antibiotics and have a potential to cause infections in patients with indwelling medical devices. Materials and Methods: To determine the efficiency of an antibiotic against the biofilm it is inappropriate to use traditional technique of determining Minimum Inhibitory Concentration (MIC on the free floating laboratory phenotype. Thus we have induced formation of biofilm in two strains (Pseudomonas aeruginosa and Staphylococcus aureus, which showed heavy growth of biofilm in screening by Tube method in a flow cell system and determined their antibiotic susceptibility against ciprofloxacin by agar dilution method in the range (0.25 mg/ml to 8 mg/ml. The MIC value of ciprofloxacin for the biofilm produced organism was compared with its free form and a standard strain as control on the same plates. Observations: Both the biofilm produced strains showed a higher resistance (MIC > 8 mg/ml than its free form, which were 2 μg/ml for Pseudomonas aeruginosa and 4 mg/ml for Staphylococcus aureus. Thus biofilm can pose a threat in the patient treatment.

Effect of biosurfactants on Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a BioFlux channel.

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Diaz De Rienzo, M A; Stevenson, P S; Marchant, R; Banat, I M

2016-07-01

Recent studies have indicated that biosurfactants play a role both in maintaining channels between multicellular structures in biofilms and in dispersal of cells from biofilms. A combination of caprylic acid (0.01 % v/v) together with rhamnolipids (0.04 % v/v) was applied to biofilms of Pseudomonas aeruginosa ATCC 15442, Staphylococcus aureus ATCC 9144 and a mixed culture under BioFlux flowthrough conditions and caused disruption of the biofilms. The biofilms were also treated with a combination of rhamnolipids (0.04 % v/v) and sophorolipids (0.01 %). Control treatments with PBS 1× had no apparent effect on biofilm disruption. The Gram-positive bacterium (S. aureus ATCC 9144) was more sensitive than P. aeruginosa ATCC 15442 in terms of disruption and viability as shown by Live/Dead staining. Disruption of biofilms of P. aeruginosa ATCC 15442 was minimal. Oxygen consumption by biofilms, after different treatments with biosurfactants, confirms that sophorolipid on its own is unable to kill/inhibit cells of P. aeruginosa ATCC 15442, and even when used in combination with rhamnolipids, under static conditions, no decrease in the cell viability was observed. Cells in biofilms exposed to mono-rhamnolipids (0.04 % v/v) showed behaviour typical of exposure to bacteriostatic compounds, but when exposed to di-rhamnolipids (0.04 % v/v), they displayed a pattern characteristic of bactericidal compounds.

Does Extracellular DNA Production Vary in Staphylococcal Biofilms Isolated From Infected Implants versus Controls?

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Zatorska, Beata; Groger, Marion; Moser, Doris; Diab-Elschahawi, Magda; Lusignani, Luigi Segagni; Presterl, Elisabeth

2017-08-01

Prosthetic implant infections caused by Staphylococcus aureus and epidermidis are major challenges for early diagnosis and treatment owing to biofilm formation on the implant surface. Extracellular DNA (eDNA) is actively excreted from bacterial cells in biofilms, contributing to biofilm stability, and may offer promise in the detection or treatment of such infections. (1) Does DNA structure change during biofilm formation? (2) Are there time-dependent differences in eDNA production during biofilm formation? (3) Is there differential eDNA production between clinical and control Staphylococcal isolates? (4) Is eDNA production correlated to biofilm thickness? We investigated eDNA presence during biofilm formation in 60 clinical and 30 control isolates of S aureus and S epidermidis. The clinical isolates were isolated from patients with infections of orthopaedic prostheses and implants: 30 from infected hip prostheses and 30 from infected knee prostheses. The control isolates were taken from healthy volunteers who had not been exposed to antibiotics and a hospital environment during the previous 3 and 12 months, respectively. Control S epidermidis was isolated from the skin of the antecubital fossa, and control S aureus was isolated from the nares. For the biofilm experiments the following methods were used to detect eDNA: (1) fluorescent staining with 4',6-diamidino-2-phenylindole (DAPI), (2) eDNA extraction using a commercial kit, and (3) confocal laser scanning microscopy for 24-hour biofilm observation using propidium iodide and concanavalin-A staining; TOTO ® -1 and SYTO ® 60 staining were used for observation and quantification of eDNA after 6 and 24 hours of biofilm formation. Additionally antibiotic resistance was described. eDNA production as observed by confocal laser scanning microscopy was greater in clinical isolates than controls (clinical isolates mean ± SD: 1.84% ± 1.31%; control mean ± SD: 1.17% ± 1.37%; p biofilm formation. After 24 hours, the

Nisin and lysostaphin activity against preformed biofilm of Staphylococcus aureus involved in bovine mastitis.

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Ceotto-Vigoder, H; Marques, S L S; Santos, I N S; Alves, M D B; Barrias, E S; Potter, A; Alviano, D S; Bastos, M C F

2016-07-01

The biofilm produced by Staphylococcus aureus isolates involved in clinical or subclinical bovine mastitis and the activity of nisin and lysostaphin against the preformed biofilm produced by these strains were investigated. Eighteen strains were tested and all produced biofilm. Eight strains with distinct biofilm composition were selected for the antimicrobial activity assays. The minimal inhibitory concentration of each bacteriocin was determined against the planktonic cells and ranged from 15·6 to 500 μg ml(-1) for nisin, and from 3·9 to 50 μg ml(-1) , for lysostaphin. Lysostaphin treatment (0·4 μg ml(-1) ) for 4 h caused a strong Staph. aureus 4181 biofilm detachment and death of the majority of the sessile cells, while nisin treatment (100 μg ml(-1) ) for the same time caused only a great reduction in cell viability. Additionally, combination of both bacteriocins for 4 h resulted in significant death of the sessile cells but no biofilm detachment. The treatment with lysostaphin alone or in combination with nisin was effective in killing most biofilm sessile cells. The action of lysostaphin, either alone or in combination with nisin, against established staphylococcal biofilm may represent an alternative to bovine mastitis control. However, the duration of the treatment should be considered for its application so that the best effectiveness can be achieved. © 2016 The Society for Applied Microbiology.

Staphylococcus aureus Clinical Isolates: Antibiotic Susceptibility, Molecular Characteristics, and Ability to Form Biofilm

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N. Indrawattana

2013-01-01

Full Text Available Periodic monitoring of Staphylococcus aureus characteristics in a locality is imperative as their drug-resistant variants cause treatment problem. In this study, antibiograms, prevalence of toxin genes (sea-see, seg-ser, seu, tsst-1, eta, etb, and etd, PFGE types, accessory gene regulator (agr groups, and ability to form biofilm of 92 S. aureus Thailand clinical isolates were investigated. They were classified into 10 drug groups: groups 1–7 (56 isolates were methicillin resistant (MRSA and 8–10 (36 isolates were methicillin sensitive (MSSA. One isolate did not have any toxin gene, 4 isolates carried one toxin gene (seq, and 87 isolates had two or more toxin genes. No isolate had see, etb, or tsst-1; six isolates had eta or etd. Combined seg-sei-sem-sen-seo of the highly prevalent egc locus was 26.1%. The seb, sec, sel, seu, and eta associated significantly with MSSA; sek was more in MRSA. The sek-seq association was 52.17% while combined sed-sej was not found. Twenty-three PFGE types were revealed, no association of toxin genes with PFGE types. All four agr groups were present; agr group 1 was predominant (58.70% but agr group 2 strains carried more toxin genes and were more frequent toxin producers. Biofilm formation was found in 72.83% of the isolates but there was no association with antibiograms. This study provides insight information on molecular and phenotypic markers of Thailand S. aureus clinical isolates which should be useful for future active surveillance that aimed to control a spread of existing antimicrobial resistant bacteria and early recognition of a newly emerged variant.

Effects of Miramistin and Phosprenil on Microbial Biofilms.

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Danilova, T A; Danilina, G A; Adzhieva, A A; Minko, A G; Nikolaeva, T N; Zhukhovitskii, V G; Pronin, A V

2017-08-01

Effects of Miramistin and Phosprenil on biofilms of S. pyogenes, S. aureus, E. coli, L. acidophilus, and L. plantarum were studied. Significant differences in the effects of these substances on mature biofilms of microorganisms and the process of their formation were observed. Miramistin had significant inhibiting effects on the forming of biofilms and on the formed biofilms of all studied microorganisms. Treatment with Miramistin inhibited biofilm formation by 2-3 times compared to the control. This effect was found already after using of Miramistin in the low doses (3.12 μg/ml). Inhibition of the growth of a formed biofilm was observed only after treatment with Miramistin in the high doses (25-50 μg/ml). Phosprenil in the high doses (15-30 mg/ml) inhibited the forming of biofilms, especially the biofilms of S. pyogenes and L. plantarum (by 3-4.5 times). Treatment of formed biofilms with the agent in doses of 6.0 and 0.6 mg/ml was associated with pronounced stimulation of its growth in S. pyogenes, S. aureus, and L. acidophilus.

Bisphosphonates enhance bacterial adhesion and biofilm formation on bone hydroxyapatite.

Science.gov (United States)

Kos, Marcin; Junka, Adam; Smutnicka, Danuta; Szymczyk, Patrycja; Gluza, Karolina; Bartoszewicz, Marzenna

2015-07-01

Because of the suspicion that bisphosphonates enhance bacterial colonization, this study evaluated adhesion and biofilm formation by Streptococcus mutans 25175, Staphylococcus aureus 6538, and Pseudomonas aeruginosa 14454 reference strains on hydroxyapatite coated with clodronate, pamidronate, or zoledronate. Bacterial strains were cultured on bisphosphonate-coated and noncoated hydroxyapatite discs. After incubation, nonadhered bacteria were removed by centrifugation. Biofilm formation was confirmed by scanning electron microscopy. Bacterial colonization was estimated using quantitative cultures compared by means with Kruskal-Wallis and post-hoc Student-Newman-Keuls tests. Modeling of the interactions between bisphosphonates and hydroxyapatite was performed using the Density Functional Theory method. Bacterial colonization of the hydroxyapatite discs was significantly higher for all tested strains in the presence of bisphosphonates vs. Adherence in the presence of pamidronate was higher than with other bisphosphonates. Density Functional Theory analysis showed that the protonated amine group of pamidronate, which are not present in clodronate or zoledronate, forms two additional hydrogen bonds with hydroxyapatite. Moreover, the reactive cationic amino group of pamidronate may attract bacteria by direct electrostatic interaction. Increased bacterial adhesion and biofilm formation can promote osteomyelitis, cause failure of dental implants or bisphosphonate-coated joint prostheses, and complicate bone surgery in patients on bisphosphonates. Copyright © 2015 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

A novel in vitro model for hematogenous spreading of S. aureus device biofilms demonstrating clumping dispersal as an advantageous dissemination mechanism

DEFF Research Database (Denmark)

Grønnemose, Rasmus Birkholm; Lindhardt Sæderup (Madsen), Kirstine; Kolmos, Hans Jørn

2017-01-01

Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life-threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics in the proc......Staphylococcus aureus is able to disseminate from vascular device biofilms to the blood and organs, resulting in life-threatening infections such as endocarditis. The mechanisms behind spreading are largely unknown, especially how the bacterium escapes immune effectors and antibiotics...... the ability to adhere to and initiate colonization of endothelial cell layers under flow. In vivo experiments showed that the released biofilm material reached the heart similarly as ordinary broth-grown bacteria, but also that clumps to some extend were trapped in the lungs. The clumping dispersal of S....... aureus from in vivo-like vascular biofilms and their specific properties demonstrated here help explain the pathophysiology associated with S. aureus bloodstream infections....

Relevant Role of Fibronectin-Binding Proteins in Staphylococcus aureus Biofilm-Associated Foreign-Body Infections▿ †

Science.gov (United States)

Vergara-Irigaray, Marta; Valle, Jaione; Merino, Nekane; Latasa, Cristina; García, Begoña; Ruiz de los Mozos, Igor; Solano, Cristina; Toledo-Arana, Alejandro; Penadés, José R.; Lasa, Iñigo

2009-01-01

Staphylococcus aureus can establish chronic infections on implanted medical devices due to its capacity to form biofilms. Analysis of the factors that assemble cells into a biofilm has revealed the occurrence of strains that produce either a polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) exopolysaccharide- or a protein-dependent biofilm. Examination of the influence of matrix nature on the biofilm capacities of embedded bacteria has remained elusive, because a natural strain that readily converts between a polysaccharide- and a protein-based biofilm has not been studied. Here, we have investigated the clinical methicillin (meticillin)-resistant Staphylococcus aureus strain 132, which is able to alternate between a proteinaceous and an exopolysaccharidic biofilm matrix, depending on environmental conditions. Systematic disruption of each member of the LPXTG surface protein family identified fibronectin-binding proteins (FnBPs) as components of a proteinaceous biofilm formed in Trypticase soy broth-glucose, whereas a PIA/PNAG-dependent biofilm was produced under osmotic stress conditions. The induction of FnBP levels due to a spontaneous agr deficiency present in strain 132 and the activation of a LexA-dependent SOS response or FnBP overexpression from a multicopy plasmid enhanced biofilm development, suggesting a direct relationship between the FnBP levels and the strength of the multicellular phenotype. Scanning electron microscopy revealed that cells growing in the FnBP-mediated biofilm formed highly dense aggregates without any detectable extracellular matrix, whereas cells in a PIA/PNAG-dependent biofilm were embedded in an abundant extracellular material. Finally, studies of the contribution of each type of biofilm matrix to subcutaneous catheter colonization revealed that an FnBP mutant displayed a significantly lower capacity to develop biofilm on implanted catheters than the isogenic PIA/PNAG-deficient mutant. PMID:19581398

Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing.

Science.gov (United States)

Chino, T; Nukui, Y; Morishita, Y; Moriya, K

2017-01-01

The bactericidal effect of disinfectants against biofilms is essential to reduce potential endoscopy-related infections caused by contamination. Here, we investigated the bactericidal effect of a high-level disinfectant, peracetic acid (PAA), against Staphylococcus aureus and Pseudomonas aeruginosa biofilm models in vitro. S. aureus and P. aeruginosa biofilms were cultured at 35 °C for 7 days with catheter tubes. The following high-level disinfectants (HLDs) were tested: 0.3% PAA, 0.55% ortho-phthalaldehyde (OPA), and 2.0% alkaline-buffered glutaraldehyde (GA). Biofilms were exposed to these agents for 1-60 min and observed after 5 min and 30 min by transmission and scanning electron microscopy. A Student's t test was performed to compare the exposure time required for bactericidal effectiveness of the disinfectants. PAA and GA were active within 1 min and 5 min, respectively, against S. aureus and P. aeruginosa biofilms. OPA took longer than 10 min and 30 min to act against S. aureus and P. aeruginosa biofilms, respectively ( p  < 0.01). Treatment with PAA elicited changes in cell shape after 5 min and structural damage after 30 min. Amongst the HLDs investigated, PAA elicited the most rapid bactericidal effects against both biofilms. Additionally, treatment with PAA induced morphological alterations in the in vitro biofilm models, suggesting that PAA exerts fast-acting bactericidal effects against biofilms associated with endoscopy-related infections. These findings indicate that the exposure time for bactericidal effectiveness of HLDs for endoscope reprocessing in healthcare settings should be reconsidered.

Crenarchaeal biofilm formation under extreme conditions.

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Andrea Koerdt

Full Text Available BACKGROUND: Biofilm formation has been studied in much detail for a variety of bacterial species, as it plays a major role in the pathogenicity of bacteria. However, only limited information is available for the development of archaeal communities that are frequently found in many natural environments. METHODOLOGY: We have analyzed biofilm formation in three closely related hyperthermophilic crenarchaeotes: Sulfolobus acidocaldarius, S. solfataricus and S. tokodaii. We established a microtitre plate assay adapted to high temperatures to determine how pH and temperature influence biofilm formation in these organisms. Biofilm analysis by confocal laser scanning microscopy demonstrated that the three strains form very different communities ranging from simple carpet-like structures in S. solfataricus to high density tower-like structures in S. acidocaldarius in static systems. Lectin staining indicated that all three strains produced extracellular polysaccharides containing glucose, galactose, mannose and N-acetylglucosamine once biofilm formation was initiated. While flagella mutants had no phenotype in two days old static biofilms of S. solfataricus, a UV-induced pili deletion mutant showed decreased attachment of cells. CONCLUSION: The study gives first insights into formation and development of crenarchaeal biofilms in extreme environments.

Influence of biofilm-forming lactic acid bacteria against methicillin-resistant Staphylococcus aureus (MRSA S547

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Laavanya M. Kumar

2017-12-01

Full Text Available Objective: To investigate the antibacterial effect of selected lactic acid bacteria (LAB biofilms on the planktonic and biofilm population of methicillin-resistant Staphylococcus aureus (MRSA (S547. Methods: In this study, biofilm-forming LAB were isolated from tairu and kefir. Isolate Y1 and isolate KF were selected based on their prominent inhibition against test pathogens (using spot-on-agar method and agar-well-diffusion assay and efficient biofilm production (using tissue culture plate method. They were then identified as Lactobacillus casei (L. casei Y1 and Lactobacillus plantarum (L. plantarum KF, respectively using 16S rDNA gene sequencing. The influence of incubation time, temperature and aeration on the biofilm production of L. casei Y1 and L. plantarum KF was also investigated using tissue culture plate method. The inhibitory activity of both the selected LAB biofilms was evaluated against MRSA (Institute for Medical Research code: S547 using L. plantarum ATCC 8014 as the reference strain. Results: L. casei Y1 showed the highest reduction of MRSA biofilms, by 3.53 log at 48 h while L. plantarum KF records the highest reduction of 2.64 log at 36 h. In inhibiting planktonic population of MRSA (S547, both L. casei Y1 and L. plantarum KF biofilms recorded their maximum reduction of 4.13 log and 3.41 log at 24 h, respectively. Despite their inhibitory effects being time-dependent, both LAB biofilms exhibited good potential in controlling the biofilm and planktonic population of MRSA (S547. Conclusions: The results from this study could highlight the importance of analysing biofilms of LAB to enhance their antibacterial efficacy. Preferably, these protective biofilms of LAB could also be a better alternative to control the formation of biofilms by pathogens such as MRSA. Keywords: MRSA, Biofilms, Lactic acid bacteria, Antibacterial

Commensal coagulase-negative Staphylococcus from the udder of healthy cows inhibits biofilm formation of mastitis-related pathogens.

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Isaac, Paula; Bohl, Luciana Paola; Breser, María Laura; Orellano, María Soledad; Conesa, Agustín; Ferrero, Marcela Alejandra; Porporatto, Carina

2017-08-01

Bovine mastitis, considered the most important cause of economic losses in the dairy industry, is a major concern in veterinary medicine. Staphylococcus aureus and coagulase-negative staphylococci (CNS) are the main pathogens associated with intramammary infections, and bacterial biofilms are suspected to be responsible for the persistence of this disease. CNS from the udder are not necessarily associated with intramammary infections. In fact, some commensal CNS have been shown to have biological activities. This issue led us to screen exoproducts from commensal Staphylococcus chromogenes for anti-biofilm activity against different mastitis pathogens. The cell-free supernatant from S. chromogenes LN1 (LN1-CFS) was confirmed to display a non-biocidal inhibition of pathogenic biofilms. The supernatant was subjected to various treatments to estimate the nature of the biofilm-inhibiting compounds. The results showed that the bioactive compound >5KDa in mass is sensitive to thermal treatment and proteinase K digestion, suggesting its protein properties. LN1-CFS was able to significantly inhibit S. aureus and CNS biofilm formation in a dose-independent manner and without affecting the viability of bovine cells. These findings reveal a new activity of the udder microflora of healthy animals. Studies are underway to purify and identify the anti-biofilm biocompound and to evaluate its biological activity in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing

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T. Chino

2017-12-01

Full Text Available Abstract Background The bactericidal effect of disinfectants against biofilms is essential to reduce potential endoscopy-related infections caused by contamination. Here, we investigated the bactericidal effect of a high-level disinfectant, peracetic acid (PAA, against Staphylococcus aureus and Pseudomonas aeruginosa biofilm models in vitro. Methods S. aureus and P. aeruginosa biofilms were cultured at 35 °C for 7 days with catheter tubes. The following high-level disinfectants (HLDs were tested: 0.3% PAA, 0.55% ortho-phthalaldehyde (OPA, and 2.0% alkaline-buffered glutaraldehyde (GA. Biofilms were exposed to these agents for 1–60 min and observed after 5 min and 30 min by transmission and scanning electron microscopy. A Student’s t test was performed to compare the exposure time required for bactericidal effectiveness of the disinfectants. Results PAA and GA were active within 1 min and 5 min, respectively, against S. aureus and P. aeruginosa biofilms. OPA took longer than 10 min and 30 min to act against S. aureus and P. aeruginosa biofilms, respectively (p < 0.01. Treatment with PAA elicited changes in cell shape after 5 min and structural damage after 30 min. Conclusions Amongst the HLDs investigated, PAA elicited the most rapid bactericidal effects against both biofilms. Additionally, treatment with PAA induced morphological alterations in the in vitro biofilm models, suggesting that PAA exerts fast-acting bactericidal effects against biofilms associated with endoscopy-related infections. These findings indicate that the exposure time for bactericidal effectiveness of HLDs for endoscope reprocessing in healthcare settings should be reconsidered.

Silver-Palladium Surfaces Inhibit Biofilm Formation

DEFF Research Database (Denmark)

Chiang, Wen-Chi; Schroll, Casper; Hilbert, Lisbeth Rischel

2009-01-01

Undesired biofilm formation is a major concern in many areas. In the present study, we investigated biofilm-inhibiting properties of a silver-palladium surface that kills bacteria by generating microelectric fields and electrochemical redox processes. For evaluation of the biofilm inhibition...... efficacy and study of the biofilm inhibition mechanism, the silver-sensitive Escherichia coli J53 and the silver-resistant E. coli J53[pMG101] strains were used as model organisms, and batch and flow chamber setups were used as model systems. In the case of the silver-sensitive strain, the silver......-palladium surfaces killed the bacteria and prevented biofilm formation under conditions of low or high bacterial load. In the case of the silver-resistant strain, the silver-palladium surfaces killed surface-associated bacteria and prevented biofilm formation under conditions of low bacterial load, whereas under...

Effect of Carvacrol on Salmonella Saintpaul Biofilms on Stainless ...

African Journals Online (AJOL)

2025 ... carvacrol on S. saintpaul biofilms on stainless steel surface was evaluated on ... cultures S. saintpaul at 35 ºC were diluted 1:100 .... characteristics of biofilm formation that occur in .... aureus and Salmonella enterica serovar Typhmurium.

Antibiofilm Effect of Octenidine Hydrochloride on Staphylococcus aureus, MRSA and VRSA

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Mary Anne Roshni Amalaradjou

2014-05-01

Full Text Available Millions of indwelling devices are implanted in patients every year, and staphylococci (S. aureus, MRSA and vancomycin-resistant S. aureus (VRSA are responsible for a majority of infections associated with these devices, thereby leading to treatment failures. Once established, staphylococcal biofilms become resistant to antimicrobial treatment and host response, thereby serving as the etiological agent for recurrent infections. This study investigated the efficacy of octenidine hydrochloride (OH for inhibiting biofilm synthesis and inactivating fully-formed staphylococcal biofilm on different matrices in the presence and absence of serum protein. Polystyrene plates and stainless steel coupons inoculated with S. aureus, MRSA or VRSA were treated with OH (zero, 0.5, one, 2 mM at 37 °C for the prevention of biofilm formation. Additionally, the antibiofilm effect of OH (zero, 2.5, five, 10 mM on fully-formed staphylococcal biofilms on polystyrene plates, stainless steel coupons and urinary catheters was investigated. OH was effective in rapidly inactivating planktonic and biofilm cells of S. aureus, MRSA and VRSA on polystyrene plates, stainless steel coupons and urinary catheters in the presence and absence of serum proteins. The use of two and 10 mM OH completely inactivated S. aureus planktonic cells and biofilm (>6.0 log reduction on all matrices tested immediately upon exposure. Further, confocal imaging revealed the presence of dead cells and loss in biofilm architecture in the OH-treated samples when compared to intact live biofilm in the control. Results suggest that OH could be applied as an effective antimicrobial to control biofilms of S. aureus, MRSA and VRSA on appropriate hospital surfaces and indwelling devices.

Antibacterial activity of Thymoquinone, an active principle of Nigella sativa and its potency to prevent bacterial biofilm formation

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Bakhrouf Amina

2011-04-01

Full Text Available Abstract Background Thymoquinone is an active principle of Nigella sativa seed known as "Habbah Al-Sauda" in Arabic countries and "Sinouj" in Tunisia. Bacterial biofilms tend to exhibit significant tolerance to antimicrobials drugs during infections. Methods The antibacterial activity of Thymoquinone (TQ and its biofilm inhibition potencies were investigated on 11 human pathogenic bacteria. The growth and development of the biofilm were assessed using the crystal violet (CV and the 2, 3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT reduction assay. Results TQ exhibited a significant bactericidal activity against the majority of the tested bacteria (MICs values ranged from 8 to 32 μg/ml especially Gram positive cocci (Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis CIP 106510. Crystal violet assay demonstrated that the minimum biofilm inhibition concentration (BIC50 was reached with 22 and 60 μg/ml for Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis CIP 106510 respectively. In addition our data revealed that cells oxidative activity was influenced by TQ supplementation. In the same way, TQ prevented cell adhesion to glass slides surface. Conclusion The ability of TQ to prevent biofilm formation warrants further investigation to explore its use as bioactive substances with antibiofilm potential.

Single- and Multispecies Biofilms by Escherichia coli, Staphylococcus aureus, and Salmonella spp. Isolated from Raw Fish and a Fish Processing Unit

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Jesieli Braz Frozi

2017-09-01

Full Text Available ABSTRACT: The consumption of fish by the Brazilian population is increasing. However, fish and seafood are highly perishable and can be contaminated with several microorganisms. In addition, the possibility of biofilm formation is a greater cause for concern. In this study, biofilm formation was evaluated in single- and multispecies cultures at 25°C for incubation periods of 0, 4, 8, 24, and 48h in stainless steel coupons (size, 1.0×1.0cm immersed in tryptic soy broth. The characteristics of the formed biofilms after sanitizing by immersing the coupons in 200ppm sodium hypochlorite solution for 10min were also evaluated under the same experimental conditions but with some modifications. Biofilm structure was evaluated using scanning electron microscopy. Analysis of single-species biofilms indicated that all bacterial strains formed biofilms at different intervals without any statistically significant difference. However, comparison of the growth of single- and multispecies cultures indicated a significantly higher biofilm formation by the pure cultures. In multispecies biofilms, compared with the other microorganisms, growth of Salmonella spp. was significantly lower for all tested incubation periods; whereas, of Staphylococcus aureus was significantly higher than that of E. coli until 8h of incubation; the differences in growth were not significantly different after this incubation period. Sanitizing with sodium hypochlorite was effective because no cell growth was observed in the coupons that were treated with 200ppm sodium hypochlorite for 10min. This study demonstrated the ability of isolated microorganisms to form biofilms, reinforcing the need for food handling establishments to adopt good manufacturing practices, developing adequate protocols for cleaning and disinfecting surfaces and equipment used in food production, maintaining and replacing equipment when necessary.

Physics of biofilms: the initial stages of biofilm formation and dynamics

International Nuclear Information System (INIS)

Lambert, Guillaume; Bergman, Andrew; Zhang, Qiucen; Bortz, David; Austin, Robert

2014-01-01

One of the physiological responses of bacteria to external stress is to assemble into a biofilm. The formation of a biofilm greatly increases a bacterial population's resistance to a hostile environment by shielding cells, for example, from antibiotics. In this paper, we describe the conditions necessary for the emergence of biofilms in natural environments and relate them to the emergence of biofilm formation inside microfluidic devices. We show that competing species of Escherichia coli bacteria form biofilms to spatially segregate themselves in response to starvation stress, and use in situ methods to characterize the physical properties of the biofilms. Finally, we develop a microfluidic platform to study the inter-species interactions and show how biofilm-mediated genetic interactions can improve a species’ resistance to external stress. (paper)

Chemoinformatics-assisted development of new anti-biofilm compounds

DEFF Research Database (Denmark)

Dürig, Anna; Kouskoumvekaki, Irene; Vejborg, Rebecca Munk

2010-01-01

Bacterial biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Here, we use a novel cross-disciplinary approach combining microbiology and chemoinformatics...... to identify new and efficient anti-biofilm drugs. We found that ellagic acid (present in green tea) significantly inhibited biofilm formation of Streptococcus dysgalactiae. Based on ellagic acid, we performed in silico screening of the Chinese Natural Product Database to predict a 2nd-generation list...... of compounds with similar characteristics. One of these, esculetin, proved to be more efficient in preventing biofilm formation by Staphylococcus aureus. From esculetin a 3rd-generation list of compounds was predicted. One of them, fisetin, was even better to abolish biofilm formation than the two parent...

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

Science.gov (United States)

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

2015-06-01

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

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

Science.gov (United States)

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

2015-01-01

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

Controlled Release of Plectasin NZ2114 from a Hybrid Silicone-Hydrogel Material for Inhibition of Staphylococcus aureus Biofilm

DEFF Research Database (Denmark)

Klein, Kasper; Grønnemose, Rasmus Birkholm; Alm, Martin

2017-01-01

this system with plectasin derivate NZ2114 in an attempt to design an S. aureus biofilm-resistant catheter. The material demonstrated promising antibiofilm properties, including properties against methicillin-resistant S. aureus, thus suggesting a novel application of this antimicrobial peptide....

Antibiotic-Loaded Synthetic Calcium Sulfate Beads for Prevention of Bacterial Colonization and Biofilm Formation in Periprosthetic Infections

Science.gov (United States)

Howlin, R. P.; Brayford, M. J.; Webb, J. S.; Cooper, J. J.; Aiken, S. S.

2014-01-01

Periprosthetic infection (PI) causes significant morbidity and mortality after fixation and joint arthroplasty and has been extensively linked to the formation of bacterial biofilms. Poly(methyl methacrylate) (PMMA), as a cement or as beads, is commonly used for antibiotic release to the site of infection but displays variable elution kinetics and also represents a potential nidus for infection, therefore requiring surgical removal once antibiotics have eluted. Absorbable cements have shown improved elution of a wider range of antibiotics and, crucially, complete biodegradation, but limited data exist as to their antimicrobial and antibiofilm efficacy. Synthetic calcium sulfate beads loaded with tobramycin, vancomycin, or vancomycin-tobramycin dual treatment (in a 1:0.24 [wt/wt] ratio) were assessed for their abilities to eradicate planktonic methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus epidermidis relative to that of PMMA beads. The ability of the calcium sulfate beads to prevent biofilm formation over multiple days and to eradicate preformed biofilms was studied using a combination of viable cell counts, confocal microscopy, and scanning electron microscopy of the bead surface. Biofilm bacteria displayed a greater tolerance to the antibiotics than their planktonic counterparts. Antibiotic-loaded beads were able to kill planktonic cultures of 106 CFU/ml, prevent bacterial colonization, and significantly reduce biofilm formation over multiple days. However, established biofilms were harder to eradicate. These data further demonstrate the difficulty in clearing established biofilms; therefore, early preventive measures are key to reducing the risk of PI. Synthetic calcium sulfate loaded with antibiotics has the potential to reduce or eliminate biofilm formation on adjacent periprosthetic tissue and prosthesis material and, thus, to reduce the rates of periprosthetic infection. PMID:25313221

Pattern formation in Pseudomonas aeruginosa biofilms

DEFF Research Database (Denmark)

Parsek, Matthew R.; Tolker-Nielsen, Tim

2008-01-01

Bacteria are capable of forming elaborate multicellular communities called biofilms. Pattern formation in biofilms depends on cell proliferation and cellular migration in response to the available nutrients and other external cues, as well as on self-generated intercellular signal molecules...... and the production of an extracellular matrix that serves as a structural 'scaffolding' for the biofilm cells. Pattern formation in biofilms allows cells to position themselves favorably within nutrient gradients and enables buildup and maintenance of physiologically distinct subpopulations, which facilitates...... survival of one or more subpopulations upon environmental insult, and therefore plays an important role in the innate tolerance displayed by biofilms toward adverse conditions....

Effects of Tween 80 on growth and biofilm formation in laboratory media

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Christina Krogsård Nielsen

2016-11-01

Full Text Available Tween 80 is a widely used nonionic emulsifier that is added to cosmetics, pharmaceuticals and foods. Because of its widespread use we need to understand how it affects bacteria on our skin, in our gut, and in food products. The aim of this study is to investigate how Tween 80 affects the growth and antimicrobial susceptibility of Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens, which are common causes of spoilage and foodborne illnesses. Addition of 0.1% Tween 80 to laboratory growth media increased the growth rate of planktonic S. aureus batch cultures, and it also increased the total biomass when S. aureus was grown as biofilms. In contrast, Tween 80 had no effect on batch cultures of L. monocytogenes, it slowed the growth rate of P. fluorescens, and it led to formation of less biofilm by both L. monocytogenes and P. fluorescens. Furthermore, Tween 80 lowered the antibacterial efficacy of two hydrophobic antimicrobials: rifampicin and the essential oil isoeugenol. Our findings underline the importance of documenting indirect effects of emulsifiers when studying the efficacy of hydrophobic antimicrobials that are dispersed in solution by emulsification, or when antimicrobials are applied in food matrixes that include emulsifiers. Furthermore, the species-specific effects on microbial growth suggests that Tween 80 in cosmetics and food products could affect the composition of skin and gut microbiota, and the effect of emulsifiers on the human microbiome should therefore be explored to uncover potential health effects.

Inhibited Bacterial Adhesion and Biofilm Formation on Quaternized Chitosan-Loaded Titania Nanotubes with Various Diameters

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Wen-tao Lin

2016-03-01

Full Text Available Titania nanotube-based local drug delivery is an attractive strategy for combating implant-associated infection. In our previous study, we demonstrated that the gentamicin-loaded nanotubes could dramatically inhibit bacterial adhesion and biofilm formation on implant surfaces. Considering the overuse of antibiotics may lead to the evolution of antibiotic-resistant bacteria, we synthesized a new quaternized chitosan derivative (hydroxypropyltrimethyl ammonium chloride chitosan, HACC with a 27% degree of substitution (DS; referred to as 27% HACC that had a strong antibacterial activity and simultaneously good biocompatibility with osteogenic cells. Titania nanotubes with various diameters (80, 120, 160, and 200 nm and 200 nm length were loaded with 2 mg of HACC using a lyophilization method and vacuum drying. Two standard strain, methicillin-resistant Staphylococcus aureus (American Type Culture Collection 43300 and Staphylococcus epidermidis (American Type Culture Collection 35984, and two clinical isolates, S. aureus 376 and S. epidermidis 389, were selected to investigate the bacterial adhesion at 6 h and biofilm formation at 24, 48, and 72 h on the HACC-loaded nanotubes (NT-H using the spread plate method, confocal laser scanning microscopy (CLSM, and scanning electron microscopy (SEM. Smooth titanium (Smooth Ti was also investigated and compared. We found that NT-H could significantly inhibit bacterial adhesion and biofilm formation on its surface compared with Smooth Ti, and the NT-H with 160 nm and 200 nm diameters had stronger antibacterial activity because of the extended HACC release time of NT-H with larger diameters. Therefore, NT-H can significantly improve the antibacterial ability of orthopedic implants and provide a promising strategy to prevent implant-associated infections.

Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

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Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

2016-05-03

β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.

Integrating fluid dynamic and biologic effects on staphylococci bacteria biofilms

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Sherman, Erica; Endres, Jennifer; Bayles, Kenneth; Wei, Timothy

2017-11-01

Staphylococcus aureus bacteria are able to form biofilms and distinctive tower structures that facilitate their ability to tolerate treatment and to spread within the human body. The formation of towers, which break off, get carried downstream and serve to initiate biofilms in other parts of the body are of particular interest here. In previous work on biofilm growth and evolution in steady, laminar microchannel flows, it has been established that tower formation occurs around a very limited range of applied shear stresses centered on 0.6 dynes/cm2. Quantifying cell density characteristics as a function of time during biofilm formation reveals indicators of tower development hours before towers actually form and become visible. The next step in this research is to explore biological factors that might explain why this specific shear is so important. Additional studies with mutants, e.g. ica-A, that have been tied to tower formation have been conducted. The shear dependence of these mutants and their correlation to the behavior of wild type S. aureus is examined.

IMPACTS OF BIOFILM FORMATION ON CELLULOSE FERMENTATION

Energy Technology Data Exchange (ETDEWEB)

Leschine, Susan

2009-10-31

This project addressed four major areas of investigation: i) characterization of formation of Cellulomonas uda biofilms on cellulose; ii) characterization of Clostridium phytofermentans biofilm development; colonization of cellulose and its regulation; iii) characterization of Thermobifida fusca biofilm development; colonization of cellulose and its regulation; and iii) description of the architecture of mature C. uda, C. phytofermentans, and T. fusca biofilms. This research is aimed at advancing understanding of biofilm formation and other complex processes involved in the degradation of the abundant cellulosic biomass, and the biology of the microbes involved. Information obtained from these studies is invaluable in the development of practical applications, such as the single-step bioconversion of cellulose-containing residues to fuels and other bioproducts. Our results have clearly shown that cellulose-decomposing microbes rapidly colonize cellulose and form complex structures typical of biofilms. Furthermore, our observations suggest that, as cells multiply on nutritive surfaces during biofilms formation, dramatic cell morphological changes occur. We speculated that morphological changes, which involve a transition from rod-shaped cells to more rounded forms, might be more apparent in a filamentous microbe. In order to test this hypothesis, we included in our research a study of biofilm formation by T. fusca, a thermophilic cellulolytic actinomycete commonly found in compost. The cellulase system of T. fusca has been extensively detailed through the work of David Wilson and colleagues at Cornell, and also, genome sequence of a T. fusca strain has been determine by the DOE Joint Genome Institute. Thus, T. fusca is an excellent subject for studies of biofilm development and its potential impacts on cellulose degradation. We also completed a study of the chitinase system of C. uda. This work provided essential background information for understanding how C. uda

Activity of essential oil-based microemulsions against Staphylococcus aureus biofilms developed on stainless steel surface in different culture media and growth conditions.

Science.gov (United States)

Campana, Raffaella; Casettari, Luca; Fagioli, Laura; Cespi, Marco; Bonacucina, Giulia; Baffone, Wally

2017-01-16

Food safety is a fundamental concern for both consumers and the food industry, especially as the numbers of reported cases of food-associated infections continue to increase. Industrial surfaces can provide a suitable substrate for the development and persistence of bacterial organized in biofilms that represent a potential source of food contamination. The negative consumer perception of chemical disinfectants has shifted the attention to natural substances, such as plant extracts. The aim of this study was to investigate the possibility of using the essential oils (EOs) in the fight against S. aureus biofilms. First, the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Minimum Biofilm Inhibitory Concentration (MBIC), Minimum Biofilm Eradication Concentration (MBEC) of eleven EOs against S. aureus were determined. Cinnamomum cassia and Salvia officinalis EOs showed the greatest antibacterial properties with 1.25% MIC and MBC, 1.25% MBIC and 2.5% MBEC respectively. Gas Chromatography/Mass Spectrometry analysis revealed cinnamaldehyde (82.66%) and methoxy cinnamaldehyde (10.12%) as the most abundant substances of C. cassia, while cis-thujone (23.90%), camphor (19.22%) and 1.8-cineole (10.62%) of S. officinalis. Three different microemulsions, formulated with C. cassia, S. officinalis or both, were finally tested against S. aureus biofilms in different culture media and growth conditions, causing a >3 logarithmic reductions in S. aureus 24h-old biofilms and desiccated biofilms, and up to 68% of biofilm removal after 90min of exposure. The obtained data suggest the potential use of EOs, alone or in combination, for the formulation of sanitizers as alternative or in support in the disinfection of contaminated surfaces. Copyright © 2016 Elsevier B.V. All rights reserved.

Biofilm formation in attached microalgal reactors.

Science.gov (United States)

Shen, Y; Zhu, W; Chen, C; Nie, Y; Lin, X

2016-08-01

The objective of this study was to investigate the fundamental question of biofilm formation. First, a drum biofilm reactor was introduced. The drums were coated with three porous substrates (cotton rope, canvas, and spandex), respectively. The relationships among the substrate, extracellular polymeric substances (EPS), and adhesion ratio were analyzed. Second, a plate biofilm reactor (PBR) was applied by replacing the drum with multiple parallel vertical plates to increase the surface area. The plates were coated with porous substrates on each side, and the nutrients were delivered to the cells by diffusion. The influence of nitrogen source and concentration on compositions of EPS and biofilm formation was analyzed using PBR under sunlight. The results indicated that both substrate and nitrogen were critical on the EPS compositions and biofilm formation. Under the optimal condition (glycine with concentration of 1 g l(-1) and substrate of canvas), the maximum biofilm productivity of 54.46 g m(-2) d(-1) with adhesion ratio of 84.4 % was achieved.

The Effect of Negative Pressure Wound Therapy With Antiseptic Instillation on Biofilm Formation in a Porcine Model of Infected Spinal Instrumentation.

Science.gov (United States)

Singh, Devinder P; Gowda, Arvind U; Chopra, Karan; Tholen, Michael; Chang, Sarah; Mavrophilipos, Vasilios; Semsarzadeh, Nina; Rasko, Yvonne; Holton Iii, Luther

2017-06-01

This study evaluates the effect of negative pressure wound therapy with antiseptic instillation (NPWTi) in the clearance of infection and biofilm formation in an in vivo model of infected spinal implants compared to traditional treatment modalities. Five pigs underwent titanium rod implantation of their spinous processes followed by injection of 1 x 106 CFUs/100μL of methicillin-resistant Staphylococcus aureus through the fascia at each site. At 1 week postoperatively, an experimental arm of 3 pigs received NPWTi, and a control arm of 2 pigs received wet-to-dry dressings. The persistence of local infection in the experimental group was compared to the control group using tissue cultures. Biofilm development on spinal implants was evaluated using scanning electron microscopy. Mean bacterial count showed a statistical difference between the experimental and the control groups (P < .05). Scanning electron microscopy revealed the presence of uniform biofilm formation across the surface of control group instrumentation, whereas the experimental group showed interrupted areas between biofilm formations. The authors concluded that NPWTi is associated with decreased bacterial load and biofilm formation compared to wet-to-dry dressings in an in vivo porcine model of infected spinal instrumentation.

Bacteriophage-Derived Peptidase CHAPK Eliminates and Prevents Staphylococcal Biofilms

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Mark Fenton

2013-01-01

Full Text Available New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.

Biofilm Removal and Antimicrobial Activities of Agar Hydrogel Containing Colloid Nano-Silver against Staphylococcus aureus and Salmonella typhimurium

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Leyla Sadat Bouryabaf

2017-10-01

Full Text Available Background:    Antibacterial and biofilm removal effects of agar hydrogel incorporating silver nanoparticles (SNP at various concentrations were studied against Staphylococcus aureus and Salmonella typhimurium in vitro.Methods:      The minimum inhibitory concentrations (MIC of SNP was determined by agar dilution method. Then, hydrogels were prepared by mixing of 0.5% w/v agar and SNP (1/2 MIC, MIC, and 2 MIC and their inhibitory efficacies against planktonic and biofilm forms of bacteria were measured using agar spot and microtiter test, respectively.Results:    The MIC value was 125 µg/ mL for both bacteria. All SNP hydrogels represented antibacterial activity against Staphylococcus aureus and S. typhimurium on agar culture, which was significant compared to control group (silver sulfadiazine cream. The developed biofilm of S. aureus and S. typhimurium were strongly (85% reduction and modernly affected (60% reduction by SNP hydrogels during 15 min contact time, respectively. A dose-dependent biofilm reduction was not demonstrated when different SNP concentrations were tested. Moreover, the results from this study confirmed the moderate sanitizing ability of SNP loaded hydrogel against planktonic forms of both bacteria, which SNP (2MIC hydrogel decreased only 2.3 log10 CFU/ mL in a primary population of S. typhimurium during 15 min exposure time.Conclusion:     We recommended SNP incorporated agar hydrogel as an effective biofilm removal sanitizer.

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

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Katheryne Benini Martins

2015-06-01

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

A C-Type Lectin from Bothrops jararacussu Venom Disrupts Staphylococcal Biofilms

Science.gov (United States)

Klein, Raphael Contelli; Fabres-Klein, Mary Hellen; de Oliveira, Leandro Licursi; Feio, Renato Neves; Malouin, François; Ribon, Andréa de Oliveira Barros

2015-01-01

Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16) showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model. PMID:25811661

A C-type lectin from Bothrops jararacussu venom disrupts Staphylococcal biofilms.

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Raphael Contelli Klein

Full Text Available Bovine mastitis is a major threat to animal health and the dairy industry. Staphylococcus aureus is a contagious pathogen that is usually associated with persistent intramammary infections, and biofilm formation is a relevant aspect of the outcome of these infections. Several biological activities have been described for snake venoms, which led us to screen secretions of Bothrops jararacussu for antibiofilm activity against S. aureus NRS155. Crude venom was fractionated by size-exclusion chromatography, and the fractions were tested against S. aureus. Biofilm growth, but not bacterial growth, was affected by several fractions. Two fractions (15 and 16 showed the best activities and were also assayed against S. epidermidis NRS101. Fraction 15 was identified by TripleTOF mass spectrometry as a galactose-binding C-type lectin with a molecular weight of 15 kDa. The lectin was purified from the crude venom by D-galactose affinity chromatography, and only one peak was observed. This pure lectin was able to inhibit 75% and 80% of S. aureus and S. epidermidis biofilms, respectively, without affecting bacterial cell viability. The lectin also exhibited a dose-dependent inhibitory effect on both bacterial biofilms. The antibiofilm activity was confirmed using scanning electron microscopy. A pre-formed S. epidermidis biofilm was significantly disrupted by the C-type lectin in a time-dependent manner. Additionally, the lectin demonstrated the ability to inhibit biofilm formation by several mastitis pathogens, including different field strains of S. aureus, S. hyicus, S. chromogenes, Streptococcus agalactiae, and Escherichia coli. These findings reveal a new activity for C-type lectins. Studies are underway to evaluate the biological activity of these lectins in a mouse mastitis model.

Biofilm Formation As a Response to Ecological Competition.

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Nuno M Oliveira

2015-07-01

Full Text Available Bacteria form dense surface-associated communities known as biofilms that are central to their persistence and how they affect us. Biofilm formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: biofilm formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation. We show that strain mixing reliably increases biofilm formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate biofilm formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in biofilm occurs due to a general response to cellular damage: a comparable biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase biofilm formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

Evaluation of biofilm formation by bacterial strains isolated from milking equipment and milk samples from cows with mastitis

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Laura Gonçalves da Silva Chagas

2017-08-01

Full Text Available The presence of biofilm-forming bacteria from the mammary gland of dairy cows adhered to equipment in the milking environment represents one of the major causes of bacterial resistance during mastitis treatment. The aim of this study was to identify strains of Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli in milk samples from cows with mastitis, as well as in the expansion tank and milking set liners. We aimed to quantify the extracellular proteins and polysaccharides in the biofilm produced by each strain. A total of 294 samples were collected from a dairy farm in the municipality of Uberlândia, Minas Gerais. To identify the S. aureus, S. epidermidis and E. coli isolates responsible for biofilm production, we tested the phenotype using the Congo red agar (CRA and microplate adhesion tests. Protein quantification was performed with a Bicinchoninic Acid Protein Assay Kit (BCA kit, and polysaccharides were quantified by the phenol sulfuric acid method. We identified eight strains of S. aureus, one strain of S. epidermidis and 11 strains of E. coli responsible for biofilm production, all of which showed a higher concentration of polysaccharides than proteins in the matrix. Escherichia coli was considered the most prevalent bacterium among the samples, and S. aureus was determined to be the largest biofilm producer. The results of the CRA and microplate adhesion tests were similar in regard to identification of the biofilm-producing strains according to their phenotype and matrix composition. The classification of S. aureus strains as major biofilm producers is of great concern for producers, as such bacteria are considered one of the predominant contagious etiological agents that cause bovine mastitis. In addition, our observation that E. coli and S. epidermidis can produce biofilms highlights the need to reassess prophylactic measures to avoid the adhesion of biofilm-producing bacteria.

Rapid in Vitro Quantification of S. aureus Biofilms on Vascular Graft Surfaces

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Monika Herten

2017-12-01

Full Text Available Objectives: Increasing resistance of microorganisms and particularly tolerance of bacterial biofilms against antibiotics require the need for alternative antimicrobial substances. S. aureus is the most frequent pathogen causing vascular graft infections. In order to evaluate the antimicrobial efficacy, quantification of the bacterial biofilms is necessary. Aim of the present study was the validation of an in vitro model for quantification of bacterial biofilm on vascular graft surfaces using three different assays.Methods: Standardized discs of vascular graft material (Dacron or PTFE or polystyrene (PS as control surface with 0.25 cm2 surface area were inoculated with 10−3 diluted overnight culture of three biofilm-producing S. aureus isolates (BEB-029, BEB-295, SH1000 in 96-well PS culture plates. After incubation for 4 and 18 h, the biofilm was determined by three different methods: (a mitochondrial ATP concentration as measure of bacterial viability (ATP, (b crystal violet staining (Cry, and (c vital cell count by calculation of colony-forming units (CFU. The experiments were performed three times. Quadruplicates were used for each isolate, time point, and method. In parallel, bacterial biofilms were documented via scanning electron microscopy.Results: All three methods could quantify biofilms on the PS control. Time needed was 0:40, 13:10, and 14:30 h for ATP, Cry, and CFU, respectively. The Cry assay could not be used for vascular graft surfaces due to high unspecific background staining. However, ATP assay and CFU count showed comparable results on vascular graft material and control. The correlations between ATP and CFU assay differed according to the surface and incubation time and were significant only after 4 h on Dacron (BEB-029, p = 0.013 and on PS (BEB-029, p < 0.001. Between ATP and Cry assay on PS, a significant correlation could be detected after 4 h (BEB-295, p = 0.027 and after 18 h (all three strains, p < 0.026. The

Propolis-Sahara honeys preparation exhibits antibacterial and anti-biofilm activity against bacterial biofims formed on urinary catheters

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Saad Aissat

2016-11-01

Full Text Available Objective: To evaluate the antibacterial effect of Sahara honeys (SHs against bacterial biofilms formed on urinary catheters in combination with propolis-Sahara honeys (P-SHs. Methods: Three clinical isolates were subjected to biofilm detection methods. The antibacterial and anti-biofilm activity for SHs and P-SHs were determined using agar well diffusion and the percentage of biofilm inhibition (PBI methods. Results: The PBI for Gram-positive bacteria [Staphylococcus aureus (S. aureus] was in the range of 0%–20%, while PBI for Gram-negative bacteria [Pseudomonas aeruginosa and Escherichia coli (E. coli] were in range of 17%–57% and 16%–65%, respectively. The highest PBI (65% was produced by SH2 only on E. coli. In agar well diffusion assay, zones of inhibition ranged from 11–20 mm (S. aureus, 9–19 mm (Pseudomonas aeruginosa and 11–19 mm (E. coli. The highest inhibition (20 mm was produced by SH1 only on S. aureus. In addition, the treatment of SHs and P-SHs catheters with a polymicrobial biofilms reduced biofilm formation after 48 h exposure period. Conclussions: SHs and P-SHs applied as a natural agent can be used as a prophylactic agent to prevent the formation of in vitro biofilm.

Association of biofilm production with colonization among clinical isolates of Acinetobacter baumannii.

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Ryu, Seong Yeol; Baek, Won-Ki; Kim, Hyun Ah

2017-03-01

The pathogen Acinetobacter baumannii is increasingly causing healthcare-associated infections worldwide, particularly in intensive care units. Biofilm formation, a factor contributing to the virulence of A. baumannii , is associated with long-term persistence in hospital environments. The present study investigates the clinical impact of biofilm production on colonization and acquisition after patient admission. Forty-nine A. baumannii isolates were obtained between August and November 2013 from Keimyung University Dongsan Medical Center, Daegu, Korea. All isolates were obtained from sputum samples of new patients infected or colonized by A. baumannii . The microtiter plate assay was used to determine biofilm formation. Twenty-four A. baumannii isolates (48%) demonstrated enhanced biofilm formation capacity than that of the standard A. baumannii strain (ATCC 19606). All isolates were resistant to carbapenem, 38 isolates (77%) were collected from patients in an intensive care unit, and 47 isolates (95%) were from patients who had been exposed to antibiotics in the previous month. The median duration of colonization was longer for biofilm-producing isolates than that of the biofilm non-biofilm producing isolates (18 days vs. 12 days, p < 0.05). Simultaneous colonization with other bacteria was more common for biofilm-producing isolates than that for the non-biofilm producing isolates. The most prevalent co-colonizing bacteria was Staphylococcus aureus . Biofilm-producing isolates seem to colonize the respiratory tract for longer durations than the non-biofilm producing isolates. During colonization, biofilm producers promote co-colonization by other bacteria, particularly S. aureus . Additional research is required to determine possible links between biofilm formation and nosocomial infection.

Development of a biofilm inhibitor molecule against multidrug resistant Staphylococcus aureus associated with gestational urinary tract infections

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Balamurugan eP

2015-08-01

Full Text Available Urinary Tract Infection (UTI is a globally widespread human infection caused by an infestation of uropathogens. Eventhough, Escherichia coli is often quoted as being the chief among them, Staphylococcus aureus involvement in UTI especially in gestational UTI is often understated. Staphylococcal accessory regulator A (SarA is a quorum regulator of S. aureus that controls the expression of various virulence and biofilm phenotypes. Since SarA had been a focussed target for antibiofilm agent development, the study aims to develop a potential drug molecule targeting the SarA of S. aureus to combat biofilm associated infections in which it is involved. In our previous studies, we have reported the antibiofilm activity of SarA based biofilm inhibitor, (SarABI with a 50% minimum biofilm inhibitory concentration (MBIC50 value of 200 µg/mL against S. aureus associated with vascular graft infections and also the antibiofilm activity of the root ethanolic extracts of Melia dubia against uropathogenic E. coli. In the present study, in silico design of a hybrid molecule composed of a molecule screened from M. dubia root ethanolic extracts and a modified SarA based inhibitor (SarABIM was undertaken. SarABIM is a modified form of SarABI where the fluorine groups are absent in SarABIM. Chemical synthesis of the hybrid molecule, 4-(Benzylaminocyclohexyl 2-hydroxycinnamate (henceforth referred to as UTI Quorum-Quencher, UTIQQ was then performed, followed by in vitro and in vivo validation. The MBIC¬50 and MBIC90 of UTIQQ were found to be 15 µg/mL and 65 µg/mL respectively. Confocal laser scanning microscopy (CLSM images witnessed biofilm reduction and bacterial killing in either UTIQQ or in combined use of antibiotic gentamicin and UTIQQ. Similar results were observed with in vivo studies of experimental UTI in rat model. So, we propose that the drug UTIQQ would be a promising candidate when used alone or, in combination with an antibiotic for staphylococcal

Specific plant induced biofilm formation in Methylobacterium species

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Rossetto, Priscilla B.; Dourado, Manuella N.; Quecine, Maria C.; Andreote, Fernando D.; Araújo, Welington L.; Azevedo, João L.; Pizzirani-Kleiner, Aline A.

2011-01-01

Two endophytic strains of Methylobacterium spp. were used to evaluate biofilm formation on sugarcane roots and on inert wooden sticks. Results show that biofilm formation is variable and that plant surface and possibly root exudates have a role in Methylobacterium spp. host recognition, biofilm formation and successful colonization as endophytes. PMID:24031703

Effect of Algae and Plant Lectins on Planktonic Growth and Biofilm Formation in Clinically Relevant Bacteria and Yeasts

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Mayron Alves Vasconcelos

2014-01-01

Full Text Available This study aimed to evaluate the abilities of plant and algae lectins to inhibit planktonic growth and biofilm formation in bacteria and yeasts. Initially, ten lectins were tested on Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella oxytoca, Pseudomonas aeruginosa, Candida albicans, and C. tropicalis at concentrations of 31.25 to 250 μg/mL. The lectins from Cratylia floribunda (CFL, Vatairea macrocarpa (VML, Bauhinia bauhinioides (BBL, Bryothamnion seaforthii (BSL, and Hypnea musciformis (HML showed activities against at least one microorganism. Biofilm formation in the presence of the lectins was also evaluated; after 24 h of incubation with the lectins, the biofilms were analyzed by quantifying the biomass (by crystal violet staining and by enumerating the viable cells (colony-forming units. The lectins reduced the biofilm biomass and/or the number of viable cells to differing degrees depending on the microorganism tested, demonstrating the different characteristics of the lectins. These findings indicate that the lectins tested in this study may be natural alternative antimicrobial agents; however, further studies are required to better elucidate the functional use of these proteins.

Paired methods to measure biofilm killing and removal: a case study with Penicillin G treatment of Staphylococcus aureus biofilm.

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Ausbacher, D; Lorenz, L; Pitts, B; Stewart, P S; Goeres, D M

2018-03-01

Biofilms are microbial aggregates that show high tolerance to antibiotic treatments in vitro and in vivo. Killing and removal are both important in biofilm control, therefore methods that measure these two mechanisms were evaluated in a parallel experimental design. Kill was measured using the single tube method (ASTM method E2871) and removal was determined by video microscopy and image analysis using a new treatment flow cell. The advantage of the parallel test design is that both methods used biofilm covered coupons harvested from a CDC biofilm reactor, a well-established and standardized biofilm growth method. The control Staphylococcus aureus biofilms treated with growth medium increased by 0·6 logs during a 3-h contact time. Efficacy testing showed biofilms exposed to 400 μmol l -1 penicillin G decreased by only 0·3 logs. Interestingly, time-lapse confocal scanning laser microscopy revealed that penicillin G treatment dispersed the biofilm despite being an ineffective killing agent. In addition, no biofilm removal was detected when assays were performed in 96-well plates. These results illustrate that biofilm behaviour and impact of treatments can vary substantially when assayed by different methods. Measuring both killing and removal with well-characterized methods will be crucial for the discovery of new anti-biofilm strategies. Biofilms are tolerant to antimicrobial treatments and can lead to persistent infections. Finding new anti-biofilm strategies and understanding their mode-of-action is therefore of high importance. Historically, antimicrobial testing has focused on measuring the decrease in viability. While kill data are undeniably important, measuring biofilm disruption provides equally useful information. Starting with biofilm grown in the same reactor, we paired assessment of biofilm removal using a new treatment-flow-cell and real-time microscopy with kill data collected using the single tube method (ASTM E2871). Pairing these two methods

Sexual Biofilm Formation in Candida tropicalis Opaque Cells

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Jones, Stephen K.; Hirakawa, Matthew P.; Bennett, Richard J.

2014-01-01

Summary Candida albicans and Candida tropicalis are opportunistic fungal pathogens that can transition between white and opaque phenotypic states. White and opaque cells differ both morphologically and in their responses to environmental signals. In C. albicans, opaque cells respond to sexual pheromones by undergoing conjugation, while white cells are induced by pheromones to form sexual biofilms. Here, we show that sexual biofilm formation also occurs in C. tropicalis but, unlike C. albicans, biofilms are formed exclusively by opaque cells. C. tropicalis biofilm formation was dependent on the pheromone receptors Ste2 and Ste3, confirming the role of pheromone signaling in sexual biofilm development. Structural analysis of C. tropicalis sexual biofilms revealed stratified communities consisting of a basal layer of yeast cells and an upper layer of filamentous cells, together with an extracellular matrix. Transcriptional profiling showed that genes involved in pheromone signaling and conjugation were upregulated in sexual biofilms. Furthermore, FGR23, which encodes an agglutinin-like protein, was found to enhance both mating and sexual biofilm formation. Together, these studies reveal that C. tropicalis opaque cells form sexual biofilms with a complex architecture, and suggest a conserved role for sexual agglutinins in mediating mating, cell cohesion and biofilm formation. PMID:24612417

Inactivation of Efflux Pumps Abolishes Bacterial Biofilm Formation

DEFF Research Database (Denmark)

Kvist, Malin; Hancock, Viktoria; Klemm, Per

2008-01-01

Bacterial biofilms cause numerous problems in health care and industry; notably, biofilms are associated with a large number of infections. Biofilm-dwelling bacteria are particularly resistant to antibiotics, making it hard to eradicate biofilm-associated infections. Bacteria rely on efflux pumps...... to get rid of toxic substances. We discovered that efflux pumps are highly active in bacterial biofilms, thus making efflux pumps attractive targets for antibiofilm measures. A number of efflux pump inhibitors (EPIs) are known. EPIs were shown to reduce biofilm formation, and in combination they could...... abolish biofilm formation completely. Also, EPIs were able to block the antibiotic tolerance of biofilms. The results of this feasibility study might pave the way for new treatments for biofilm-related infections and may be exploited for prevention of biofilms in general....

Effect of biocides on biofilms of some multidrug resistant clinical ...

African Journals Online (AJOL)

The ability of Escherichia coli and Klebsiella aerogenes to form biofilms was most affected. There was little inhibition of biofilm formation by the biocides on Staphylococcus aureus. This study has shown a relationship between biocide and multidrug resistance. Keywords: Biocides, Multi drug resistance, sodium hypochlorite, ...

(68)Ga-labeled phage-display selected peptides as tracers for positron emission tomography imaging of Staphylococcus aureus biofilm-associated infections: Selection, radiolabelling and preliminary biological evaluation

DEFF Research Database (Denmark)

Nielsen, Karin M; Kyneb, Majbritt H; Alstrup, Aage K O

2016-01-01

, while the in vivo plasma stability was analyzed in mice and pigs. Additionally, the whole-body distribution kinetics of (68)Ga-A9-K-DOTA was measured in vivo by PET imaging of pigs and ex vivo in excised mice tissues. RESULTS: The (68)Ga-A9-K-DOTA and (68)Ga-A11-GSGK-DOTA remained stable in product......INTRODUCTION: Staphylococcus aureus is a major cause of skin and deep-sited infections, often associated with the formation of biofilms. Early diagnosis and initiated therapy is essential to prevent disease progression and to reduce complications that can be serious. Imaging techniques are helpful...... combining anatomical with functional data in order to describe and characterize site, extent and activity of the disease. The purpose of the study was to identify and (68)Ga-label peptides with affinity for S. aureus biofilm and evaluate their potential as bacteria-specific positron emission tomography (PET...

Quantitative characterization of the influence of the nanoscale morphology of nanostructured surfaces on bacterial adhesion and biofilm formation.

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Ajay Vikram Singh

Full Text Available Bacterial infection of implants and prosthetic devices is one of the most common causes of implant failure. The nanostructured surface of biocompatible materials strongly influences the adhesion and proliferation of mammalian cells on solid substrates. The observation of this phenomenon has led to an increased effort to develop new strategies to prevent bacterial adhesion and biofilm formation, primarily through nanoengineering the topology of the materials used in implantable devices. While several studies have demonstrated the influence of nanoscale surface morphology on prokaryotic cell attachment, none have provided a quantitative understanding of this phenomenon. Using supersonic cluster beam deposition, we produced nanostructured titania thin films with controlled and reproducible nanoscale morphology respectively. We characterized the surface morphology; composition and wettability by means of atomic force microscopy, X-ray photoemission spectroscopy and contact angle measurements. We studied how protein adsorption is influenced by the physico-chemical surface parameters. Lastly, we characterized Escherichia coli and Staphylococcus aureus adhesion on nanostructured titania surfaces. Our results show that the increase in surface pore aspect ratio and volume, related to the increase of surface roughness, improves protein adsorption, which in turn downplays bacterial adhesion and biofilm formation. As roughness increases up to about 20 nm, bacterial adhesion and biofilm formation are enhanced; the further increase of roughness causes a significant decrease of bacterial adhesion and inhibits biofilm formation. We interpret the observed trend in bacterial adhesion as the combined effect of passivation and flattening effects induced by morphology-dependent protein adsorption. Our findings demonstrate that bacterial adhesion and biofilm formation on nanostructured titanium oxide surfaces are significantly influenced by nanoscale morphological

Protection from Staphylococcus aureus mastitis associated with poly-N-acetyl β-1,6 glucosamine specific antibody production using biofilm-embedded bacteria

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Pérez, M. M.; Prenafeta, A.; Valle, J.; Penadés, J.; Rota, C.; Solano, C.; Marco, J.; Grilló, M.J.; Lasa, I.; Irache, J.M.; Maira-Litran, T.; Jiménez-Barbero, J.; Costa, L.; Pier, G.B.; de Andrés, D.; Amorena, B.

2010-01-01

Staphylococcus aureus vaccines based on bacterins surrounded by slime, surface polysaccharides coupled to protein carriers and polysaccharides embedded in liposomes administered together with non-biofilm bacterins confer protection against mastitis. However, it remains unknown whether protective antibodies are directed to slime-associated known exopolysaccharides and could be produced in the absence of bacterin immunizations. Here, a sheep mastitis vaccination study was carried out using bacterins, crude bacterial extracts or a purified exopolysaccharide from biofilm bacteria delivered in different vehicles. This polysaccharide reacted specifically with antibodies to poly-N-acetyl-β-1,6-glucosamine (PNAG) and not with antibodies to other capsular antigens or bacterial components. Following intra-mammary challenge with biofilm-producing bacteria, antibody production against the polysaccharide, milk bacterial counts and mastitis lesions were determined. Bacterins from strong biofilm-producing bacteria triggered the highest production of antibodies to PNAG and conferred the highest protection against infection and mastitis, compared with weak biofilm-producing bacteria and non-cellular inocula. Thus, bacterins from strong biofilm bacteria, rather than purified polysaccharide, are proposed as a cost-efficient vaccination against S. aureus ruminant mastitis. PMID:19428854

Biofilm Formation Characteristics of Pseudomonas lundensis Isolated from Meat.

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Liu, Yong-Ji; Xie, Jing; Zhao, Li-Jun; Qian, Yun-Fang; Zhao, Yong; Liu, Xiao

2015-12-01

Biofilms formations of spoilage and pathogenic bacteria on food or food contact surfaces have attracted increasing attention. These events may lead to a higher risk of food spoilage and foodborne disease transmission. While Pseudomonas lundensis is one of the most important bacteria that cause spoilage in chilled meat, its capability for biofilm formation has been seldom reported. Here, we investigated biofilm formation characteristics of P. lundensis mainly by using crystal violet staining, and confocal laser scanning microscopy (CLSM). The swarming and swimming motility, biofilm formation in different temperatures (30, 10, and 4 °C) and the protease activity of the target strain were also assessed. The results showed that P. lundensis showed a typical surface-associated motility and was quite capable of forming biofilms in different temperatures (30, 10, and 4 °C). The strain began to adhere to the contact surfaces and form biofilms early in the 4 to 6 h. The biofilms began to be formed in massive amounts after 12 h at 30 °C, and the extracellular polysaccharides increased as the biofilm structure developed. Compared with at 30 °C, more biofilms were formed at 4 and 10 °C even by a low bacterial density. The protease activity in the biofilm was significantly correlated with the biofilm formation. Moreover, the protease activity in biofilm was significantly higher than that of the corresponding planktonic cultures after cultured 12 h at 30 °C. © 2015 Institute of Food Technologists®

Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation

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2011-01-01

Background Staphylococcus epidermidis (SE) has emerged as one of the most important causes of nosocomial infections. The SaeRS two-component signal transduction system (TCS) influences virulence and biofilm formation in Staphylococcus aureus. The deletion of saeR in S. epidermidis results in impaired anaerobic growth and decreased nitrate utilization. However, the regulatory function of SaeRS on biofilm formation and autolysis in S. epidermidis remains unclear. Results The saeRS genes of SE1457 were deleted by homologous recombination. The saeRS deletion mutant, SE1457ΔsaeRS, exhibited increased biofilm formation that was disturbed more severely (a 4-fold reduction) by DNase I treatment compared to SE1457 and the complementation strain SE1457saec. Compared to SE1457 and SE1457saec, SE1457ΔsaeRS showed increased Triton X-100-induced autolysis (approximately 3-fold) and decreased cell viability in planktonic/biofilm states; further, SE1457ΔsaeRS also released more extracellular DNA (eDNA) in the biofilms. Correlated with the increased autolysis phenotype, the transcription of autolysis-related genes, such as atlE and aae, was increased in SE1457ΔsaeRS. Whereas the expression of accumulation-associated protein was up-regulated by 1.8-fold in 1457ΔsaeRS, the expression of an N-acetylglucosaminyl transferase enzyme (encoded by icaA) critical for polysaccharide intercellular adhesin (PIA) synthesis was not affected by the deletion of saeRS. Conclusions Deletion of saeRS in S. epidermidis resulted in an increase in biofilm-forming ability, which was associated with increased eDNA release and up-regulated Aap expression. The increased eDNA release from SE1457ΔsaeRS was associated with increased bacterial autolysis and decreased bacterial cell viability in the planktonic/biofilm states. PMID:21702925

Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation

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Francois Patrice

2011-06-01

Full Text Available Abstract Background Staphylococcus epidermidis (SE has emerged as one of the most important causes of nosocomial infections. The SaeRS two-component signal transduction system (TCS influences virulence and biofilm formation in Staphylococcus aureus. The deletion of saeR in S. epidermidis results in impaired anaerobic growth and decreased nitrate utilization. However, the regulatory function of SaeRS on biofilm formation and autolysis in S. epidermidis remains unclear. Results The saeRS genes of SE1457 were deleted by homologous recombination. The saeRS deletion mutant, SE1457ΔsaeRS, exhibited increased biofilm formation that was disturbed more severely (a 4-fold reduction by DNase I treatment compared to SE1457 and the complementation strain SE1457saec. Compared to SE1457 and SE1457saec, SE1457ΔsaeRS showed increased Triton X-100-induced autolysis (approximately 3-fold and decreased cell viability in planktonic/biofilm states; further, SE1457ΔsaeRS also released more extracellular DNA (eDNA in the biofilms. Correlated with the increased autolysis phenotype, the transcription of autolysis-related genes, such as atlE and aae, was increased in SE1457ΔsaeRS. Whereas the expression of accumulation-associated protein was up-regulated by 1.8-fold in 1457ΔsaeRS, the expression of an N-acetylglucosaminyl transferase enzyme (encoded by icaA critical for polysaccharide intercellular adhesin (PIA synthesis was not affected by the deletion of saeRS. Conclusions Deletion of saeRS in S. epidermidis resulted in an increase in biofilm-forming ability, which was associated with increased eDNA release and up-regulated Aap expression. The increased eDNA release from SE1457ΔsaeRS was associated with increased bacterial autolysis and decreased bacterial cell viability in the planktonic/biofilm states.

Bacterial and fungal biofilm formation on contact lenses and their susceptibility to lens care solutions

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Siddharth Kackar

2017-01-01

Full Text Available Background: Microbial biofilm formation on contact lenses and lens storage cases may be a risk factor for contact lens-associated corneal infections. Various types of contact lens care solutions are used to reduce microbial growths on lenses. Objectives: The present study aimed at comparing the growths of biofilms on the different contact lenses and lens cases. The study also aimed at determining the effect of lens care solutions and bacteriophage on these biofilms. Materials and Methods: One type of hard lens and two types of soft lenses were used for the study. The organisms used were Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Candida albicans ATCC 60193 and Escherichia coli ATCC 25922. Biofilm production was performed by modified O'Toole and Kolter method and effect of lens cleaning solutions and a crude coliphage on biofilms was also studied. Results were visualised using scanning electron microscopy and quantitated by colony counting method and spectrophotometric measurement of optical density (OD. Statistical analysis was done by SPSS 11.5, Kruskal–Wallis test and Chi-square test. Results: Soft lens cleaning solutions had a significant inhibitory effect (P = 0.020 on biofilm formation on soft lenses and also lens cases (P < 0.001. Soft lens cleaning solution 2 was more efficient than solution 1. However, no such inhibitory effect was observed with regard to hard lens cleaning solution, but for a significant reduction in the OD values (P < 0.001. There was no significant inhibitory effect by bacteriophages. Conclusion: This study showed the importance of selecting the appropriate lens cleaning solution to prevent biofilm production on contact lenses.

Biofilm formation on nanostructured titanium oxide surfaces and a micro/nanofabrication-based preventive strategy using colloidal lithography

International Nuclear Information System (INIS)

Singh, Ajay Vikram; Vyas, Varun; Salve, Tushar S; Dellasega, David; Cortelli, Daniele; Podestà, Alessandro; Milani, Paolo; Gade, W N

2012-01-01

The contamination of implant devices as a result of biofilm formation through bacterial infection has instigated major research in this area, particularly to understand the mechanism of bacterial cell/implant surface interactions and their preventions. In this paper, we demonstrate a controlled method of nanostructured titanium oxide surface synthesis using supersonic cluster beam depositions. The nanoscale surface characterization using atomic force microscopy and a profilometer display a regulated evolution in nanomorphology and physical properties. X-ray photoelectron spectroscopy analyses display a stoichiometric nanostructured TiO 2 film. Measurement of the water contact angle shows a nominal increase in the hydrophilic nature of ns-TiO 2 films, whereas the surface energy increases with decreasing contact angle. Bacterial species Staphylococcus aureus and Escherichia coli interaction with nanostructured surfaces shows an increase in adhesion and biofilm formation with increasing nanoscale morphological properties. Conversely, limiting ns-TiO 2 film distribution to micro/nanopatterned designed substrates integrated with bovine serum albumin functionalization leads to a reduction in biofilm formations due to a globally decreased bacterial cell–surface interaction area. The results have potential implications in inhibiting bacterial colonization and promoting mammalian cell–implant interactions. (paper)

Antibiofilm activity of Vetiveria zizanioides root extract against methicillin-resistant Staphylococcus aureus.

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Kannappan, Arunachalam; Gowrishankar, Shanmugaraj; Srinivasan, Ramanathan; Pandian, Shunmugiah Karutha; Ravi, Arumugam Veera

2017-09-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading human pathogen responsible for causing chronic clinical manifestation worldwide. In addition to antibiotic resistance genes viz. mecA and vanA, biofilm formation plays a prominent role in the pathogenicity of S. aureus by enhancing its resistance to existing antibiotics. Considering the role of folk medicinal plants in the betterment of human health from the waves of multidrug resistant bacterial infections, the present study was intended to explore the effect of Vetiveria zizanioides root on the biofilm formation of MRSA and its clinical counterparts. V. zizanioides root extract (VREX) showed a concentration-dependent reduction in biofilm formation without hampering the cellular viability of the tested strains. Micrographs of scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) portrayed the devastating impact of VREX on biofilm formation. In addition to antibiofilm activity, VREX suppresses the production of biofilm related phenotypes such as exopolysaccharide, slime and α-hemolysin toxin. Furthermore, variation in FT-IR spectra evidenced the difference in cellular factors of untreated and VREX treated samples. Result of mature biofilm disruption assay and down regulation of genes like fnbA, fnbB, clfA suggested that VREX targets these adhesin genes responsible for initial adherence. GC-MS analysis revealed the presence of sesquiterpenes as a major constituent in VREX. Thus, the data of present study strengthen the ethnobotanical value of V. zizanioides and concludes that VREX contain bioactive molecules that have beneficial effect over the biofilm formation of MRSA and its clinical isolates. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biofilm Formation of Listeria monocytogenes on Various Surfaces

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M Mahdavi

2007-10-01

Full Text Available Introduction & Objective: Listeria monocytogenes is considered as a ubiquitous foodborne pathogen which can lead to serious infections, especially in newborns, elderly, pregnant, and immunocompromised people. The organism has been isolated from many foods and may cause meningitis, septicemia and abortion in pregnant women. Also L. monocytogenes forms biofilms on many food contact surface materials and medical devices. Development of biofilms on many surfaces is a potential source of contamination of foods that may lead to spoilage or transmission of foodborne pathogens. Materials & Methods: Biofilm formation of L. monocytogenes (RITCC 1293 serotype 4a was investigated. Hydrophobicity of L. monocytogenes was measured by MATH method. Then biofilm formation of the organism was assessed at 2, 4, 8, 16 and 20 hours on stainless steel (type 304 no 2B, polyethylene and glass by drop plate method. Results: Results indicated that L. monocytogenes with 85% of hydrophobicity formed biofilm on each of three surfaces. Biofilm formation on stainless steel surfaces was significantly more than other surfaces (p<0.05. Conclusion: The ability of biofilm formation of L. monocytogenes on medical devices and food containers is very important as far as hygiene and disease outbreaks are concerned.

Inhibitory effect of farnesol on biofilm formation by Candida tropicalis

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E Zibafar

2009-03-01

Full Text Available ABSTRACT Background: Candidiasis associated with indwelling medical devices is especially problematic since they can act as substrates for biofilm growth which are highly resistant to antifungal drugs. Farnesol is a quorum-sensing molecule that inhibits filamentation and biofilm formation in Candida albicans. Since in recent years Candida tropicalis have been reported as an important and common non-albicans Candida species with high drug resistance pattern, the inhibitory effect of farnesol on biofilm formation by Candida tropicalis was evaluated. Methods: Five Candida tropicalis strains were treated with different concentration of farnesol (0, 30 and 300 µM after 0, 1 and 4 hrs of adherence and then they were maintained under biofilm formation condition in polystyrene, 96-well microtiter plates at 37°C for 48 hrs. Biofilm formation was measured by a semiquantitative colorimetric technique based on reduction assay of 2,3- bis  -2H-tetrazolium- 5- carboxanilide (XTT. Results: The results indicated that the initial adherence time had no effect on biofilm formation and low concentration of farnesol (30 µM could not inhibit biofilm formation. However the presence of non-adherent cells increased biofilm formation significantly and the high concentration of farnesol (300 µM could inhibit biofilm formation. Conclusion: Results of this study showed that the high concentration of farnesol could inhibit biofilm formation and may be used as an adjuvant in prevention and in therapeutic strategies with antifungal drugs.

N-halamine-based rechargeable antimicrobial and biofilm-controlling polyurethane

Science.gov (United States)

Sun, Xinbo; Cao, Zhengbing; Porteous, Nuala; Sun, Yuyu

2012-01-01

An N-halamine precursor, 5, 5-dimethyl hydantoin (DMH), was covalently linked to the surface of polyurethane (PU) with 1,6-hexamethylene diisocyanate (HDI) as a coupling agent. The reaction pathways were investigated using propyl isocyanate (PI) as a model compound, and the results suggested that the imide and amide groups of DMH had very similar reactivity toward the isocyanate groups on PU surfaces activated with HDI. After bleach treatment, the covalently bound DMH moieties were transformed into N-halamines. The new N-halmaine-based PU provided potent antimicrobial effects against Staphylococcus aureus (S. aureus, Gram-positive), Escherichia coli (E. coli, Gram-negative), methicillin-resistant staphylococcus aureus (MRSA, drug resistant Gram-positive bacteria), vancomycin-resistant enterococcus (VRE, drug resistant Gram-positive bacteria), and Candida albicans (C. ablicans, fungi), and successfully prevented bacterial and fungal biofilm formation. The antimicrobial and biofilm-controlling effects were stable for longer than 6 months under normal storage in open air. Furthermore, if the functions were lost due to prolonged use, they could be recharged by another chlorination treatment. The recharging could be repeated as needed to achieve long-term protection against microbial contamination and biofilm-formation. PMID:22244984

Biofilm formation in a hot water system

DEFF Research Database (Denmark)

Bagh, L.K.; Albrechtsen, Hans-Jørgen; Arvin, Erik

2002-01-01

The biofilm formation rate was measured in situ in a hot water system in an apartment building by specially designed sampling equipment, and the net growth of the suspended bacteria was measured by incubation of water samples with the indigeneous bacteria. The biofilm formation rate reached......, in the sludge, or in the water from the distribution system was negligible. This indicated that bacterial growth took place on the inner surfaces in the hot water system and biofilm formation and detachment of bacteria could account for most of the suspended bacteria actually measured in hot water. Therefore...

Prevention of biofilm formation and removal of existing biofilms by extracellular DNases of Campylobacter jejuni.

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Brown, Helen L; Reuter, Mark; Hanman, Kate; Betts, Roy P; van Vliet, Arnoud H M

2015-01-01

The fastidious nature of the foodborne bacterial pathogen Campylobacter jejuni contrasts with its ability to survive in the food chain. The formation of biofilms, or the integration into existing biofilms by C. jejuni, is thought to contribute to food chain survival. As extracellular DNA (eDNA) has previously been proposed to play a role in C. jejuni biofilms, we have investigated the role of extracellular DNases (eDNases) produced by C. jejuni in biofilm formation. A search of 2791 C. jejuni genomes highlighted that almost half of C. jejuni genomes contains at least one eDNase gene, but only a minority of isolates contains two or three of these eDNase genes, such as C. jejuni strain RM1221 which contains the cje0256, cje0566 and cje1441 eDNase genes. Strain RM1221 did not form biofilms, whereas the eDNase-negative strains NCTC 11168 and 81116 did. Incubation of pre-formed biofilms of NCTC 11168 with live C. jejuni RM1221 or with spent medium from a RM1221 culture resulted in removal of the biofilm. Inactivation of the cje1441 eDNase gene in strain RM1221 restored biofilm formation, and made the mutant unable to degrade biofilms of strain NCTC 11168. Finally, C. jejuni strain RM1221 was able to degrade genomic DNA from C. jejuni NCTC 11168, 81116 and RM1221, whereas strain NCTC 11168 and the RM1221 cje1441 mutant were unable to do so. This was mirrored by an absence of eDNA in overnight cultures of C. jejuni RM1221. This suggests that the activity of eDNases in C. jejuni affects biofilm formation and is not conducive to a biofilm lifestyle. These eDNases do however have a potential role in controlling biofilm formation by C. jejuni strains in food chain relevant environments.

XF-70 and XF-73, novel antibacterial agents active against slow-growing and non-dividing cultures of Staphylococcus aureus including biofilms.

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Ooi, Nicola; Miller, Keith; Randall, Christopher; Rhys-Williams, William; Love, William; Chopra, Ian

2010-01-01

Slow-growing and non-dividing bacteria exhibit tolerance to many antibiotics. However, membrane-active agents may act against bacteria in all growth phases. We sought to examine whether the novel porphyrin antibacterial agents XF-70 and XF-73, which have rapid membrane-perturbing activity against Staphylococcus aureus, retained antistaphylococcal activity against growth-attenuated cells. The killing kinetics of XF-70, XF-73 and various comparator agents against exponential phase cultures of S. aureus SH1000 were compared with effects on cells held at 4 degrees C, non-growing cultures expressing the stringent response induced by mupirocin and bacteria in the stationary phase. Biofilms of S. aureus SH1000 were generated with the Calgary device to examine the activities of XF-70 and XF-73 under a further system exhibiting diminished bacterial growth. Cold culture, stringent response and stationary phase cultures remained susceptible to XF-70 and XF-73, which caused > or =5 log reductions in viability over 2 h. During this period the most active comparator agents (chlorhexidine and cetyltrimethylammonium bromide) only promoted a 3 log drop in viability. XF-70 and XF-73 were also highly active against biofilms, with both agents exhibiting low biofilm MICs (1 mg/L) and minimum biofilm eradication concentrations (2 mg/L). XF-70 and XF-73 remained highly active against various forms of slow-growing or non-dividing S. aureus. The results support the hypothesis that membrane-active agents may be particularly effective in eradicating slow- or non-growing bacteria and suggest that XF-70 and XF-73 could be utilized to treat staphylococcal infections where the organisms are only dividing slowly, such as biofilm-associated infections of prosthetic devices.

Flagellar motility is critical for Listeria monocytogenes biofilm formation.

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Lemon, Katherine P; Higgins, Darren E; Kolter, Roberto

2007-06-01

The food-borne pathogen Listeria monocytogenes attaches to environmental surfaces and forms biofilms that can be a source of food contamination, yet little is known about the molecular mechanisms of its biofilm development. We observed that nonmotile mutants were defective in biofilm formation. To investigate how flagella might function during biofilm formation, we compared the wild type with flagellum-minus and paralyzed-flagellum mutants. Both nonmotile mutants were defective in biofilm development, presumably at an early stage, as they were also defective in attachment to glass during the first few hours of surface exposure. This attachment defect could be significantly overcome by providing exogenous movement toward the surface via centrifugation. However, this centrifugation did not restore mature biofilm formation. Our results indicate that it is flagellum-mediated motility that is critical for both initial surface attachment and subsequent biofilm formation. Also, any role for L. monocytogenes flagella as adhesins on abiotic surfaces appears to be either minimal or motility dependent under the conditions we examined.

Topical colloidal silver as an anti-biofilm agent in a Staphylococcus aureus chronic rhinosinusitis sheep model.

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Rajiv, Sukanya; Drilling, Amanda; Bassiouni, Ahmed; James, Craig; Vreugde, Sarah; Wormald, Peter-John

2015-04-01

Treatment of recalcitrant chronic rhinosinusitis (CRS) is a challenge with increasing antibiotic resistance, leading to re-emergence of topical therapies. The aim of this study was to assess safety and efficacy of topical colloidal silver solution for the treatment of Staphylococcus aureus biofilms in a sheep model. In the safety study, normal saline (control) and 30-ppm colloidal silver solution (test) was used to flush the frontal sinuses for 14 days in 8 sheep (4 sheep each). In the efficacy study, following frontal sinus infection with Staphylococcus aureus, sheep were treated with either control saline or topical silver solution of varying concentrations (30 ppm/20 ppm/10 ppm/5 ppm) for 5 days, with 4 sheep in each group. Blood silver level, full blood counts, and biochemical parameters were analyzed in both safety and efficacy studies. Sinus tissue was harvested for histological examination and ciliary structure analysis in safety and for biofilm biomass quantification by fluorescence in situ hybridization (FISH) technique and COMSTAT 2 software in the efficacy study. Results were analyzed using appropriate statistical tests. Sheep treated with silver showed a significant decrease in biofilm biomass (0.004, 0.004, 0.004, and 0.007, in the 4 silver-treated groups, respectively) compared to saline control (0.175), p silver levels were higher in the treated groups compared to controls (p colloidal silver solution has effective antibiofilm activity in Staphylococcus aureus CRS in a sheep model and appears safe. © 2015 ARS-AAOA, LLC.

Lactobacillus plantarum lipoteichoic acid inhibits biofilm formation of Streptococcus mutans

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Ahn, Ki Bum; Baik, Jung Eun; Park, Ok-Jin; Yun, Cheol-Heui

2018-01-01

Dental caries is a biofilm-dependent oral disease and Streptococcus mutans is the known primary etiologic agent of dental caries that initiates biofilm formation on tooth surfaces. Although some Lactobacillus strains inhibit biofilm formation of oral pathogenic bacteria, the molecular mechanisms by which lactobacilli inhibit bacterial biofilm formation are not clearly understood. In this study, we demonstrated that Lactobacillus plantarum lipoteichoic acid (Lp.LTA) inhibited the biofilm formation of S. mutans on polystyrene plates, hydroxyapatite discs, and dentin slices without affecting the bacterial growth. Lp.LTA interferes with sucrose decomposition of S. mutans required for the production of exopolysaccharide, which is a main component of biofilm. Lp.LTA also attenuated the biding of fluorescein isothiocyanate-conjugated dextran to S. mutans, which is known to have a high affinity to exopolysaccharide on S. mutans. Dealanylated Lp.LTA did not inhibit biofilm formation of S. mutans implying that D-alanine moieties in the Lp.LTA structure were crucial for inhibition. Collectively, these results suggest that Lp.LTA attenuates S. mutans biofilm formation and could be used to develop effective anticaries agents. PMID:29420616

Kaffir lime leaves extract inhibits biofilm formation by Streptococcus mutans.

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Kooltheat, Nateelak; Kamuthachad, Ludthawun; Anthapanya, Methinee; Samakchan, Natthapon; Sranujit, Rungnapa Pankla; Potup, Pachuen; Ferrante, Antonio; Usuwanthim, Kanchana

2016-04-01

Although kaffir lime has been reported to exhibit antioxidant and antileukemic activity, little is known about the antimicrobial effect of kaffir lime extract. Because Streptococcus mutans has been known to cause biofilm formation, it has been considered the most important causative pathogen of dental caries. Thus, the effective control of its effects on the oral biofilm is the key to the prevention of dental caries. The aims of the present study were to investigate the effect of kaffir lime leaves extract on biofilm formation and its antibacterial activity on S. mutans. We examined the effect of kaffir lime leaves extract on growth and biofilm formation of S. mutans. For the investigation we used a kaffir lime extract with high phenolic content. The minimum inhibitory concentration of the extract was determined by broth microdilution assay. The inhibitory effect of the test substances on biofilm formation was also investigated by biofilm formation assay and qRT-PCR of biofilm formation-associated genes. Kaffir lime leaves extract inhibits the growth of S. mutans, corresponding to the activity of an antibiotic, ampicillin. Formation of biofilm by S. mutans was also inhibited by the extract. These results were confirmed by the down-regulation of genes associated with the biofilm formation. The findings highlight the ability of kaffir lime leaves extract to inhibit S. mutans activity, which may be beneficial in the prevention of biofilm formation on dental surface, reducing dental plaque and decreasing the chance of dental carries. Copyright © 2016 Elsevier Inc. All rights reserved.

Inactivation of the Autolysis-Related Genes lrgB and yycI in Staphylococcus aureus Increases Cell Lysis-Dependent eDNA Release and Enhances Biofilm Development In Vitro and In Vivo.

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Cristiana Ossaille Beltrame

Full Text Available Staphylococcus aureus ica-independent biofilms are multifactorial in nature, and various bacterial proteins have been associated with biofilm development, including fibronectin-binding proteins A and B, protein A, surface protein SasG, proteases, and some autolysins. The role of extracellular DNA (eDNA has also been demonstrated in some S. aureus biofilms. Here, we constructed a Tn551 library, and the screening identified two genes that affected biofilm formation, lrgB and yycI. The repressive effect of both genes on the development of biofilm was also confirmed in knockout strains constructed by allelic recombination. In contrast, the superexpression of either lrgB or yycI by a cadmium-inducible promoter led to a decrease in biofilm accumulation. Indeed, a significant increase in the cell-lysis dependent eDNA release was detected when lrgB or yycI were inactivated, explaining the enhanced biofilm formed by these mutants. In fact, lrgB and yycI genes belong to distinct operons that repress bacterial autolysis through very different mechanisms. LrgB is associated with the synthesis of phage holin/anti-holin analogues, while YycI participates in the activation/repression of the two-component system YycGF (WalKR. Our in vivo data suggest that autolysins activation lead to increased bacterial virulence in the foreign body animal model since a higher number of attached cells was recovered from the implanted catheters inoculated with lrgB or yycI knockout mutants.

Several Virulence Factors of Multidrug-Resistant Staphylococcus aureus Isolates From Hospitalized Patients in Tehran

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Abdolmajid Ghasemian

2015-05-01

Full Text Available Background: Biofilm formation plays an important role in resistance of Staphylococcus aureus isolates; especially multidrug-resistant isolates are a threat to healthcare settings. Objectives: The aims of this study were to detect biofilm formation and presence of several related genes among multidrug-resistant (MDR isolates of Staphylococcus aureus. Patients and Methods: A total Of 209 S. aureus strains were isolated from patients and identified by conventional diagnostic tests. The multidrug-resistant MRSA isolates were detected by antibiotic susceptibility test. The phenotypic biofilm formation was detected by micro-titre tissue plate assay. The polymerase chain reaction (PCR was performed to detect the mecA, Staphylococcal Cassette Chromosome mec (SCCmec types, accessory gene regulatory (agr genes, the icaADBC and several genes encoding staphylococcal surface proteins including clfAB, fnbAB, fib, eno, can, ebps and bbp genes with specific primers. Results: Sixty-four (30.6% isolates were methicillin-resistant, among which thirty-six (56.2% were MDR. These isolates were resistant to amoxicillin, tetracycline, ciprofloxacin, gentamicin, erythromycin and trimethoprim-sulfamethoxazole (except to 6 isolates. All the isolates were susceptible to vancomycin and linezolid. All the MDR-MRSA harbored SCCmec type III. All the MDR- MRSA isolates were strong biofilm producers in the phenotypic test. The majority of MDR- MRSA was belonged to agrI (67%, n = 24, followed by agr II (17%, n = 6, agrIV (11%, n = 4 and agrIII (5.5%, n = 2. The frequency of icaADBC genes were 75% (n = 27, 61% (n = 22, 72% (n = 26 and 72% (n = 26, respectively. Furthermore, the prevalence of clfA, clfB, fnbA, fnbB, fib, can, eno, ebps and bbp genes was 100%, 100%, 67%, 56%, 80%, 63%, 78%, 7% and 0%, respectively. Furthermore, approximately all the MRSA was strong biofilm producers. Conclusions: Multidrug-resistant isolates produced biofilm strongly and the majority harbored most

Organic compounds inhibiting S. epidermidis adhesion and biofilm formation

International Nuclear Information System (INIS)

Qin, Zhiqiang; Zhang, Jingdong; Hu, Yifan; Chi, Qijin; Mortensen, Ninell P.; Qu, Di; Molin, Soren; Ulstrup, Jens

2009-01-01

The formation of biofilms on surfaces of indwelling medical devices is a serious medical problem. Staphylococcus epidermidis is a common pathogen found to colonize implanted devices and as a biofilm is more resistant to the host immune system as well as to antibiotic treatments. Combating S. epidermidis infections by preventing or eradicating biofilm formation of the bacterium is therefore a medically important challenge. We report here a study of biofilm formation of S. epidermidis on solid surfaces using a combination of confocal laser scanning (CLSM) and atomic force microscopy (AFM) in both air and aqueous environments. We have investigated the inhibitory effects of surfaces treated with four organic compounds, two benzoate derivatives denoted as compound 59 and 75 and two carboxamide derivatives denoted as compound 47 and 73, on S. epidermidis adhesion and biofilm formation. All four compounds evoke significant inhibitory effects on the formation of S. epidermidis biofilms with compounds 47 and 73 being most effective. None of the compounds were found to inhibit growth of S. epidermidis in liquid cultures. Bacteria attached to the substrate when exposed to the compounds were not affected indicating that these compounds inhibit initial adhesion. These results suggest a pretreatment for medically implanted surfaces that can prevent the biofilm formation and reduce infection.

Organic compounds inhibiting S. epidermidis adhesion and biofilm formation

Energy Technology Data Exchange (ETDEWEB)

Qin, Zhiqiang [Department of Systems Biology, Technical University of Denmark, Dk-2800 Kgs. Lyngby (Denmark); Key Laboratory of Medical Molecular Virology of Ministry of Education and Public Health, Institute of Medical Microbiology and Institutes of Biomedical Science, Shanghai Medical School of Fudan University, Yi Xue Yuan Road 138, Shanghai 200032 (China); Division of Infectious Diseases, Department of Medicine, Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas Street, Charleston, SC 29425 (United States); Zhang, Jingdong; Hu, Yifan; Chi, Qijin [Department of Chemistry, Building 207, NanoDTU, Technical University of Denmark, DK-2800 Kgs. Lyngby (Denmark); Mortensen, Ninell P. [Department of Systems Biology, Technical University of Denmark, Dk-2800 Kgs. Lyngby (Denmark); Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, TN 37932 (United States); Qu, Di [Key Laboratory of Medical Molecular Virology of Ministry of Education and Public Health, Institute of Medical Microbiology and Institutes of Biomedical Science, Shanghai Medical School of Fudan University, Yi Xue Yuan Road 138, Shanghai 200032 (China); Molin, Soren [Department of Systems Biology, Technical University of Denmark, Dk-2800 Kgs. Lyngby (Denmark); Ulstrup, Jens, E-mail: ju@kemi.dtu.dk [Department of Chemistry, Building 207, NanoDTU, Technical University of Denmark, DK-2800 Kgs. Lyngby (Denmark)

2009-07-15

The formation of biofilms on surfaces of indwelling medical devices is a serious medical problem. Staphylococcus epidermidis is a common pathogen found to colonize implanted devices and as a biofilm is more resistant to the host immune system as well as to antibiotic treatments. Combating S. epidermidis infections by preventing or eradicating biofilm formation of the bacterium is therefore a medically important challenge. We report here a study of biofilm formation of S. epidermidis on solid surfaces using a combination of confocal laser scanning (CLSM) and atomic force microscopy (AFM) in both air and aqueous environments. We have investigated the inhibitory effects of surfaces treated with four organic compounds, two benzoate derivatives denoted as compound 59 and 75 and two carboxamide derivatives denoted as compound 47 and 73, on S. epidermidis adhesion and biofilm formation. All four compounds evoke significant inhibitory effects on the formation of S. epidermidis biofilms with compounds 47 and 73 being most effective. None of the compounds were found to inhibit growth of S. epidermidis in liquid cultures. Bacteria attached to the substrate when exposed to the compounds were not affected indicating that these compounds inhibit initial adhesion. These results suggest a pretreatment for medically implanted surfaces that can prevent the biofilm formation and reduce infection.

Biofilm Formation by Staphylococcus epidermidis on Foldable and Rigid Intraocular Lenses.

Science.gov (United States)

Fazly Bazzaz, Bibi Sedigheh; Jalalzadeh, Monireh; Sanati, Maryam; Zarei-Ghanavati, Syamak; Khameneh, Bahman

2014-05-01

Biofilm formation of Staphylococcus epidermidis is a major etiological factor of inducing device-related infections. The ability of biofilm formation by the S. epidermidis was assessed in vitro on two brands of foldable (hydrophilic) and two brands of rigid (hydrophobic) intraocular lens materials in order to investigate the role of lens material in postoperative endophthalmitis. To ensure reproducibility of biofilm formation on intraocular lenses, two strains of S. epidermidis and three quantification methods were performed. The S. epidermidis strains, DSMZ3270 (biofilm-producer) and ATCC12228 (non-biofilm-producer) were applied. Organisms were cultivated on disks of different brands of foldable hydrophilic Intra Ocular Lens (IOL) made of acrylic (Didar, Iran; (A) and Omni, India; (B)), and rigid hydrophobic IOL made of polymethyl methacrylate (PMMA; Didar, Iran; (C) and Hexavision, France; (D)). Biofilms were stained with crystal violet (CV) dye, which is an index of biofilm formation. The bacterial population was counted after biofilm homogenization. Scanning electron microscopy (SEM) was performed to examine the extent of biofilm formation. Adherence of DSMZ3270 strain on both types of foldable and rigid IOLs, was significantly more than ATCC12228 (P brands of foldable and PMMA IOLs. According to statistical analyses the incubation time influenced the biofilm formation on both types of IOLs which meant that by increasing incubation time, the biofilm formation increased. According to the SEM pictures, biofilm seems to be lysed at 72 hours. These data demonstrated that the attachment of bacteria to hydrophilic acrylic IOLs was more than hydrophobic PMMA ones independent of the brand. According to these results the bacterial strain might have more hydrophilic properties. Augmenting the biomass of biofilm by passing of time demonstrated the key role of time in biofilm formation on the IOL surfaces. The differences between IOL brands in the biofilm formation

Fractal analysis of Xylella fastidiosa biofilm formation

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Moreau, A. L. D.; Lorite, G. S.; Rodrigues, C. M.; Souza, A. A.; Cotta, M. A.

2009-07-01

We have investigated the growth process of Xylella fastidiosa biofilms inoculated on a glass. The size and the distance between biofilms were analyzed by optical images; a fractal analysis was carried out using scaling concepts and atomic force microscopy images. We observed that different biofilms show similar fractal characteristics, although morphological variations can be identified for different biofilm stages. Two types of structural patterns are suggested from the observed fractal dimensions Df. In the initial and final stages of biofilm formation, Df is 2.73±0.06 and 2.68±0.06, respectively, while in the maturation stage, Df=2.57±0.08. These values suggest that the biofilm growth can be understood as an Eden model in the former case, while diffusion-limited aggregation (DLA) seems to dominate the maturation stage. Changes in the correlation length parallel to the surface were also observed; these results were correlated with the biofilm matrix formation, which can hinder nutrient diffusion and thus create conditions to drive DLA growth.

Influence of Streptococcus mutans on Enterococcus faecalis Biofilm Formation

NARCIS (Netherlands)

Deng, Dong Mei; Hoogenkamp, Michel A.; Exterkate, Rob A. M.; Jiang, Lei Meng; van der Sluis, Lucas W. M.; ten Cate, Jacob M.; Crielaard, Wim

Introduction: An important virulence factor of Enterococcus faecalis is its ability to form biofilms. Most studies on biofilm formation have been carried out by using E. faecalis monocultures. Given the polymicrobial nature of root canal infections, it is important to understand biofilm formation of

Investigating Microbial Biofilm Formations on Crustal Rock Substrates

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Weiser, M.; D'Angelo, T.; Carr, S. A.; Orcutt, B.

2017-12-01

Ocean crust hosts microbial life that, in some cases, alter the component rocks as a means of obtaining energy. Variations in crust lithology, included trace metal and mineral content, as well as the chemistry of the fluids circulating through them, provide substrates for some microbes to metabolize, leading to formation of biofilm community structures. Microbes have different parameters for the situations in which they will form biofilms, but they must have some source of energy in excess at the site of biofilm formation for them to become stationary and form the carbohydrate-rich structures connecting the cells to one another and the substrate. Generally, the requirements for microbes to form biofilms on crustal minerals are unclear. We designed two experiments to test (1) mineral preference and biofilm formation rates by natural seawater microbial communities, and (2) biofilm development as a function of phosphate availability for an organism isolated from subseafloor ocean crust. In Experiment 1, we observed that phyric basalt groundmass is preferentially colonized over aphyric basalt or metal sulfides in a shallow water and oxic seawater environment. In experiment 2, tests of the anaerobic heterotroph Thalassospira bacteria isolated from oceanic crustal fluids showed that they preferentially form biofilms, lose motility, and increase exponentially in number over time in higher-PO4 treatments (50 micromolar), including with phosphate-doped basalts, than in treatments with low phosphate concentrations (0.5 micromolar) often found in crustal fluids. These observations suggest phosphate as a main driver of biofilm formation in subsurface crust. Overall, these data suggest that the drivers of microbial biofilm formation on crustal substrates are selective to the substrate conditions, which has important implications for estimating the global biomass of life harbored in oceanic crust.

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

DEFF Research Database (Denmark)

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

2012-01-01

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

Biofilm formation and determination of minimum biofilm eradication concentration of antibiotics in Mycoplasma hyopneumoniae.

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Tassew, Dereje Damte; Mechesso, Abraham Fikru; Park, Na-Hye; Song, Ju-Beom; Shur, Joo-Woon; Park, Seung-Chun

2017-10-20

The study was aimed to investigate biofilm forming ability of Mycoplasma hyopneumoniae and to determine the minimum biofilm eradication concentrations of antibiotics. Biofilm forming ability of six strains of M. hyopneumoniae was examined using crystal violet staining on coverslips. The results demonstrated an apparent line of biofilm growth in 3 of the strains isolated from swine with confirmed cases of enzootic pneumonia. BacLight bacterial viability assay revealed that the majority of the cells were viable after 336 hr of incubation. Moreover, M. hyopneumoniae persists in the biofilm after being exposed to 10 fold higher concentration of antibiotics than the minimum inhibitory concentrations in planktonic cells. To the best of our knowledge, this is the first report of biofilm formation in M. hyopneumoniae. However, comprehensive studies on the mechanisms of biofilm formation are needed to combat swine enzootic pneumonia caused by resistant M. hyopneumoniae.

Effects of substrates on biofilm formation observed by atomic force microscopy

International Nuclear Information System (INIS)

Oh, Y.J.; Lee, N.R.; Jo, W.; Jung, W.K.; Lim, J.S.

2009-01-01

Formation of biofilm is known to be strongly dependent on substrates including topography, materials, and chemical treatment. In this study, a variety of substrates are tested for understanding biofilm formation. Sheets of aluminum, steel, rubber, and polypropylene have been used to examine their effects on formation of Pseudomonas aeruginosa biofilm. In particular, the morphological variation, transition, and adhesiveness of biofilm were investigated through local measurement by atomic force microscopy (AFM). Mechanism of removing biofilm from adhering to substrate is also analyzed, thus the understanding of the mechanism can be potentially useful to prevent the biofilm formation. The results reveal that formation of biofilm can remain on rough surface regardless of substrates in hot water, which may easily induce extra-polymeric substances detachment from bacterial surface. By probing using AFM, local force-distance characterization of extra-cellular materials extracted from the bacteria can exhibit the progress of the biofilm formation and functional complexities.

Increased resistance of contact lens related bacterial biofilms to antimicrobial activity of soft contact lens care solutions

Science.gov (United States)

Szczotka-Flynn, Loretta B.; Imamura, Yoshifumi; Chandra, Jyotsna; Yu, Changping; Mukherjee, Pranab K.; Pearlman, Eric; Ghannoum, Mahmoud A.

2014-01-01

PURPOSE To determine if clinical and reference strains of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus form biofilms on silicone hydrogel contact lenses, and ascertain antimicrobial activities of contact lens care solutions. METHODS Clinical and American Type Culture Collection (ATCC) reference strains of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus were incubated with lotrafilcon A lenses under conditions that facilitate biofilm formation. Biofilms were quantified by quantitative culturing (colony forming units, CFUs), and gross morphology and architecture were evaluated using scanning electron microscopy (SEM) and confocal microscopy. Susceptibilities of the planktonic and biofilm growth phases of the bacteria to five common multipurpose contact lens care solutions and one hydrogen peroxide care solution were assessed. RESULTS P. aeruginosa, S. marcescens, and S. aureus reference and clinical strains formed biofilms on lotrafilcon A silicone hydrogel contact lenses, as dense networks of cells arranged in multiple layers with visible extracellular matrix. The biofilms were resistant to commonly used biguanide preserved multipurpose care solutions. P. aeruginosa and S. aureus biofilms were susceptible to a hydrogen peroxide and a polyquaternium preserved care solution, whereas S. marcescens biofilm was resistant to a polyquaternium preserved care solution but susceptible to hydrogen peroxide disinfection. In contrast, the planktonic forms were always susceptible. CONCLUSIONS P. aeruginosa, S. marcescens, and S. aureus form biofilms on lotrafilcon A contact lenses, which in contrast to planktonic cells, are resistant to the antimicrobial activity of several soft contact lens care products. PMID:19654521

The adhesive properties of the Staphylococcus lugdunensis multifunctional autolysin AtlL and its role in biofilm formation and internalization.

Science.gov (United States)

Hussain, Muzaffar; Steinbacher, Tim; Peters, Georg; Heilmann, Christine; Becker, Karsten

2015-01-01

Although it belongs to the group of coagulase-negative staphylococci, Staphylococcus lugdunensis has been known to cause aggressive courses of native and prosthetic valve infective endocarditis with high mortality similar to Staphylococcus aureus. In contrast to S. aureus, only little is known about the equipment of S. lugdunensis with virulence factors including adhesins and their role in mediating attachment to extracellular matrix and plasma proteins and host cells. In this study, we show that the multifunctional autolysin/adhesin AtlL of S. lugdunensis binds to the extracellular matrix and plasma proteins fibronectin, fibrinogen, and vitronectin as well as to human EA.hy926 endothelial cells. Furthermore, we demonstrate that AtlL also plays an important role in the internalization of S. lugdunensis by eukaryotic cells: The atlL-deficient mutant Mut17 adheres to and becomes internalized by eukaryotic cells to a lesser extent than the isogenic wild-type strain Sl253 and the complemented mutant Mut17 (pCUatlL) shows an increased internalization level in comparison to Mut17. Thus, surface localized AtlL that exhibits a broad binding spectrum also mediates the internalization of S. lugdunensis by eukaryotic cells. We therefore propose an internalization pathway for S. lugdunensis, in which AtlL plays a major role. Investigating the role of AtlL in biofilm formation of S. lugdunensis, Mut17 shows a significantly reduced ability for biofilm formation, which is restored in the complemented mutant. Thus, our data provide evidence for a significant role for AtlL in adherence and internalization processes as well as in biofilm formation of S. lugdunensis. Copyright © 2014 Elsevier GmbH. All rights reserved.

Extract from the fermented soybean product Natto inhibits Vibrio biofilm formation and reduces shrimp mortality from Vibrio harveyi infection.

Science.gov (United States)

Yatip, Pattanan; Nitin Chandra Teja, D; Flegel, Timothy W; Soowannayan, Chumporn

2018-01-01

Many bacteria, including Vibrio pathogens of shrimp, need to colonize and/or form biofilms in hosts or the environment to cause disease. Thus, one possible control strategy for shrimp Vibriosis is biofilm inhibition. With this objective, an extract from the Japanese fermented soybean product, Natto was tested with the luminescent shrimp pathogen Vibrio harveyi (VH) for its ability to inhibit or degrade biofilm and to interfere with cell growth in broth. Natto is a traditional fermentation product of Bacillus subtilis var Natto (BSN1). Using 96 well microtiter plates coated with 0.4% chitosan, we found that biofilm formation by VH was inhibited, while growth in parallel broth cultures was not. When an extract from Natto prepared using BSN1 was mixed with feed for the whiteleg shrimp Penaeus vannamei before immersion challenge with V. harveyi at 10 6  cfu/ml, survival was significantly higher (p≤0.05) than for control shrimp given feed without these additives. Further work done to test whether d-amino acids were involved in biofilm formation as previously reported for B. subtilis, Staphylococus aureus and Pseudomonas aeruginosa gave negative results. In conclusion, we discovered that Natto extract can inhibit Vibrio biofilm formation and that it or BSN1 alone added to shrimp feed can significantly reduce shrimp mortality in immersion challenges with pathogenic VH. This shows some promise for possible application against Vibriosis in shrimp since Natto is generally regarded as safe (GRAS) for human consumption. Copyright © 2017 Elsevier Ltd. All rights reserved.

Organic compounds inhibiting S. epidermidis adhesion and biofilm formation

DEFF Research Database (Denmark)

Qin, Zhiqiang; Zhang, Jingdong; Hu, Yifan

2009-01-01

The formation of biofilms on surfaces of indwelling medical devices is a serious medical problem. Staphylococcus epidermidis is a common pathogen found to colonize implanted devices and as a biofilm is more resistant to the host immune system as well as to antibiotic treatments. Combating S....... epidermidis infections by preventing or eradicating biofilm formation of the bacterium is therefore a medically important challenge. We report here a study of biofilm formation of S. epidermidis on solid surfaces using a combination of confocal laser scanning (CLSM) and atomic force microscopy (AFM) in both...... air and aqueous environments. We have investigated the inhibitory effects of surfaces treated with four organic compounds, two benzoate derivatives denoted as compound 59 and 75 and two carboxamicle derivatives denoted as compound 47 and 73, on S. epidermidis adhesion and biofilm formation. All four...

Systematic Exploration of Natural and Synthetic Flavonoids for the Inhibition of Staphylococcus aureus Biofilms

Science.gov (United States)

Manner, Suvi; Skogman, Malena; Goeres, Darla; Vuorela, Pia; Fallarero, Adyary

2013-01-01

When single-cell (or suspended) bacteria switch into the biofilm lifestyle, they become less susceptible to antimicrobials, imposing the need for anti-biofilms research. Flavonoids are among the most extensively studied natural compounds with an unprecedented amount of bioactivity claims. Most studies focus on the antibacterial effects against suspended cells; fewer reports have researched their anti-biofilm properties. Here, a high throughput phenotypic platform was utilized to screen for the inhibitory activity of 500 flavonoids, including natural and synthetic derivatives, against Staphylococcus aureus biofilms. Since discrepancies among results from earlier antibacterial studies on flavonoids had been noted, the current study aimed to minimize sources of variations. After the first screen, flavonoids were classified as inactive (443), moderately active (47) or highly active (10). Further, exclusion criteria combining bioactivity and selectivity identified two synthetic flavans as the most promising. The body of data reported here serves three main purposes. First, it offers an improved methodological workflow for anti-biofilm screens of chemical libraries taking into account the (many times ignored) connections between anti-biofilm and antibacterial properties. This is particularly relevant for the study of flavonoids and other natural products. Second, it provides a large and freely available anti-biofilm bioactivity dataset that expands the knowledge on flavonoids and paves the way for future structure-activity relationship studies and structural optimizations. Finally, it identifies two new flavans that can successfully act on biofilms, as well as on suspended bacteria and represent more feasible antibacterial candidates. PMID:24071942

Patterned biofilm formation reveals a mechanism for structural heterogeneity in bacterial biofilms.

Science.gov (United States)

Gu, Huan; Hou, Shuyu; Yongyat, Chanokpon; De Tore, Suzanne; Ren, Dacheng

2013-09-03

Bacterial biofilms are ubiquitous and are the major cause of chronic infections in humans and persistent biofouling in industry. Despite the significance of bacterial biofilms, the mechanism of biofilm formation and associated drug tolerance is still not fully understood. A major challenge in biofilm research is the intrinsic heterogeneity in the biofilm structure, which leads to temporal and spatial variation in cell density and gene expression. To understand and control such structural heterogeneity, surfaces with patterned functional alkanthiols were used in this study to obtain Escherichia coli cell clusters with systematically varied cluster size and distance between clusters. The results from quantitative imaging analysis revealed an interesting phenomenon in which multicellular connections can be formed between cell clusters depending on the size of interacting clusters and the distance between them. In addition, significant differences in patterned biofilm formation were observed between wild-type E. coli RP437 and some of its isogenic mutants, indicating that certain cellular and genetic factors are involved in interactions among cell clusters. In particular, autoinducer-2-mediated quorum sensing was found to be important. Collectively, these results provide missing information that links cell-to-cell signaling and interaction among cell clusters to the structural organization of bacterial biofilms.

Ginger Extract Inhibits Biofilm Formation by Pseudomonas aeruginosa PA14

Science.gov (United States)

Kim, Han-Shin; Park, Hee-Deung

2013-01-01

Bacterial biofilm formation can cause serious problems in clinical and industrial settings, which drives the development or screening of biofilm inhibitors. Some biofilm inhibitors have been screened from natural products or modified from natural compounds. Ginger has been used as a medicinal herb to treat infectious diseases for thousands of years, which leads to the hypothesis that it may contain chemicals inhibiting biofilm formation. To test this hypothesis, we evaluated ginger’s ability to inhibit Pseudomonas aeruginosa PA14 biofilm formation. A static biofilm assay demonstrated that biofilm development was reduced by 39–56% when ginger extract was added to the culture. In addition, various phenotypes were altered after ginger addition of PA14. Ginger extract decreased production of extracellular polymeric substances. This finding was confirmed by chemical analysis and confocal laser scanning microscopy. Furthermore, ginger extract formed noticeably less rugose colonies on agar plates containing Congo red and facilitated swarming motility on soft agar plates. The inhibition of biofilm formation and the altered phenotypes appear to be linked to a reduced level of a second messenger, bis-(3′-5′)-cyclic dimeric guanosine monophosphate. Importantly, ginger extract inhibited biofilm formation in both Gram-positive and Gram-negative bacteria. Also, surface biofilm cells formed with ginger extract detached more easily with surfactant than did those without ginger extract. Taken together, these findings provide a foundation for the possible discovery of a broad spectrum biofilm inhibitor. PMID:24086697

Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology

Science.gov (United States)

Gutiérrez, Diana; Hidalgo-Cantabrana, Claudio; Rodríguez, Ana; García, Pilar

2016-01-01

Bacteria found in diverse ecosystems grow in a community of aggregated cells that favors their survival and colonization. Different extracellular polymeric substances are used to entrap this multispecies community forming a biofilm, which can be associated to biotic and abiotic surfaces. This widespread and successful way of bacterial life, however, can lead to negative effects for human activity since many pathogen and spoiling bacteria form biofilms which are not easy to eradicate. Therefore, the search for novel anti-biofilm bio-active molecules is a very active research area for which simple, reliable, and fast screening methods are demanded. In this work we have successfully validated an impedance-based method, initially developed for the study of adherent eukaryotic cells, to monitor the formation of single-species biofilms of three model bacteria in real time. The xCelligence real time cell analyzer (RTCA) equipment uses specific microtiter E-plates coated with gold-microelectrodes that detect the attachment of adherent cells, thus modifying the impedance signal. In the current study, this technology allowed the distinction between biofilm-producers and non-producers of Staphylococcus aureus and Staphylococcus epidermidis, as well as the formation of Streptococcus mutans biofilms only when sucrose was present in the culture medium. Besides, different impedance values permitted discrimination among the biofilm-producing strains tested regardless of the nature of the polymeric biofilm matrix. Finally, we have continuously monitored the inhibition of staphylococcal biofilm formation by the bacteriophage phi-IPLA7 and the bacteriophage-encoded endolysin LysH5, as well as the removal of a preformed biofilm by this last antimicrobial treatment. Results observed with the impedance-based method showed high correlation with those obtained with standard approaches, such as crystal violet staining and bacteria enumeration, as well as with those obtained upon other

Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology.

Directory of Open Access Journals (Sweden)

Diana Gutiérrez

Full Text Available Bacteria found in diverse ecosystems grow in a community of aggregated cells that favors their survival and colonization. Different extracellular polymeric substances are used to entrap this multispecies community forming a biofilm, which can be associated to biotic and abiotic surfaces. This widespread and successful way of bacterial life, however, can lead to negative effects for human activity since many pathogen and spoiling bacteria form biofilms which are not easy to eradicate. Therefore, the search for novel anti-biofilm bio-active molecules is a very active research area for which simple, reliable, and fast screening methods are demanded. In this work we have successfully validated an impedance-based method, initially developed for the study of adherent eukaryotic cells, to monitor the formation of single-species biofilms of three model bacteria in real time. The xCelligence real time cell analyzer (RTCA equipment uses specific microtiter E-plates coated with gold-microelectrodes that detect the attachment of adherent cells, thus modifying the impedance signal. In the current study, this technology allowed the distinction between biofilm-producers and non-producers of Staphylococcus aureus and Staphylococcus epidermidis, as well as the formation of Streptococcus mutans biofilms only when sucrose was present in the culture medium. Besides, different impedance values permitted discrimination among the biofilm-producing strains tested regardless of the nature of the polymeric biofilm matrix. Finally, we have continuously monitored the inhibition of staphylococcal biofilm formation by the bacteriophage phi-IPLA7 and the bacteriophage-encoded endolysin LysH5, as well as the removal of a preformed biofilm by this last antimicrobial treatment. Results observed with the impedance-based method showed high correlation with those obtained with standard approaches, such as crystal violet staining and bacteria enumeration, as well as with those

Monitoring in Real Time the Formation and Removal of Biofilms from Clinical Related Pathogens Using an Impedance-Based Technology.

Science.gov (United States)

Gutiérrez, Diana; Hidalgo-Cantabrana, Claudio; Rodríguez, Ana; García, Pilar; Ruas-Madiedo, Patricia

2016-01-01

Bacteria found in diverse ecosystems grow in a community of aggregated cells that favors their survival and colonization. Different extracellular polymeric substances are used to entrap this multispecies community forming a biofilm, which can be associated to biotic and abiotic surfaces. This widespread and successful way of bacterial life, however, can lead to negative effects for human activity since many pathogen and spoiling bacteria form biofilms which are not easy to eradicate. Therefore, the search for novel anti-biofilm bio-active molecules is a very active research area for which simple, reliable, and fast screening methods are demanded. In this work we have successfully validated an impedance-based method, initially developed for the study of adherent eukaryotic cells, to monitor the formation of single-species biofilms of three model bacteria in real time. The xCelligence real time cell analyzer (RTCA) equipment uses specific microtiter E-plates coated with gold-microelectrodes that detect the attachment of adherent cells, thus modifying the impedance signal. In the current study, this technology allowed the distinction between biofilm-producers and non-producers of Staphylococcus aureus and Staphylococcus epidermidis, as well as the formation of Streptococcus mutans biofilms only when sucrose was present in the culture medium. Besides, different impedance values permitted discrimination among the biofilm-producing strains tested regardless of the nature of the polymeric biofilm matrix. Finally, we have continuously monitored the inhibition of staphylococcal biofilm formation by the bacteriophage phi-IPLA7 and the bacteriophage-encoded endolysin LysH5, as well as the removal of a preformed biofilm by this last antimicrobial treatment. Results observed with the impedance-based method showed high correlation with those obtained with standard approaches, such as crystal violet staining and bacteria enumeration, as well as with those obtained upon other

Molecular mechanisms involved in Bacillus subtilis biofilm formation

Science.gov (United States)

Mielich-Süss, Benjamin; Lopez, Daniel

2014-01-01

Summary Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil-dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programs of cellular specialization and cell-cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis. In particular, a special emphasis is placed on summarizing the most recent discoveries in the field and integrating them into the general view of these truly sophisticated microbial communities. PMID:24909922

Efficacy of Linezolid and Fosfomycin in Catheter-Related Biofilm Infection Caused by Methicillin-Resistant Staphylococcus aureus

Science.gov (United States)

Chai, Dong; Liu, Xu; Wang, Rui; Bai, Yan; Cai, Yun

2016-01-01

As long-standing clinical problems, catheter-related infections and other chronic biofilm infections are more difficult to treat due to the high antibiotic resistance of biofilm. Therefore, new treatments are needed for more effective bacteria clearance. In this study, we evaluated the antibacterial activities of several common antibiotics alone and their combinations against biofilm-embedded methicillin-resistant staphylococcus aureus (MRSA) infections, both in vitro and in vivo. In brief, fosfomycin, levofloxacin, and rifampin alone or in combination with linezolid were tested in vitro against planktonic and biofilm-embedded MRSA infection in three MRSA stains. The synergistic effects between linezolid and the other three antibiotics were assessed by fractional inhibitory concentration index (FICI) and time-kill curves, where the combination of linezolid plus fosfomycin showed the best synergistic effect in all strains. For further evaluation in vivo, we applied the combination of linezolid and fosfomycin in a catheter-related biofilm rat model and found that viable bacteria counts in biofilm were significantly reduced after treatment (P linezolid and fosfomycin treatment had improved therapeutic effects on biofilm-embedded MRSA infection both in vitro and in vivo, which provided important basis for new clinical therapy development. PMID:27366751

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent.

Science.gov (United States)

Schuch, Raymond; Khan, Babar K; Raz, Assaf; Rotolo, Jimmy A; Wittekind, Michael

2017-07-01

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC 90 ) value of ≤0.25 μg/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes , and Streptococcus agalactiae were also sensitive to disruption, with MBEC 90 values ranging from 0.25 to 8 μg/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component. Copyright © 2017 American Society for Microbiology.

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent

KAUST Repository

Schuch, Raymond; Khan, Babar Khalid; Raz, Assaf; Rotolo, Jimmy A.; Wittekind, Michael

2017-01-01

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of <= 0.25 mu g/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 mu g/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

Bacteriophage Lysin CF-301, a Potent Antistaphylococcal Biofilm Agent

KAUST Repository

Schuch, Raymond

2017-05-02

Biofilms pose a unique therapeutic challenge because of the antibiotic tolerance of constituent bacteria. Treatments for biofilm-based infections represent a major unmet medical need, requiring novel agents to eradicate mature biofilms. Our objective was to evaluate bacteriophage lysin CF-301 as a new agent to target Staphylococcus aureus biofilms. We used minimum biofilm-eradicating concentration (MBEC) assays on 95 S. aureus strains to obtain a 90% MBEC (MBEC90) value of <= 0.25 mu g/ml for CF-301. Mature biofilms of coagulase-negative staphylococci, Streptococcus pyogenes, and Streptococcus agalactiae were also sensitive to disruption, with MBEC90 values ranging from 0.25 to 8 mu g/ml. The potency of CF-301 was demonstrated against S. aureus biofilms formed on polystyrene, glass, surgical mesh, and catheters. In catheters, CF-301 removed all biofilm within 1 h and killed all released bacteria by 6 h. Mixed-species biofilms, formed by S. aureus and Staphylococcus epidermidis on several surfaces, were removed by CF-301, as were S. aureus biofilms either enriched for small-colony variants (SCVs) or grown in human synovial fluid. The antibacterial activity of CF-301 was further demonstrated against S. aureus persister cells in exponential-phase and stationary-phase populations. Finally, the antibiofilm activity of CF-301 was greatly improved in combinations with the cell wall hydrolase lysostaphin when tested against a range of S. aureus strains. In all, the data show that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria, and, as such, it may be an efficient new agent for treating staphylococcal infections with a biofilm component.

Origanum vulgare subsp. hirtum essential oil prevented biofilm formation and showed antibacterial activity against planktonic and sessile bacterial cells.

Science.gov (United States)

Schillaci, Domenico; Napoli, Edoardo Marco; Cusimano, Maria Grazia; Vitale, Maria; Ruberto, Andgiuseppe

2013-10-01

Essential oils from six different populations of Origanum vulgare subsp. hirtum were compared for their antibiofilm properties. The six essential oils (A to F) were characterized by a combination of gas chromatography with flame ionization detector and gas chromatography with mass spectrometer detector analyses. All oils showed weak activity against the planktonic form of a group of Staphylococcus aureus strains and against a Pseudomonas aeruginosa ATCC 15442 reference strain. The ability to inhibit biofilm formation was investigated at sub-MIC levels of 200, 100, and 50 m g/ml by staining sessile cells with safranin. Sample E showed the highest average effectiveness against all tested strains at 50 m g/ml and had inhibition percentages ranging from 30 to 52%. In the screening that used preformed biofilm from the reference strain P. aeruginosa, essential oils A through E were inactive at 200 m g/ml; F was active with a percentage of inhibition equal to 53.2%. Oregano essential oil can inhibit the formation of biofilms of various food pathogens and food spoilage organisms.

Motility of Pseudomonas aeruginosa contributes to SOS-inducible biofilm formation.

Science.gov (United States)

Chellappa, Shakinah T; Maredia, Reshma; Phipps, Kara; Haskins, William E; Weitao, Tao

2013-12-01

DNA-damaging antibiotics such as ciprofloxacin induce biofilm formation and the SOS response through autocleavage of SOS-repressor LexA in Pseudomonas aeruginosa. However, the biofilm-SOS connection remains poorly understood. It was investigated with 96-well and lipid biofilm assays. The effects of ciprofloxacin were examined on biofilm stimulation of the SOS mutant and wild-type strains. The stimulation observed in the wild-type in which SOS was induced was reduced in the mutant in which LexA was made non-cleavable (LexAN) and thus SOS non-inducible. Therefore, the stimulation appeared to involve SOS. The possible mechanisms of inducible biofilm formation were explored by subproteomic analysis of outer membrane fractions extracted from biofilms. The data predicted an inhibitory role of LexA in flagellum function. This premise was tested first by functional and morphological analyses of flagellum-based motility. The flagellum swimming motility decreased in the LexAN strain treated with ciprofloxacin. Second, the motility-biofilm assay was performed, which tested cell migration and biofilm formation. The results showed that wild-type biofilm increased significantly over the LexAN. These results suggest that LexA repression of motility, which is the initial event in biofilm development, contributes to repression of SOS-inducible biofilm formation. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Denitrification-derived nitric oxide modulates biofilm formation in Azospirillum brasilense.

Science.gov (United States)

Arruebarrena Di Palma, Andrés; Pereyra, Cintia M; Moreno Ramirez, Lizbeth; Xiqui Vázquez, María L; Baca, Beatriz E; Pereyra, María A; Lamattina, Lorenzo; Creus, Cecilia M

2013-01-01

Azospirillum brasilense is a rhizobacterium that provides beneficial effects on plants when they colonize roots. The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with surfaces in response to appropriate signals. Nitric oxide (NO) is a signaling molecule implicated in numerous processes in bacteria, including biofilm formation or dispersion, depending on genera and lifestyle. Azospirillum brasilense Sp245 produces NO by denitrification having a role in root growth promotion. We analyzed the role of endogenously produced NO on biofilm formation in A. brasilense Sp245 and in a periplasmic nitrate reductase mutant (napA::Tn5; Faj164) affected in NO production. Cells were statically grown in media with nitrate or ammonium as nitrogen sources and examined for biofilm formation using crystal violet and by confocal laser microscopy. Both strains formed biofilms, but the mutant produced less than half compared with the wild type in nitrate medium showing impaired nitrite production in this condition. NO measurements in biofilm confirmed lower values in the mutant strain. The addition of a NO donor showed that NO influences biofilm formation in a dose-dependent manner and reverses the mutant phenotype, indicating that Nap positively regulates the formation of biofilm in A. brasilense Sp245. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Synergy in biofilm formation between Fusobacterium nucleatum and Prevotella species.

Science.gov (United States)

Okuda, Tamaki; Kokubu, Eitoyo; Kawana, Tomoko; Saito, Atsushi; Okuda, Katsuji; Ishihara, Kazuyuki

2012-02-01

The formation of biofilm by anaerobic, Gram-negative bacteria in the subgingival crevice plays an important role in the development of chronic periodontitis. The aim of this study was to characterize the role of coaggregation between Fusobacterium nucleatum and Prevotella species in biofilm formation. Coaggregation between F. nucleatum and Prevotella species was determined by visual assay. Effect of co-culture of the species on biofilm formation was assessed by crystal violet staining. Effect of soluble factor on biofilm formation was also examined using culture supernatant and two-compartment co-culture separated by a porous membrane. Production of autoinducer-2 (AI-2) by the organisms was evaluated using Vibrio harveyi BB170. Cells of all F. nucleatum strains coaggregated with Prevotella intermedia or Prevotella nigrescens with a score of 1-4. Addition of ethylenediamine tetraacetic acid or l-lysine inhibited coaggregation. Coaggregation disappeared after heating of P. intermedia or P. nigrescens cells, or Proteinase K treatment of P. nigrescens cells. Co-culture of F. nucleatum ATCC 25586 with P. intermedia or P. nigrescens strains increased biofilm formation compared with single culture (p culture with culture supernatant of these strains, however, did not enhance biofilm formation by F. nucleatum. Production of AI-2 in Prevotella species was not related to enhancement of biofilm formation by F. nucleatum. These findings indicate that physical contact by coaggregation of F. nucleatum strains with P. intermedia or P. nigrescens plays a key role in the formation of biofilm by these strains. Copyright © 2011 Elsevier Ltd. All rights reserved.

Biofilm Formation on Dental Restorative and Implant Materials

NARCIS (Netherlands)

Busscher, H. J.; Rinastiti, M.; Siswomihardjo, W.; van der Mei, H. C.

Biomaterials for the restoration of oral function are prone to biofilm formation, affecting oral health. Oral bacteria adhere to hydrophobic and hydrophilic surfaces, but due to fluctuating shear, little biofilm accumulates on hydrophobic surfaces in vivo. More biofilm accumulates on rough than on

Candida albicans survival and biofilm formation under starvation conditions.

Science.gov (United States)

Ning, Y; Hu, X; Ling, J; Du, Y; Liu, J; Liu, H; Peng, Z

2013-01-01

To investigate the survival and biofilm formation capacity of Candida albicans in starvation and under anaerobic conditions. Candida albicans growth and survival were monitored in vitro for up to 8 months. Fungal suspensions from late exponential, stationary and starvation phases were incubated on human dentine, polystyrene and glass slides. Scanning electron microscopy (SEM) was used to observe the process of biofilm formation. 2,3-bis(2-Methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide inner salt (XTT) reduction assay was performed to quantify the biofilm formation capability, and confocal laser scanning microscopy (CLSM) was used to study and make semi-quantitative comparisons of the ultrastructure of biofilms formed on human dentine. 'XTT bioactivity' and 'COMSTAT results' were analysed by two-way analysis of variance (ANOVA) and one-way ANOVA, respectively. Candida albicans survived for over six months. SEM demonstrated that starving C. albicans produced mature biofilms on different substrata. C. albicans of the same growth phase incubated on human dentine displayed significantly higher biofilm formation capability than on polystyrene or glass slides (P roughness coefficient and surface/volume ratio (P < 0.05). Candida albicans cells can survive and form biofilms in anaerobic and nutrient-limited conditions and may pose a treatment challenge. © 2012 International Endodontic Journal.

Alginate production affects Pseudomonas aeruginosa biofilm development and architecture, but is not essential for biofilm formation

DEFF Research Database (Denmark)

Stapper, A.P.; Narasimhan, G.; Oman, D.E.

2004-01-01

of their biofilm formation using confocal laser scanning microscopy. Biofilm Image Processing (BIP) and Community Statistics (COMSTAT) software programs were used to provide quantitative measurements of the two-dimensional biofilm images. All three strains formed distinguishable biofilm architectures, indicating...

Genetic Control of Conventional and Pheromone-Stimulated Biofilm Formation in Candida albicans

Science.gov (United States)

Lin, Ching-Hsuan; Kabrawala, Shail; Fox, Emily P.; Nobile, Clarissa J.; Johnson, Alexander D.; Bennett, Richard J.

2013-01-01

Candida albicans can stochastically switch between two phenotypes, white and opaque. Opaque cells are the sexually competent form of C. albicans and therefore undergo efficient polarized growth and mating in the presence of pheromone. In contrast, white cells cannot mate, but are induced – under a specialized set of conditions – to form biofilms in response to pheromone. In this work, we compare the genetic regulation of such “pheromone-stimulated” biofilms with that of “conventional” C. albicans biofilms. In particular, we examined a network of six transcriptional regulators (Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1) that mediate conventional biofilm formation for their potential roles in pheromone-stimulated biofilm formation. We show that four of the six transcription factors (Bcr1, Brg1, Rob1, and Tec1) promote formation of both conventional and pheromone-stimulated biofilms, indicating they play general roles in cell cohesion and biofilm development. In addition, we identify the master transcriptional regulator of pheromone-stimulated biofilms as C. albicans Cph1, ortholog of Saccharomyces cerevisiae Ste12. Cph1 regulates mating in C. albicans opaque cells, and here we show that Cph1 is also essential for pheromone-stimulated biofilm formation in white cells. In contrast, Cph1 is dispensable for the formation of conventional biofilms. The regulation of pheromone- stimulated biofilm formation was further investigated by transcriptional profiling and genetic analyses. These studies identified 196 genes that are induced by pheromone signaling during biofilm formation. One of these genes, HGC1, is shown to be required for both conventional and pheromone-stimulated biofilm formation. Taken together, these observations compare and contrast the regulation of conventional and pheromone-stimulated biofilm formation in C. albicans, and demonstrate that Cph1 is required for the latter, but not the former. PMID:23637598

Neutrophil extracellular trap formation in supragingival biofilms.

Science.gov (United States)

Hirschfeld, Josefine; Dommisch, Henrik; Skora, Philipp; Horvath, Gabor; Latz, Eicke; Hoerauf, Achim; Waller, Tobias; Kawai, Toshihisa; Jepsen, Søren; Deschner, James; Bekeredjian-Ding, Isabelle

2015-01-01

Oral biofilms are the causative agents of the highly prevalent oral diseases periodontitis and caries. Additionally, the host immune response is thought to play a critical role in disease onset. Neutrophils are known to be a key host response factor to bacterial challenge on host surfaces. Release of neutrophil extracellular traps (NETs) as a novel antimicrobial defense strategy has gained increasing attention in the past years. Here, we investigated the influx of neutrophils into the dental plaque and the ability of oral bacteria to trigger intra-biofilm release of NETs and intracellular proteins. Supragingival biofilms and whole saliva were sampled from systemically healthy subjects participating in an experimental gingivitis study. Biofilms were analysed by immunofluorescence followed by confocal and fluorescence microscopy. Moreover, concentrations of cytokines and immune-associated proteins in biofilm suspensions and saliva were assessed by ELISA. Neutrophils obtained from blood were stimulated with twelve bacterial species isolated from cultured biofilms or with lipopolysaccharide to monitor NET formation. Neutrophils, NETs, neutrophil-associated proteins (myeloperoxidase, elastase-2, cathepsin G, cathelicidin LL-37), interleukin-8, interleukin-1β and tumor necrosis factor were detected within plaque samples and saliva. All tested bacterial species as well as the polymicrobial samples isolated from the plaque of each donor induced release of NETs and interleukin-8. The degree of NET formation varied among different subjects and did not correlate with plaque scores or clinical signs of local inflammation. Our findings indicate that neutrophils are attracted towards dental biofilms, in which they become incorporated and where they are stimulated by microbes to release NETs and immunostimulatory proteins. Thus, neutrophils and NETs may be involved in host biofilm control, although their specific role needs to be further elucidated. Moreover, inter

Photodynamic inactivation of Staphylococcus aureus and Escherichia coli biofilms by malachite green and phenothiazine dyes: an in vitro study.

Science.gov (United States)

Vilela, Simone Furgeri Godinho; Junqueira, Juliana Campos; Barbosa, Junia Oliveira; Majewski, Marta; Munin, Egberto; Jorge, Antonio Olavo Cardoso

2012-06-01

The organization of biofilms in the oral cavity gives them added resistance to antimicrobial agents. The action of phenothiazinic photosensitizers on oral biofilms has already been reported. However, the action of the malachite green photosensitizer upon biofilm-organized microorganisms has not been described. The objective of the present work was to compare the action of malachite green with the phenothiazinic photosensitizers (methylene blue and toluidine blue) on Staphylococcus aureus and Escherichia coli biofilms. The biofilms were grown on sample pieces of acrylic resin and subjected to photodynamic therapy using a 660-nm diode laser and photosensitizer concentrations ranging from 37.5 to 3000 μM. After photodynamic therapy, cells from the biofilms were dispersed in a homogenizer and cultured in Brain Heart Infusion broth for quantification of colony-forming units per experimental protocol. For each tested microorganism, two control groups were maintained: one exposed to the laser radiation without the photosensitizer (L+PS-) and other treated with the photosensitizer without exposure to the red laser light (L-PS+). The results were subjected to descriptive statistical analysis. The best results for S. aureus and E. coli biofilms were obtained with photosensitizer concentrations of approximately 300 μM methylene blue, with microbial reductions of 0.8-1.0 log(10); 150 μM toluidine blue, with microbial reductions of 0.9-1.0 log(10); and 3000 μM malachite green, with microbial reductions of 1.6-4.0 log(10). Greater microbial reduction was achieved with the malachite green photosensitizer when used at higher concentrations than those employed for the phenothiazinic dyes. Copyright © 2011 Elsevier Ltd. All rights reserved.

Enhancement of Biofilm Formation on Pyrite by Sulfobacillus thermosulfidooxidans

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Qian Li

2016-07-01

Full Text Available Bioleaching is the mobilization of metal cations from insoluble ores by microorganisms. Biofilms can enhance this process. Since Sulfobacillus often appears in leaching heaps or reactors, this genus has aroused attention. In this study, biofilm formation and subsequent pyrite dissolution by the Gram-positive, moderately thermophilic acidophile Sulfobacillus thermosulfidooxidans were investigated. Five strategies, including adjusting initial pH, supplementing an extra energy source or ferric ions, as well as exchanging exhausted medium with fresh medium, were tested for enhancement of its biofilm formation. The results show that regularly exchanging exhausted medium leads to a continuous biofilm development on pyrite. By this way, multiply layered biofilms were observed on pyrite slices, while only monolayer biofilms were visible on pyrite grains. In addition, biofilms were proven to be responsible for pyrite leaching in the early stages.

Helicobacter pylori-coccoid forms and biofilm formation

DEFF Research Database (Denmark)

Andersen, Leif Percival; Rasmussen, Lone

2009-01-01

be detected by PCR in water supplies. There is no substantial evidence for viable H. pylori persisting in water supplies. Epidemiological studies suggest that environmental water is a risk factor for H. pylori infection when compared with tap water, and formation of H. pylori biofilm cannot be excluded....... Helicobacter pylori does not seem to take part in biofilm formation in the oral cavity even though the bacterium may be detected....

Inhibitory effect of Ti-Ag alloy on artificial biofilm formation.

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Nakajo, Kazuko; Takahashi, Masatoshi; Kikuchi, Masafumi; Takada, Yukyo; Okuno, Osamu; Sasaki, Keiichi; Takahashi, Nobuhiro

2014-01-01

Titanium-silver (Ti-Ag) alloy has been improved for machinability and mechanical properties, but its anti-biofilm properties have not been elucidated yet. Thus, this study aimed to evaluate the effects of Ti-Ag alloy on biofilm formation and bacterial viability in comparison with pure Ti, pure Ag and silver-palladium (Ag-Pd) alloy. Biofilm formation on the metal plates was evaluated by growing Streptococcus mutans and Streptococcus sobrinus in the presence of metal plates. Bactericidal activity was evaluated using a film contact method. There were no significant differences in biofilm formation between pure Ti, pure Ag and Ag-Pd alloy, while biofilm amounts on Ti-20% Ag and Ti-25% Ag alloys were significantly lower (p<0.05). In addition, Ti-Ag alloys and pure Ti were not bactericidal, although pure Ag and Ag-Pd alloy killed bacteria. These results suggest that Ti-20% Ag and Ti-25% Ag alloys are suitable for dental material that suppresses biofilm formation without disturbing healthy oral microflora.

How can in vitro models best reflect in vivo Staphylococcus biofilms?

DEFF Research Database (Denmark)

Meyer, Rikke Louise

In vitro biofilm models are the basis for most studies of biofilm biology because they enable high-throughput analyses without the expenditure of animals. But how do we ensure that what we learn from in vitro studies is relevant in vivo? Biofilms grown in standard laboratory media do not interact...... with host factors and are thus profoundly different from in vivo biofilms. We therefore need in vitro models that are as in vivo-like as possible. We investigated how the addition of divalent cations and human plasma to brain heart infusion broth affected biofilm formation by Staphylococcus aureus...

Dual Roles of FmtA in Staphylococcus aureus Cell Wall Biosynthesis and Autolysis

Science.gov (United States)

Qamar, Aneela

2012-01-01

The fmtA gene is a member of the Staphylococcus aureus core cell wall stimulon. The FmtA protein interacts with β-lactams through formation of covalent species. Here, we show that FmtA has weak d-Ala-d-Ala-carboxypeptidase activity and is capable of covalently incorporating C14-Gly into cell walls. The fluorescence microscopy study showed that the protein is localized to the cell division septum. Furthermore, we show that wall teichoic acids interact specifically with FmtA and mediate recruitment of FmtA to the S. aureus cell wall. Subjection of S. aureus to FmtA concentrations of 0.1 μM or less induces autolysis and biofilm production. This effect requires the presence of wall teichoic acids. At FmtA concentrations greater than 0.2 μM, autolysis and biofilm formation in S. aureus are repressed and growth is enhanced. Our findings indicate dual roles of FmtA in S. aureus growth, whereby at low concentrations, FmtA may modulate the activity of the major autolysin (AtlA) of S. aureus and, at high concentrations, may participate in synthesis of cell wall peptidoglycan. These two roles of FmtA may reflect dual functions of FmtA in the absence and presence of cell wall stress, respectively. PMID:22564846

Spaceflight promotes biofilm formation by Pseudomonas aeruginosa.

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Wooseong Kim

Full Text Available Understanding the effects of spaceflight on microbial communities is crucial for the success of long-term, manned space missions. Surface-associated bacterial communities, known as biofilms, were abundant on the Mir space station and continue to be a challenge on the International Space Station. The health and safety hazards linked to the development of biofilms are of particular concern due to the suppression of immune function observed during spaceflight. While planktonic cultures of microbes have indicated that spaceflight can lead to increases in growth and virulence, the effects of spaceflight on biofilm development and physiology remain unclear. To address this issue, Pseudomonas aeruginosa was cultured during two Space Shuttle Atlantis missions: STS-132 and STS-135, and the biofilms formed during spaceflight were characterized. Spaceflight was observed to increase the number of viable cells, biofilm biomass, and thickness relative to normal gravity controls. Moreover, the biofilms formed during spaceflight exhibited a column-and-canopy structure that has not been observed on Earth. The increase in the amount of biofilms and the formation of the novel architecture during spaceflight were observed to be independent of carbon source and phosphate concentrations in the media. However, flagella-driven motility was shown to be essential for the formation of this biofilm architecture during spaceflight. These findings represent the first evidence that spaceflight affects community-level behaviors of bacteria and highlight the importance of understanding how both harmful and beneficial human-microbe interactions may be altered during spaceflight.

68Ga-labeled phage-display selected peptides as tracers for positron emission tomography imaging of Staphylococcus aureus biofilm-associated infections: Selection, radiolabelling and preliminary biological evaluation

International Nuclear Information System (INIS)

Nielsen, Karin M.; Kyneb, Majbritt H.; Alstrup, Aage K.O.; Jensen, Jakob J.; Bender, Dirk; Schønheyder, Henrik C.; Afzelius, Pia; Nielsen, Ole L.; Jensen, Svend B.

2016-01-01

Introduction: Staphylococcus aureus is a major cause of skin and deep-sited infections, often associated with the formation of biofilms. Early diagnosis and initiated therapy is essential to prevent disease progression and to reduce complications that can be serious. Imaging techniques are helpful combining anatomical with functional data in order to describe and characterize site, extent and activity of the disease. The purpose of the study was to identify and 68 Ga-label peptides with affinity for S. aureus biofilm and evaluate their potential as bacteria-specific positron emission tomography (PET) imaging agents. Methods: Phage-displayed dodecapeptides were selected using an in vitro grown S. aureus biofilm as target. One cyclic (A8) and two linear (A9, A11) dodecapeptides were custom synthesized with 1,4,7,10-tetraazacyclododecane-N,N′,N″,N‴-tetraacetic acid (DOTA) conjugated via a lysine linker (K), and for A11 also a glycine–serine–glycine spacer (GSG). The 68 Ga-labeling of A8-K-DOTA, A9-K-DOTA, and A11-GSGK-DOTA were optimized and in vitro bacterial binding was evaluated for 68 Ga-A9-K-DOTA and 68 Ga-A11-GSGK-DOTA. Stability of 68 Ga-A9-K-DOTA was studied in vitro in human serum, while the in vivo plasma stability was analyzed in mice and pigs. Additionally, the whole-body distribution kinetics of 68 Ga-A9-K-DOTA was measured in vivo by PET imaging of pigs and ex vivo in excised mice tissues. Results: The 68 Ga-A9-K-DOTA and 68 Ga-A11-GSGK-DOTA remained stable in product formulation, whereas 68 Ga-A8-K-DOTA was unstable. The S. aureus binding of 68 Ga-A11-GSGK-DOTA and 68 Ga-A9-K-DOTA was observed in vitro, though blocking of the binding was not possible by excess of cold peptide. The 68 Ga-A9-K-DOTA was degraded slowly in vitro, while the combined in vivo evaluation in pigs and mice showed a rapid blood clearance and renal excretion of the 68 Ga-A9-K-DOTA. Conclusion: The preliminary in vitro and in vivo studies of the phage-display S. aureus

A transposon mutant library of Bacillus cereus ATCC 10987 reveals novel genes required for biofilm formation and implicates motility as an important factor for pellicle-biofilm formation.

Science.gov (United States)

Okshevsky, Mira; Louw, Matilde Greve; Lamela, Elena Otero; Nilsson, Martin; Tolker-Nielsen, Tim; Meyer, Rikke Louise

2018-04-01

Bacillus cereus is one of the most common opportunistic pathogens causing foodborne illness, as well as a common source of contamination in the dairy industry. B. cereus can form robust biofilms on food processing surfaces, resulting in food contamination due to shedding of cells and spores. Despite the medical and industrial relevance of this species, the genetic basis of biofilm formation in B. cereus is not well studied. In order to identify genes required for biofilm formation in this bacterium, we created a library of 5000 +  transposon mutants of the biofilm-forming strain B. cereusATCC 10987, using an unbiased mariner transposon approach. The mutant library was screened for the ability to form a pellicle biofilm at the air-media interface, as well as a submerged biofilm at the solid-media interface. A total of 91 genes were identified as essential for biofilm formation. These genes encode functions such as chemotaxis, amino acid metabolism and cellular repair mechanisms, and include numerous genes not previously known to be required for biofilm formation. Although the majority of disrupted genes are not directly responsible for motility, further investigations revealed that the vast majority of the biofilm-deficient mutants were also motility impaired. This observation implicates motility as a pivotal factor in the formation of a biofilm by B. cereus. These results expand our knowledge of the fundamental molecular mechanisms of biofilm formation by B. cereus. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Efficacy of Linezolid and Fosfomycin in Catheter-Related Biofilm Infection Caused by Methicillin-Resistant Staphylococcus aureus

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Dong Chai

2016-01-01

Full Text Available As long-standing clinical problems, catheter-related infections and other chronic biofilm infections are more difficult to treat due to the high antibiotic resistance of biofilm. Therefore, new treatments are needed for more effective bacteria clearance. In this study, we evaluated the antibacterial activities of several common antibiotics alone and their combinations against biofilm-embedded methicillin-resistant staphylococcus aureus (MRSA infections, both in vitro and in vivo. In brief, fosfomycin, levofloxacin, and rifampin alone or in combination with linezolid were tested in vitro against planktonic and biofilm-embedded MRSA infection in three MRSA stains. The synergistic effects between linezolid and the other three antibiotics were assessed by fractional inhibitory concentration index (FICI and time-kill curves, where the combination of linezolid plus fosfomycin showed the best synergistic effect in all strains. For further evaluation in vivo, we applied the combination of linezolid and fosfomycin in a catheter-related biofilm rat model and found that viable bacteria counts in biofilm were significantly reduced after treatment (P<0.05. In summary, we have shown here that the combination of linezolid and fosfomycin treatment had improved therapeutic effects on biofilm-embedded MRSA infection both in vitro and in vivo, which provided important basis for new clinical therapy development.

Biofilm-Forming Methicillin-Resistant Staphylococcus aureus Survive in Kupffer Cells and Exhibit High Virulence in Mice

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Takuto Oyama

2016-06-01

Full Text Available Although Staphylococcus aureus is part of the normal body flora, heavy usage of antibiotics has resulted in the emergence of methicillin-resistant strains (MRSA. MRSA can form biofilms and cause indwelling foreign body infections, bacteremia, soft tissue infections, endocarditis, and osteomyelitis. Using an in vitro assay, we screened 173 clinical blood isolates of MRSA and selected 20 high-biofilm formers (H-BF and low-biofilm formers (L-BF. These were intravenously administered to mice and the general condition of mice, the distribution of bacteria, and biofilm in the liver, lung, spleen, and kidney were investigated. MRSA count was the highest in the liver, especially within Kupffer cells, which were positive for acid polysaccharides that are associated with intracellular biofilm. After 24 h, the general condition of the mice worsened significantly in the H-BF group. In the liver, bacterial deposition and aggregation and the biofilm-forming spot number were all significantly greater for H-BF group than for L-BF. CFU analysis revealed that bacteria in the H-BF group survived for long periods in the liver. These results indicate that the biofilm-forming ability of MRSA is a crucial factor for intracellular persistence, which could lead to chronic infections.

Investigating the link between imipenem resistance and biofilm formation by Pseudomonas aeruginosa.

Science.gov (United States)

Musafer, Hadeel K; Kuchma, Sherry L; Naimie, Amanda A; Schwartzman, Joseph D; Al-Mathkhury, Harith J Fahad; O'Toole, George A

2014-07-01

Pseudomonas aeruginosa, a ubiquitous environmental organism, is a difficult-to-treat opportunistic pathogen due to its broad-spectrum antibiotic resistance and its ability to form biofilms. In this study, we investigate the link between resistance to a clinically important antibiotic, imipenem, and biofilm formation. First, we observed that the laboratory strain P. aeruginosa PAO1 carrying a mutation in the oprD gene, which confers resistance to imipenem, showed a modest reduction in biofilm formation. We also observed an inverse relationship between imipenem resistance and biofilm formation for imipenem-resistant strains selected in vitro, as well as for clinical isolates. We identified two clinical isolates of P. aeruginosa from the sputum of cystic fibrosis patients that formed robust biofilms, but were sensitive to imipenem (MIC ≤ 2 μg/ml). To test the hypothesis that there is a general link between imipenem resistance and biofilm formation, we performed transposon mutagenesis of these two clinical strains to identify mutants defective in biofilm formation, and then tested these mutants for imipenem resistance. Analysis of the transposon mutants revealed a role for previously described biofilm factors in these clinical isolates of P. aeruginosa, including mutations in the pilY1, pilX, pilW, algC, and pslI genes, but none of the biofilm-deficient mutants became imipenem resistant (MIC ≥ 8 μg/ml), arguing against a general link between biofilm formation and resistance to imipenem. Thus, assessing biofilm formation capabilities of environmental isolates is unlikely to serve as a good predictor of imipenem resistance. We also discuss our findings in light of the limited literature addressing planktonic antibiotic resistance factors that impact biofilm formation.

Blocking of bacterial biofilm formation by a fish protein coating

DEFF Research Database (Denmark)

Vejborg, Rebecca Munk; Klemm, Per

2008-01-01

Bacterial biofilm formation on inert surfaces is a significant health and economic problem in a wide range of environmental, industrial, and medical areas. Bacterial adhesion is generally a prerequisite for this colonization process and, thus, represents an attractive target for the development......, this proteinaceous coating is characterized with regards to its biofilm-reducing properties by using a range of urinary tract infectious isolates with various pathogenic and adhesive properties. The antiadhesive coating significantly reduced or delayed biofilm formation by all these isolates under every condition...... examined. The biofilm-reducing activity did, however, vary depending on the substratum physicochemical characteristics and the environmental conditions studied. These data illustrate the importance of protein conditioning layers with respect to bacterial biofilm formation and suggest that antiadhesive...

Biofilm formation of Francisella noatunensis subsp. orientalis

Science.gov (United States)

Soto, Esteban; Halliday-Wimmonds, Iona; Francis , Stewart; Kearney, Michael T.; Hansen, John D.

2015-01-01

Francisella noatunensis subsp. orientalis (Fno) is an emergent fish pathogen in both marine and fresh water environments. The bacterium is suspected to persist in the environment even without the presence of a suitable fish host. In the present study, the influence of different abiotic factors such as salinity and temperature were used to study the biofilm formation of different isolates of Fno including intracellular growth loci C (iglC)and pathogenicity determinant protein A (pdpA) knockout strains. Finally, we compared the susceptibility of planktonic and biofilm to three disinfectants used in the aquaculture and ornamental fish industry, namely Virkon®, bleach and hydrogen peroxide. The data indicates that Fno is capable of producing biofilms within 24 h where both salinity as well as temperature plays a role in the growth and biofilm formation of Fno. Mutations in theiglC or pdpA, both known virulence factors, do not appear to affect the capacity of Fno to produce biofilms, and the minimum inhibitory concentration, and minimum biocidal concentration for the three disinfectants were lower than the minimum biofilm eradication concentration values. This information needs to be taken into account if trying to eradicate the pathogen from aquaculture facilities or aquariums.

Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation

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Samuel Takashi Saito

2012-01-01

Full Text Available Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS. Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI<3 only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications.

Bioguided Fractionation Shows Cassia alata Extract to Inhibit Staphylococcus epidermidis and Pseudomonas aeruginosa Growth and Biofilm Formation

Science.gov (United States)

Saito, Samuel Takashi; Trentin, Danielle da Silva; Macedo, Alexandre José; Pungartnik, Cristina; Gosmann, Grace; Silveira, Jaqueline de Deos; Guecheva, Temenouga Nikolova; Henriques, João Antonio Pêgas; Brendel, Martin

2012-01-01

Plant extracts have a long history to be used in folk medicine. Cassia alata extracts are known to exert antibacterial activity but details on compounds and mechanism of action remain poorly explored. We purified and concentrated the aqueous leaf extract of C. alata by reverse phase-solid phase extraction and screened the resulting CaRP extract for antimicrobial activity. CaRP extract exhibited antimicrobial activity for Pseudomonas aeruginosa, Staphylococcus epidermidis, S. aureus, and Bacillus subtilis. CaRP also inhibited biofilm formation of S. epidermidis and P. aeruginosa. Several bacterial growth-inhibiting compounds were detected when CaRP extract was fractionated by TLC chromatography coupled to bioautography agar overlay technique. HPLC chromatography of CaRP extract yielded 20 subfractions that were tested by bioautography for antimicrobial activity against S. aureus and S. epidermidis. Five bioactive fractions were detected and chemically characterized, using high-resolution mass spectrometry (qTOF-MS/MS). Six compounds from four fractions could be characterized as kaempferol, kaempferol-O-diglucoside, kaempferol-O-glucoside, quercetin-O-glucoside, rhein, and danthron. In the Salmonella/microsome assay CaRP showed weak mutagenicity (MI < 3) only in strain TA98, pointing to a frameshift mutation activity. These results indicate that C. alata leaf extract contains a minimum of 7 compounds with antimicrobial activity and that these together or as single substance are active in preventing formation of bacterial biofilm, indicating potential for therapeutic applications. PMID:22548121

Host Proteins Determine MRSA Biofilm Structure and Integrity

DEFF Research Database (Denmark)

Dreier, Cindy; Nielsen, Astrid; Jørgensen, Nis Pedersen

Human extracellular matrix (hECM) proteins aids the initial attachment and initiation of an infection, by specific binding to bacterial cell surface proteins. However, the importance of hECM proteins in structure, integrity and antibiotic resilience of a biofilm is unknown. This study aims...... to determine how specific hECM proteins affect S. aureus USA300 JE2 biofilms. Biofilms were grown in the presence of synovial fluid from rheumatoid arteritis patients to mimic in vivo conditions, where bacteria incorporate hECM proteins into the biofilm matrix. Difference in biofilm structure, with and without...... addition of hECM to growth media, was visualized by confocal laser scanning microscopy. Two enzymatic degradation experiments were used to study biofilm matrix composition and importance of hECM proteins: enzymatic removal of specific hECM proteins from growth media, before biofilm formation, and enzymatic...

Involvement of NADH Oxidase in Biofilm Formation in Streptococcus sanguinis.

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Xiuchun Ge

Full Text Available Biofilms play important roles in microbial communities and are related to infectious diseases. Here, we report direct evidence that a bacterial nox gene encoding NADH oxidase is involved in biofilm formation. A dramatic reduction in biofilm formation was observed in a Streptococcus sanguinis nox mutant under anaerobic conditions without any decrease in growth. The membrane fluidity of the mutant bacterial cells was found to be decreased and the fatty acid composition altered, with increased palmitic acid and decreased stearic acid and vaccenic acid. Extracellular DNA of the mutant was reduced in abundance and bacterial competence was suppressed. Gene expression analysis in the mutant identified two genes with altered expression, gtfP and Idh, which were found to be related to biofilm formation through examination of their deletion mutants. NADH oxidase-related metabolic pathways were analyzed, further clarifying the function of this enzyme in biofilm formation.

DNase I and proteinase K impair Listeria monocytogenes biofilm formation and induce dispersal of pre-existing biofilms.

Science.gov (United States)

Nguyen, Uyen T; Burrows, Lori L

2014-09-18

Current sanitation methods in the food industry are not always sufficient for prevention or dispersal of Listeria monocytogenes biofilms. Here, we determined if prevention of adherence or dispersal of existing biofilms could occur if biofilm matrix components were disrupted enzymatically. Addition of DNase during biofilm formation reduced attachment (biofilms with 100μg/ml of DNase for 24h induced incomplete biofilm dispersal, with biofilm remaining compared to control. In contrast, addition of proteinase K completely inhibited biofilm formation, and 72h biofilms-including those grown under stimulatory conditions-were completely dispersed with 100μg/ml proteinase K. Generally-regarded-as-safe proteases bromelain and papain were less effective dispersants than proteinase K. In a time course assay, complete dispersal of L. monocytogenes biofilms from both polystyrene and type 304H food-grade stainless steel occurred within 5min at proteinase K concentrations above 25μg/ml. These data confirm that both DNA and proteins are required for L. monocytogenes biofilm development and maintenance, and that these components of the biofilm matrix can be targeted for effective prevention and removal of biofilms. Copyright © 2014 Elsevier B.V. All rights reserved.

Capsular Polysaccharide Interferes with Biofilm Formation by Pasteurella multocida Serogroup A

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Briana Petruzzi

2017-11-01

Full Text Available Pasteurella multocida is an important multihost animal and zoonotic pathogen that is capable of causing respiratory and multisystemic diseases, bacteremia, and bite wound infections. The glycosaminoglycan capsule of P. multocida is an essential virulence factor that protects the bacterium from host defenses. However, chronic infections (such as swine atrophic rhinitis and the carrier state in birds and other animals may be associated with biofilm formation, which has not been characterized in P. multocida. Biofilm formation by clinical isolates was inversely related to capsule production and was confirmed with capsule-deficient mutants of highly encapsulated strains. Capsule-deficient mutants formed biofilms with a larger biomass that was thicker and smoother than the biofilm of encapsulated strains. Passage of a highly encapsulated, poor-biofilm-forming strain under conditions that favored biofilm formation resulted in the production of less capsular polysaccharide and a more robust biofilm, as did addition of hyaluronidase to the growth medium of all of the strains tested. The matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS, as determined by gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion. However, a putative glycogen synthesis locus was not differentially regulated when the bacteria were grown as a biofilm or planktonically, as determined by quantitative reverse transcriptase PCR. Therefore, the negatively charged capsule may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix from encasing large numbers of bacterial cells. This is the first detailed description of biofilm formation and a glycogen EPS by P. multocida.

Phenotypic Variation Is Almost Entirely Independent of the Host-Pathogen Relationship in Clinical Isolates of S. aureus.

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Adrian D Land

Full Text Available A key feature of Staphylococcus aureus biology is its ability to switch from an apparently benign colonizer of ~30% of the population to a cutaneous pathogen, to a deadly invasive pathogen. Little is known about the mechanisms driving this transition or the propensity of different S. aureus strains to engender different types of host-pathogen interactions. At the same time, significant weight has been given to the role of specific in vitro phenotypes in S. aureus virulence. Biofilm formation, hemolysis and pigment formation have all been associated with virulence in mice.To determine if there is a correlation between in vitro phenotype and the three types of host-pathogen relationships commonly exhibited by S. aureus in the context of its natural human host, we assayed 300 clinical isolates for phenotypes implicated in virulence including hemolysis, sensitivity to autolysis, and biofilm formation. For comparative purposes, we also assayed phenotype in 9 domesticated S. aureus strains routinely used for analysis of virulence determinants in laboratory settings.Strikingly, the clinical strains exhibited significant phenotypic uniformity in each of the assays evaluated in this study. One exception was a small, but significant, correlation between an increased propensity for biofilm formation and isolation from skin and soft tissue infections (SSTIs. In contrast, we observed a high degree of phenotypic variation between common laboratory strains that exhibit virulence in mouse models. These data suggest the existence of significant evolutionary pressure on the S. aureus genome and highlight a role for host factors as a strong determinant of the host-pathogen relationship. In addition, the high degree of variation between laboratory strains emphasizes the need for caution when applying data obtained in one lab strain to the analysis of another.

Fibrinogen-Induced Streptococcus mutans Biofilm Formation and Adherence to Endothelial Cells

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Telma Blanca Lombardo Bedran

2013-01-01

Full Text Available Streptococcus mutans, the predominant bacterial species associated with dental caries, can enter the bloodstream and cause infective endocarditis. The aim of this study was to investigate S. mutans biofilm formation and adherence to endothelial cells induced by human fibrinogen. The putative mechanism by which biofilm formation is induced as well as the impact of fibrinogen on S. mutans resistance to penicillin was also evaluated. Bovine plasma dose dependently induced biofilm formation by S. mutans. Of the various plasma proteins tested, only fibrinogen promoted the formation of biofilm in a dose-dependent manner. Scanning electron microscopy observations revealed the presence of complex aggregates of bacterial cells firmly attached to the polystyrene support. S. mutans in biofilms induced by the presence of fibrinogen was markedly resistant to the bactericidal effect of penicillin. Fibrinogen also significantly increased the adherence of S. mutans to endothelial cells. Neither S. mutans cells nor culture supernatants converted fibrinogen into fibrin. However, fibrinogen is specifically bound to the cell surface of S. mutans and may act as a bridging molecule to mediate biofilm formation. In conclusion, our study identified a new mechanism promoting S. mutans biofilm formation and adherence to endothelial cells which may contribute to infective endocarditis.

An update on Pseudomonas aeruginosa biofilm formation, tolerance, and dispersal

DEFF Research Database (Denmark)

Harmsen, Morten; Yang, Liang; Pamp, Sünje Johanna

2010-01-01

We review the recent advances in the understanding of the Pseudomonas aeruginosa biofilm lifestyle from studies using in vitro laboratory setups such as flow chambers and microtiter trays. Recent work sheds light on the role of nutrients, motility, and quorum sensing in structure formation in P....... aeruginosa biofilms. The second messenger, c-di-GMP, is established as an important regulator of the synthesis of polysaccharide and protein components of the biofilm matrix. Extracellular DNA is shown to be an essential component of the biofilm matrix. It has become apparent that biofilm formation involves...... interactions between different subpopulations. The molecular mechanisms underlying the tolerance of biofilm bacteria to antimicrobial agents are beginning to be unraveled, and new knowledge has been obtained regarding the environmental cues and regulatory mechanisms involved in biofilm dispersal....

The role of Proteus mirabilis cell wall features in biofilm formation.

Science.gov (United States)

Czerwonka, Grzegorz; Guzy, Anna; Kałuża, Klaudia; Grosicka, Michalina; Dańczuk, Magdalena; Lechowicz, Łukasz; Gmiter, Dawid; Kowalczyk, Paweł; Kaca, Wiesław

2016-11-01

Biofilms formed by Proteus mirabilis strains are a serious medical problem, especially in the case of urinary tract infections. Early stages of biofilm formation, such as reversible and irreversible adhesion, are essential for bacteria to form biofilm and avoid eradication by antibiotic therapy. Adhesion to solid surfaces is a complex process where numerous factors play a role, where hydrophobic and electrostatic interactions with solid surface seem to be substantial. Cell surface hydrophobicity and electrokinetic potential of bacterial cells depend on their surface composition and structure, where lipopolysaccharide, in Gram-negative bacteria, is prevailing. Our studies focused on clinical and laboratory P. mirabilis strains, where laboratory strains have determined LPS structures. Adherence and biofilm formation tests revealed significant differences between strains adhered in early stages of biofilm formation. Amounts of formed biofilm were expressed by the absorption of crystal violet. Higher biofilm amounts were formed by the strains with more negative values of zeta potential. In contrast, high cell surface hydrophobicity correlated with low biofilm amount.

Adaptation to copper stress influences biofilm formation in Alteromonas macleodii.

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Cusick, Kathleen D; Dale, Jason R; Fitzgerald, Lisa A; Little, Brenda J; Biffinger, Justin C

2017-07-01

An Alteromonas macleodii strain was isolated from copper-containing coupons incubated in surface seawater (Key West, FL, USA). In addition to the original isolate, a copper-adapted mutant was created and maintained with 0.78 mM Cu 2+ . Biofilm formation was compared between the two strains under copper-amended and low-nutrient conditions. Biofilm formation was significantly increased in the original isolate under copper amendment, while biofilm formation was significantly higher in the mutant under low-nutrient conditions. Biofilm expression profiles of diguanylate cyclase (DGC) genes, as well as genes involved in secretion, differed between the strains. Comparative genomic analysis demonstrated that both strains possessed a large number of gene attachment harboring cyclic di-GMP synthesis and/or degradation domains. One of the DGC genes, induced at very high levels in the mutant, possessed a degradation domain in the original isolate that was lacking in the mutant. The genetic and transcriptional mechanisms contributing to biofilm formation are discussed.

Experimental model of biofilm implant-related osteomyelitis to test combination biomaterials using biofilms as initial inocula.

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Williams, Dustin L; Haymond, Bryan S; Woodbury, Kassie L; Beck, J Peter; Moore, David E; Epperson, R Tyler; Bloebaum, Roy D

2012-07-01

Currently, the majority of animal models that are used to study biofilm-related infections use planktonic bacterial cells as initial inocula to produce positive signals of infection in biomaterials studies. However, the use of planktonic cells has potentially led to inconsistent results in infection outcomes. In this study, well-established biofilms of methicillin-resistant Staphylococcus aureus were grown and used as initial inocula in an animal model of a Type IIIB open fracture. The goal of the work was to establish, for the first time, a repeatable model of biofilm implant-related osteomyelitis, wherein biofilms were used as initial inocula to test combination biomaterials. Results showed that 100% of animals that were treated with biofilms developed osteomyelitis, whereas 0% of animals not treated with biofilm developed infection. The development of this experimental model may lead to an important shift in biofilm and biomaterials research by showing that when biofilms are used as initial inocula, they may provide additional insights into how biofilm-related infections in the clinic develop and how they can be treated with combination biomaterials to eradicate and/or prevent biofilm formation. Copyright © 2012 Wiley Periodicals, Inc.

Stimulated phase-shift acoustic nanodroplets enhance vancomycin efficacy against methicillin-resistant Staphylococcus aureus biofilms

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Guo H

2017-06-01

Full Text Available Hao Guo,1 Ziming Wang,1 Quanyin Du,1 Pan Li,2 Zhigang Wang,2 Aimin Wang1 1Department of Orthopedics, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, China; 2Chongqing Key Laboratory of Ultrasound Molecular Imaging, Institute of Ultrasound Imaging, Second Affiliated Hospital of Chongqing Medical University, Chongqing, China Purpose: Bacterial biofilms on the surface of prostheses are becoming a rising concern in managing prosthetic joint infections. The inherent resistant features of biofilms render traditional antimicrobial therapy unproductive and revision surgery outcomes uncertain. This situation has prompted the exploration of novel antimicrobial strategies. The synergy of ultrasound microbubbles and vancomycin has been proposed as an efficient alternative for biofilm eradication. The purpose of this study was to evaluate the anti-biofilm effect of stimulated phase-shift acoustic nanodroplets (NDs combined with vancomycin.Materials and methods: We fabricated lipid phase-shift NDs with a core of liquid perfluoropentane. A new phase change mode for NDs incorporating an initial unfocused low-intensity pulsed ultrasound for 5 minutes and a subsequent incubation at 37°C into a 24-hour duration was developed. Methicillin-resistant Staphylococcus aureus (MRSA biofilms were incubated with vancomycin and NDs under the hybrid stimulation. Biofilm morphology following treatment was determined using confocal laser scanning microscopy and scanning electron microscopy. Resazurin assay was used to quantify bactericidal efficacy against MRSA biofilm bacteria.Results: NDs treated sequentially with ultrasound and heating at 37°C achieved gradual and substantial ND vaporization and cavitation in a successive process. NDs after stimulation were capable of generating stronger destruction on biofilm structure which was best characterized by residual circular arc margins and more dead bacteria. Furthermore, NDs

Calcium-Phosphate-Osteopontin Particles Reduce Biofilm Formation and pH Drops in in situ-Grown Dental Biofilms

DEFF Research Database (Denmark)

Schlafer, Sebastian; Ibsen, Casper Jon Steenberg; Birkedal, Henrik

2017-01-01

This 2-period crossover study investigated the effect of calcium-phosphate-osteopontin particles on biofilm formation and pH in 48-h biofilms grown in situ. Bovine milk osteopontin is a highly phosphorylated glycoprotein that has been shown to interfere with bacterial adhesion to salivary......-coated surfaces. Calcium-phosphate-osteopontin particles have been shown to reduce biofilm formation and pH drops in a 5-species laboratory model of dental biofilm without affecting bacterial viability. Here, smooth surface biofilms from 10 individuals were treated ex vivo 6 times/day for 30 min with either...... calcium-phosphate-osteopontin particles or sterile saline. After growth, the amount of biofilm formed was determined by confocal microscopy, and pH drops upon exposure to glucose were monitored using confocal-microscopy-based pH ratiometry. A total of 160 biofilms were analysed. No adverse effects...

Influence of culture conditions for clinically isolated non-albicans Candida biofilm formation.

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Tan, Yulong; Leonhard, Matthias; Ma, Su; Schneider-Stickler, Berit

2016-11-01

Non-albicans Candida species have been isolated in increasing numbers in patients. Moreover, they are adept at forming biofilms. This study analyzed biofilm formation of clinically isolated non-albicans Candida, including Candida tropicalis, Candida krusei and Candida parapsilosis under the influence of different growth media (RPMI 1640, YPD and BHI) and several culture variables (inoculum concentration, incubation period and feeding conditions). The results showed that culture conditions strongly influenced non-albicans Candida species biofilm formation. YPD and BHI resulted in larger amount of biofilm formation with higher metabolic activity of biofilms. Furthermore, the growth media seems to have varying effects on adhesion and biofilm development. Growth conditions may also influence biofilm formation, which was enhanced when starting the culture with a larger inoculum, longer incubation period and using a fed-batch system. Therefore, the potential influences of external environmental factors should be considered when studying the non-albicans Candida biofilms in vitro. Copyright © 2016 Elsevier B.V. All rights reserved.

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

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Wei Wang

2018-06-01

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

Coexistence facilitates interspecific biofilm formation in complex microbial communities

DEFF Research Database (Denmark)

Madsen, Jonas Stenløkke; Røder, Henriette Lyng; Russel, Jakob

2016-01-01

Social interactions in which bacteria respond to one another by modifying their phenotype are central determinants of microbial communities. It is known that interspecific interactions influence the biofilm phenotype of bacteria; a phenotype that is central to the fitness of bacteria. However......, the underlying role of fundamental ecological factors, specifically coexistence and phylogenetic history, in biofilm formation remains unclear. This study examines how social interactions affect biofilm formation in multi-species co-cultures from five diverse environments. We found prevalence of increased...

The ability of S.aureus to form biofilm on the Ti-6Al-7Nb scaffolds produced by Selective Laser Melting and subjected to the different types of surface modifications.

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Szymczyk, Patrycja; Junka, Adam; Ziółkowski, Grzegorz; Smutnicka, Danuta; Bartoszewicz, Marzenna; Chlebus, Edward

2013-01-01

The Gram-positive coccus, Staphylococcus aureus, is the leading etiologic agent of limb and life-threatening biofilm-related infections in the patients following the orthopaedic implantations. The aim of the present paper is to estimate the ability of S. aureus to form biofilm on titanium alloy (Ti-6Al-7Nb) scaffolds produced by Selective Laser Melting (SLM) and subjected to the different types of surface modifications, including ultrasonic cleaning and chemical polishing. The results obtained indicate significantly the decreased ability of S.aureus to form biofilm on the surface of scaffolds subjected to the chemical polishing in comparison to the scaffolds cleaned ultrasonically. The data provided can be useful for future applications of the SLM technology in production of Ti-6Al-7Nb medical implants.

Increased Zinc Availability Enhances Initial Aggregation and Biofilm Formation of Streptococcus pneumoniae.

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Brown, Lindsey R; Caulkins, Rachel C; Schartel, Tyler E; Rosch, Jason W; Honsa, Erin S; Schultz-Cherry, Stacey; Meliopoulos, Victoria A; Cherry, Sean; Thornton, Justin A

2017-01-01

Bacteria growing within biofilms are protected from antibiotics and the immune system. Within these structures, horizontal transfer of genes encoding virulence factors, and promoting antibiotic resistance occurs, making biofilms an extremely important aspect of pneumococcal colonization and persistence. Identifying environmental cues that contribute to the formation of biofilms is critical to understanding pneumococcal colonization and infection. Iron has been shown to be essential for the formation of pneumococcal biofilms; however, the role of other physiologically important metals such as copper, zinc, and manganese has been largely neglected. In this study, we investigated the effect of metals on pneumococcal aggregation and early biofilm formation. Our results show that biofilms increase as zinc concentrations increase. The effect was found to be zinc-specific, as altering copper and manganese concentrations did not affect biofilm formation. Scanning electron microscopy analysis revealed structural differences between biofilms grown in varying concentrations of zinc. Analysis of biofilm formation in a mutant strain lacking the peroxide-generating enzyme pyruvate oxidase, SpxB, revealed that zinc does not protect against pneumococcal H 2 O 2 . Further, analysis of a mutant strain lacking the major autolysin, LytA, indicated the role of zinc as a negative regulator of LytA-dependent autolysis, which could affect biofilm formation. Additionally, analysis of cell-cell aggregation via plating and microscopy revealed that high concentrations of zinc contribute to intercellular interaction of pneumococci. The findings from this study demonstrate that metal availability contributes to the ability of pneumococci to form aggregates and subsequently, biofilms.

Biofilm formation in long-term central venous catheters in children with cancer

DEFF Research Database (Denmark)

Handrup, Mette Møller; Fuursted, Kurt; Funch, Peter

2012-01-01

Taurolidine has demonstrated inhibition of biofilm formation in vitro. The aim of this study was to compare the effect of catheter locking with taurolidine vs heparin in biofilm formation in central venous catheters. Forty-eight children with cancer were randomized to catheter locking by heparin (n...... = 22) or taurolidine (n = 26), respectively. After removal, catheters were examined by standardized scanning electron microscopy to assess quantitative biofilm formation. Biofilm was present if morphologically typical structures and bacterial cells were identified. Quantitative and semi...... in the intraluminal biofilm formation and the rate of bacterial colonization detected by scanning electron microscopy in the two groups....

Resistance to antimicrobials and biofilm formation in Staphylococcus spp. isolated from bovine mastitis in the Northeast of Brazil.

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da Costa Krewer, Carina; Santos Amanso, Evandro; Veneroni Gouveia, Gisele; de Lima Souza, Renata; da Costa, Mateus Matiuzzi; Aparecido Mota, Rinaldo

2015-03-01

Mastitis is the principal disease affecting dairy herds worldwide. The aim of the present study was to characterize phenotypic and genotypic features associated with resistance to antimicrobials in Staphylococcus spp. isolated from 2064 milk samples of 525 lactating cows in the Northeast of Brazil. Of the 218 isolates analyzed, 57.8% were characterized as Staphylococcus aureus, 28% as coagulase-positive staphylococci other than S. aureus (oCPS), and 14.2% as coagulase-negative staphylococci (CNS). The test for susceptibility to antimicrobials showed amoxicillin (32.6%) to be the less effective drug in vitro, and the multi-drug resistance (MDR) rate for beta-lactams varied from 0 to 0.75. The genotypic characterization showed that 93.1% of the samples were tested positive for the blaZ gene, while none amplified mecA. The antibiotic efflux mechanism was observed in 0.9% of isolates. The biofilm formation was found in 3.7 and 96.3% of samples, respectively, on Congo red agar and on the microplate adhesion test, while the icaD gene was present in 92.2% of Staphylococcus spp. The high frequency of blaZ gene observed in this study was associated with the resistance of most Staphylococcus spp. to one or more of the beta-lactams tested, which are routinely used in Brazilian herds for mastitis treatment. The biofilm formation was also detected in the isolates analyzed being an important characteristic for pathogenicity and antimicrobial resistance of bacteria.

Disinfection efficiencies of sage and spearmint essential oils against planktonic and biofilm Staphylococcus aureus cells in comparison with sodium hypochlorite.

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Vetas, Dimitrios; Dimitropoulou, Eleni; Mitropoulou, Gregoria; Kourkoutas, Yiannis; Giaouris, Efstathios

2017-09-18

Staphylococcus aureus causes human infections and foodborne intoxications. This study explored the potential antibacterial actions of sage and spearmint essential oils (EOs) against both its planktonic and biofilm cells, in comparison with sodium hypochlorite (NaOCl), a commonly applied chemical sanitizer. Initially, the minimum inhibitory and bactericidal concentrations (MICs, MBCs) of each plant mixture were determined against planktonic cultures, following growth at 30°C for 24h. Stationary phase planktonic bacteria were then individually exposed for 6min to either each EO (applied at 1-2×MBC; 2.5-5%), or NaOCl (250-450ppm). These were also left to form biofilms on 96-well polystyrene microplates, at 30°C for 96h, with medium renewal at 48h, in the presence of 10 different concentrations of each EO, expanding from sub- to super-inhibitory for planktonic growth, and the minimum biofilm inhibitory concentrations (MBICs; >90% inhibition) of each plant mixture were calculated. Formed biofilms were finally exposed for 6min to either each EO (applied at 2-6×MBC; 5-15%), or NaOCl (7500-25,000ppm; applied either alone or in combination with each EO at 5%). Results showed that both EOs presented MIC and MBC equal to 1.25 and 2.5%, respectively. As expected, their application at their MIC and above significantly inhibited biofilm formation, while spearmint EO was still able to cause this at ½ of its MIC, with MBICs equal to 1.25 and 0.63% for sage and spearmint EOs, respectively. Alarmingly, the application of both EOs at 1/8 to 1/16 of their MIC further increased biofilm formation. Regarding biofilm disinfection experiments, the individual application of each EO against the pre-established sessile communities resulted in log decrease ranges of 0.8-3logCFU/cm 2 , while in the case of NaOCl application (either alone or combined with each EO), the observed reductions never exceeded 1.7logCFU/cm 2 . These last results highlight the great antimicrobial recalcitrance of

Synergistic effect on biofilm formation between Fusobacterium nucleatum and Capnocytophaga ochracea.

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Okuda, Tamaki; Okuda, Katsuji; Kokubu, Eitoyo; Kawana, Tomoko; Saito, Atsushi; Ishihara, Kazuyuki

2012-02-01

The formation of dental plaque biofilm by specific Gram-negative rods and spirochetes plays an important role in the development of periodontal disease. The aim of this study was to characterize biofilm formation by Fusobacterium nucleatum and Capnocytophaga ochracea. Coaggregation between F. nucleatum and Capnocytophaga species was determined by visual assay. Biofilm formation was assessed by crystal violet staining. Enhancement of biofilm formation by F. nucleatum via soluble factor of C. ochracea was evaluated by addition of culture supernatant and a two-compartment separated co-culture system. Production of autoinducer-2 by the tested organisms was evaluated using Vibrio harveyi BB170. F. nucleatum strains coaggregated with C. ochracea ATCC 33596 or ONO-26 strains. Ethylenediamine tetraacetic acid, N-acetyl-d-galactosamine or lysine inhibited coaggregation. Heating or proteinase K treatment of F. nucleatum cells affected coaggregation, whereas the same treatment of C. ochracea cells did not. Co-culture of F. nucleatum with C. ochracea in the same well resulted in a statistically significant increase in biofilm formation. Enhancement of F. nucleatum biofilm formation by a soluble component of C. ochracea was observed using the two-compartment co-culture system (P culture supernatant of C. ochracea (P < 0.01). The present findings indicate that induction of coaggregation and intracellular interaction by release of a diffusible molecule by C. ochracea play a significant role in the formation of biofilm by F. nucleatum and C. ochracea. Copyright © 2012 Elsevier Ltd. All rights reserved.

The virulence regulator PrfA promotes biofilm formation by Listeria monocytogenes.

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Lemon, Katherine P; Freitag, Nancy E; Kolter, Roberto

2010-08-01

Listeria monocytogenes is a food-borne facultative intracellular pathogen. It is widespread in the environment and has several distinct life-styles. The key transcriptional activator PrfA positively regulates L. monocytogenes virulence genes to mediate the transition from extracellular, flagellum-propelled cell to intracellular pathogen. Here we report the first evidence that PrfA also has a significant positive impact on extracellular biofilm formation. Mutants lacking prfA were defective in surface-adhered biofilm formation. The DeltaprfA mutant exhibited wild-type flagellar motility, and its biofilm defect occurred after initial surface adhesion. We also observed that mutations that led to the constitutive expression of PrfA-dependent virulence genes had a minimal impact on biofilm formation. Furthermore, biofilm development was enhanced in a mutant encoding a PrfA protein variant unable to fully transition from the extracellular form to the virulent, intracellular activity conformation. These results indicate that PrfA positively regulates biofilm formation and suggest that PrfA has a global role in modulating the life-style of L. monocytogenes. The requirement of PrfA for optimal biofilm formation may provide selective pressure to maintain this critical virulence regulator when L. monocytogenes is outside host cells in the environment.

Anti-infective properties of Lactobacillus fermentum against Staphylococcus aureus and Pseudomonas aeruginosa.

Science.gov (United States)

Varma, Parvathi; Nisha, N; Dinesh, Kavitha R; Kumar, Anil V; Biswas, Raja

2011-01-01

Surgical wounds and implant-associated Staphylococcus aureus and Pseudomonas aeruginosa infections are often difficult to treat because of limited susceptibility of several of these strains to conventional antibiotics. As a result, there is a constant need for new alternative drugs. The aim of this study was to investigate the antimicrobial properties of Lactobacillus fermentum, a probiotic bacterium, which we have isolated from colonic biopsies. The inhibition of S. aureus and P. aeruginosa growth was evaluated by coincubating with L. fermentum strains. Growth inhibition was tested for several of their clinical isolates using agar well diffusion assays. For biofilm assay S. aureus and P. aeruginosa were grown on the glass slides and in 96-well plates in presence of 2.5 μg/ml culture filtrate of L. fermentum. Biofilms were photographed using confocal microscope or stained with 0.1% crystal violet. Reduction in the cytotoxicity of S. aureus and P. aeruginosa was observed in presence of 2.5 μg/ml L. fermentum-spent media. Using in vitroexperiments, we showed that L. fermentum-secreted compound(s) inhibits the growth, cytotoxicity and biofilm formation of several S. aureus and P. aeruginosa strains. Compound(s) present in the culture supernatant of L. fermentum may have promising applications in treating hospital-acquired infections. Copyright © 2011 S. Karger AG, Basel.

Chicken Juice Enhances Surface Attachment and Biofilm Formation of Campylobacter jejuni

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Brown, Helen L.; Reuter, Mark; Salt, Louise J.; Cross, Kathryn L.; Betts, Roy P.

2014-01-01

The bacterial pathogen Campylobacter jejuni is primarily transmitted via the consumption of contaminated foodstuffs, especially poultry meat. In food processing environments, C. jejuni is required to survive a multitude of stresses and requires the use of specific survival mechanisms, such as biofilms. An initial step in biofilm formation is bacterial attachment to a surface. Here, we investigated the effects of a chicken meat exudate (chicken juice) on C. jejuni surface attachment and biofilm formation. Supplementation of brucella broth with ≥5% chicken juice resulted in increased biofilm formation on glass, polystyrene, and stainless steel surfaces with four C. jejuni isolates and one C. coli isolate in both microaerobic and aerobic conditions. When incubated with chicken juice, C. jejuni was both able to grow and form biofilms in static cultures in aerobic conditions. Electron microscopy showed that C. jejuni cells were associated with chicken juice particulates attached to the abiotic surface rather than the surface itself. This suggests that chicken juice contributes to C. jejuni biofilm formation by covering and conditioning the abiotic surface and is a source of nutrients. Chicken juice was able to complement the reduction in biofilm formation of an aflagellated mutant of C. jejuni, indicating that chicken juice may support food chain transmission of isolates with lowered motility. We provide here a useful model for studying the interaction of C. jejuni biofilms in food chain-relevant conditions and also show a possible mechanism for C. jejuni cell attachment and biofilm initiation on abiotic surfaces within the food chain. PMID:25192991

Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal

DEFF Research Database (Denmark)

Gjermansen, Morten; Ragas, Paula Cornelia; Tolker-Nielsen, Tim

2006-01-01

Microbial biofilm formation often causes problems in medical and industrial settings, and knowledge about the factors that are involved in biofilm development and dispersion is useful for creating strategies to control the processes. In this report, we present evidence that proteins with GGDEF...... and EAL domains are involved in the regulation of biofilm formation and biofilm dispersion in Pseudomonas putida. Overexpression in P. putida of the Escherichia coli YedQ protein, which contains a GGDEF domain, resulted in increased biofilm formation. Overexpression in P. putida of the E. coli Yhj......H protein, which contains an EAL domain, strongly inhibited biofilm formation. Induction of YhjH expression in P. putida cells situated in established biofilms led to rapid dispersion of the biofilms. These results support the emerging theme that GGDEF-domain and EAL-domain proteins are involved...

Lactobacilli : Important in biofilm formation on voice prostheses

NARCIS (Netherlands)

Buijssen, Kevin J. D. A.; Harmsen, Hermie J. M.; van der Mei, Henny C.; Busscher, Henk J.; van der Laan, Bernard F. A. M.

OBJECTIVE: We sought to identify bacterial strains responsible for biofilm formation on silicone rubber voice prostheses. STUDY DESIGN: We conducted an analysis of the bacterial population in biofilms on used silicone rubber voice prostheses by using new microbiological methods. METHODS: Two

Biofilm inhibition activity of compounds isolated from two Eunicea species collected at the Caribbean Sea

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Yenny Martínez Díaz

Full Text Available Abstract Biofilm has a primary role in the pathogenesis of diseases and in the attachment of multicellular organisms to a fouled surface. Because of that, the control of bacterial biofilms has been identified as an important target. In the present study, five lipid compounds isolated from soft coral Eunicea sp. and three terpenoids together with a mixture of sterols from Eunicea fusca collected at the Colombian Caribbean Sea showed different effectiveness against biofilm formation by three marine bacteria associated with immersed fouled surfaces, Ochrobactrum pseudogringnonense,Alteromona macleodii and Vibrio harveyi, and against two known biofilm forming bacteria, Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923. The pure compounds were characterized by NMR, HRESI-MS, HRGC-MS and optical rotation. The most effective compounds were batyl alcohol (1 and fuscoside E peracetate (6, acting against four strains without affecting their microbial growth. Compound 1 showed biofilm inhibition greater than 30% against A. macleodii, and up to 60% against O. pseudogringnonense,V. harveyi and S. aureus. Compound 6 inhibited O. pseudogringnonense and V. harveyi between 25 and 50%, and P. aeruginosa or S. aureus up to 60% at 0.5 mg/ml. The results suggest that these compounds exhibit specific biofilm inhibition with lower antimicrobial effect against the bacterial species assayed.

The alternative sigma factor sigma B of Staphylococcus aureus modulates virulence in experimental central venous catheter-related infections.

Science.gov (United States)

Lorenz, Udo; Hüttinger, Christian; Schäfer, Tina; Ziebuhr, Wilma; Thiede, Arnulf; Hacker, Jörg; Engelmann, Susanne; Hecker, Michael; Ohlsen, Knut

2008-03-01

The impact of the alternative sigma factor sigma B (SigB) on pathogenesis of Staphylococcus aureus is not conclusively clarified. In this study, a central venous catheter (CVC) related model of multiorgan infection was used to investigate the role of SigB for the pathogenesis of S. aureus infections and biofilm formation in vivo. Analysis of two SigB-positive wild-type strains and their isogenic mutants revealed uniformly that the wild-type was significantly more virulent than the SigB-deficient mutant. The observed difference in virulence was apparently not linked to the capability of the strains to form biofilms in vivo since wild-type and mutant strains were able to produce biofilm layers inside of the catheter. The data strongly indicate that the alternative sigma factor SigB plays a role in CVC-associated infections caused by S. aureus.

A dual role of extracellular DNA during biofilm formation of Neisseria meningitidis

DEFF Research Database (Denmark)

Lappann, M.; Claus, H.; van Alen, T.

2010-01-01

formation, whereas biofilm formation of cc with low point prevalence (ST-8 cc and ST-11 cc) was eDNA-independent. For initial biofilm formation, a ST-32 cc type strain, but not a ST-11 type strain, utilized eDNA. The release of eDNA was mediated by lytic transglycosylase and cytoplasmic N......-acetylmuramyl-l-alanine amidase genes. In late biofilms, outer membrane phospholipase A-dependent autolysis, which was observed in most cc, but not in ST-8 and ST-11 strains, was required for shear force resistance of microcolonies. Taken together, N. meningitidis evolved two different biofilm formation strategies, an e....... On the contrary, spreaders (ST-11 and ST-8 cc) are unable to use eDNA for biofilm formation and might compensate for poor colonization properties by high transmission rates....

Pseudomonas aeruginosa Alters Staphylococcus aureus Sensitivity to Vancomycin in a Biofilm Model of Cystic Fibrosis Infection

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Giulia Orazi

2017-07-01

Full Text Available The airways of cystic fibrosis (CF patients have thick mucus, which fosters chronic, polymicrobial infections. Pseudomonas aeruginosa and Staphylococcus aureus are two of the most prevalent respiratory pathogens in CF patients. In this study, we tested whether P. aeruginosa influences the susceptibility of S. aureus to frontline antibiotics used to treat CF lung infections. Using our in vitro coculture model, we observed that addition of P. aeruginosa supernatants to S. aureus biofilms grown either on epithelial cells or on plastic significantly decreased the susceptibility of S. aureus to vancomycin. Mutant analyses showed that 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO, a component of the P. aeruginosa Pseudomonas quinolone signal (PQS system, protects S. aureus from the antimicrobial activity of vancomycin. Similarly, the siderophores pyoverdine and pyochelin also contribute to the ability of P. aeruginosa to protect S. aureus from vancomycin, as did growth under anoxia. Under our experimental conditions, HQNO, P. aeruginosa supernatant, and growth under anoxia decreased S. aureus growth, likely explaining why this cell wall-targeting antibiotic is less effective. P. aeruginosa supernatant did not confer additional protection to slow-growing S. aureus small colony variants. Importantly, P. aeruginosa supernatant protects S. aureus from other inhibitors of cell wall synthesis as well as protein synthesis-targeting antibiotics in an HQNO- and siderophore-dependent manner. We propose a model whereby P. aeruginosa causes S. aureus to shift to fermentative growth when these organisms are grown in coculture, leading to reduction in S. aureus growth and decreased susceptibility to antibiotics targeting cell wall and protein synthesis.

Evidence of extensive diversity in bacterial adherence mechanisms that exploit unanticipated stainless steel surface structural complexity for biofilm formation.

Science.gov (United States)

Davis, Elisabeth M; Li, Dongyang; Shahrooei, Mohammad; Yu, Bin; Muruve, Daniel; Irvin, Randall T

2013-04-01

Three protease-resistant bioorganic 304 stainless steel surfaces were created through the reaction of synthetic peptides consisting of the D-enantiomeric isomer (D-K122-4), the retro-inverso D-enantiomeric isomer (RI-K122-4), and a combination of the two peptides (D+RI) of the Pseudomonas aeruginosa PilA receptor binding domain with steel surfaces. The peptides used to produce the new materials differ only in handedness of their three-dimensional structure, but they reacted with the steel to yield materials that differed in their surface electron work function (EWF) while displaying an identical chemical composition and equivalent surface adhesive force properties. These surfaces allowed for an assessment of the relative role of surface EWF in initial biofilm formation. We examined the ability of various bacteria (selected strains of Listeria monocytogenes, L. innocua, Staphylococcus aureus and S. epidermidis) to initiate biofilm formation. The D-K1224 generated surface displayed the lowest EWF (classically associated with greater molecular interactions and more extensive biofilm formation) but was observed to be least effectively colonized by bacteria (>50% decrease in bacterial adherence of all strains). The highest surface EWF with the lowest surface free energy (RI-K122-4 generated) was more extensively colonized by bacteria, with the binding of some strains being equivalent to unmodified steel. The D+RI generated surface was least effective in minimizing biofilm formation, where some strains displayed enhanced bacterial colonization. Fluorescent microscopy revealed that the D and RI peptides displayed similar but clearly different binding patterns, suggesting that the peptides recognized different sites on the steel, and that differential binding of the peptides to the steel surfaces influences the binding of different bacterial strains and species. We have demonstrated that stainless steel surfaces can be easily modified by peptides to generate surfaces with

Emergent pattern formation in an interstitial biofilm

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Zachreson, Cameron; Wolff, Christian; Whitchurch, Cynthia B.; Toth, Milos

2017-01-01

Collective behavior of bacterial colonies plays critical roles in adaptability, survivability, biofilm expansion and infection. We employ an individual-based model of an interstitial biofilm to study emergent pattern formation based on the assumptions that rod-shaped bacteria furrow through a viscous environment and excrete extracellular polymeric substances which bias their rate of motion. Because the bacteria furrow through their environment, the substratum stiffness is a key control parameter behind the formation of distinct morphological patterns. By systematically varying this property (which we quantify with a stiffness coefficient γ ), we show that subtle changes in the substratum stiffness can give rise to a stable state characterized by a high degree of local order and long-range pattern formation. The ordered state exhibits characteristics typically associated with bacterial fitness advantages, even though it is induced by changes in environmental conditions rather than changes in biological parameters. Our findings are applicable to a broad range of biofilms and provide insights into the relationship between bacterial movement and their environment, and basic mechanisms behind self-organization of biophysical systems.

Elasticity and physico-chemical properties during drinking water biofilm formation.

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Abe, Yumiko; Polyakov, Pavel; Skali-Lami, Salaheddine; Francius, Grégory

2011-08-01

Atomic force microscope techniques and multi-staining fluorescence microscopy were employed to study the steps in drinking water biofilm formation. During the formation of a conditioning layer, surface hydrophobic forces increased and the range of characteristic hydrophobic forces diversified with time, becoming progressively complex in macromolecular composition, which in return triggered irreversible cellular adhesion. AFM visualization of 1 to 8 week drinking water biofilms showed a spatially discontinuous and heterogeneous distribution comprising an extensive network of filamentous fungi in which biofilm aggregates were embedded. The elastic modulus of 40-day-old biofilms ranged from 200 to 9000 kPa, and the biofilm deposits with a height >0.5 μm had an elastic modulus water biofilms were composed of a soft top layer and a basal layer with significantly higher elastic modulus values falling in the range of fungal elasticity.

The interconnection between biofilm formation and horizontal gene transfer

DEFF Research Database (Denmark)

Madsen, Jonas Stenløkke; Burmølle, Mette; Hansen, Lars H.

2012-01-01

Recent research has revealed that horizontal gene transfer and biofilm formation are connected processes. Although published research investigating this interconnectedness is still limited, we will review this subject in order to highlight the potential of these observations because....... Biofilms, furthermore, promote plasmid stability and may enhance the host range of mobile genetic elements that are transferred horizontally. Plasmids, on the other hand, are very well suited to promote the evolution of social traits such as biofilm formation. This, essentially, transpires because plasmids...... of their believed importance in the understanding of the adaptation and subsequent evolution of social traits in bacteria. Here, we discuss current evidence for such interconnectedness centred on plasmids. Horizontal transfer rates are typically higher in biofilm communities compared with those in planktonic states...

Wild Mushroom Extracts as Inhibitors of Bacterial Biofilm Formation

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Maria José Alves

2014-08-01

Full Text Available Microorganisms can colonize a wide variety of medical devices, putting patients in risk for local and systemic infectious complications, including local-site infections, catheter-related bloodstream infections, and endocarditis. These microorganisms are able to grow adhered to almost every surface, forming architecturally complex communities termed biofilms. The use of natural products has been extremely successful in the discovery of new medicine, and mushrooms could be a source of natural antimicrobials. The present study reports the capacity of wild mushroom extracts to inhibit in vitro biofilm formation by multi-resistant bacteria. Four Gram-negative bacteria biofilm producers (Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Acinetobacter baumannii isolated from urine were used to verify the activity of Russula delica, Fistulina hepatica, Mycena rosea, Leucopaxilus giganteus, and Lepista nuda extracts. The results obtained showed that all tested mushroom extracts presented some extent of inhibition of biofilm production. Pseudomonas aeruginosa was the microorganism with the highest capacity of biofilm production, being also the most susceptible to the extracts inhibition capacity (equal or higher than 50%. Among the five tested extracts against E. coli, Leucopaxillus giganteus (47.8% and Mycenas rosea (44.8% presented the highest inhibition of biofilm formation. The extracts exhibiting the highest inhibitory effect upon P. mirabilis biofilm formation were Sarcodon imbricatus (45.4% and Russula delica (53.1%. Acinetobacter baumannii was the microorganism with the lowest susceptibility to mushroom extracts inhibitory effect on biofilm production (highest inhibition—almost 29%, by Russula delica extract. This is a pioneer study since, as far as we know, there are no reports on the inhibition of biofilm production by the studied mushroom extracts and in particular against multi-resistant clinical isolates; nevertheless, other

Calcium-Phosphate-Osteopontin Particles Reduce Biofilm Formation and pH Drops in in situ Grown Dental Biofilms.

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Schlafer, Sebastian; Ibsen, Casper J S; Birkedal, Henrik; Nyvad, Bente

2017-01-01

This 2-period crossover study investigated the effect of calcium-phosphate-osteopontin particles on biofilm formation and pH in 48-h biofilms grown in situ. Bovine milk osteopontin is a highly phosphorylated glycoprotein that has been shown to interfere with bacterial adhesion to salivary-coated surfaces. Calcium-phosphate-osteopontin particles have been shown to reduce biofilm formation and pH drops in a 5-species laboratory model of dental biofilm without affecting bacterial viability. Here, smooth surface biofilms from 10 individuals were treated ex vivo 6 times/day for 30 min with either calcium-phosphate-osteopontin particles or sterile saline. After growth, the amount of biofilm formed was determined by confocal microscopy, and pH drops upon exposure to glucose were monitored using confocal-microscopy-based pH ratiometry. A total of 160 biofilms were analysed. No adverse effects of repeated ex vivo treatment with calcium-phosphate-osteopontin particles were observed. Particle treatment resulted in a 32% lower amount of biofilm formed (p Biofilm pH was significantly higher upon particle treatment, both shortly after the addition of glucose and after 30 min of incubation with glucose (p biofilms as well as the remineralizing potential of the particles. © 2016 S. Karger AG, Basel.

AI-2 of Aggregatibacter actinomycetemcomitans Inhibits Candida albicans Biofilm Formation

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Endang W. Bachtiar

2014-07-01

Full Text Available Aggregatibacter actinomycetemcomitans, a Gram-negative bacterium, and Candida albicans, a polymorphic fungus, are both commensals of the oral cavity but both are opportunistic pathogens that can cause oral diseases. A. actinomycetemcomitans produces a quorum-sensing molecule called autoinducer-2 (AI-2, synthesized by LuxS, that plays an important role in expression of virulence factors, in intra- but also in interspecies communication. The aim of this study was to investigate the role of AI-2 based signaling in the interactions between C. albicans and A. actinomycetemcomitans. A. actinomycetemcomitans adhered to C. albicans and inhibited biofilm formation by means of a molecule that was secreted during growth. C. albicans biofilm formation increased significantly when co-cultured with A. actinomycetemcomitans luxS, lacking AI-2 production. Addition of wild-type-derived spent medium or synthetic AI-2 to spent medium of the luxS strain, restored inhibition of C. albicans biofilm formation to wild-type levels. Addition of synthetic AI-2 significantly inhibited hypha formation of C. albicans possibly explaining the inhibition of biofilm formation. AI-2 of A. actinomycetemcomitans is synthesized by LuxS, accumulates during growth and inhibits C. albicans hypha- and biofilm formation. Identifying the molecular mechanisms underlying the interaction between bacteria and fungi may provide important insight into the balance within complex oral microbial communities.

Effect of chlorhexidine on oral airway biofilm formation of Staphylococcus epidermidis

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Ünase Büyükkoçak

2015-12-01

Full Text Available Objective: Biofilm formation of microorganisms on the surface of airways may lead to supraglottic colonization that may cause lower respiratuar tract infections. Studies searching the efficiency of local disinfectants on biofilm formation are limited. The aim of this study was to investigate the effects of chlorhexidine coated airways on biofilm formation of Staphylococcus epidermidis. Methods: Culture and electron microscopy methods were used for biofilm assessment. Airways were divided into two groups to investigate the effects of chlorhexidine on number of bacteria attached to the airway and biofilm formation. Group 1(control: naive material, S. epidermidis, Group 2: chlorhexidine coated material, S. epidermidis. No process was applied in Group 1. Chlorhexidine gluconate (0.2% was sprayed on the surface of naive material for four seconds and then left to dry in air, in Group to. Number of bacteria attached to the airway were counted by microbiological methods and biofilm formation was shown by Scanning Electron Microscope (SEM. Mann-Whitney u test was performed for statistical analyses. Results: In Group 2, bacteria numbers were 1x102-8x102 cfu/ml, whereas they were 3x103-1x104 cfu/ml in Group 1. Chlorhexidine decreased number of microorganisms attached to the airways with statistical significance (p=0.04. The results of the electron microscopic evaluation were in accordance with the acteriological findings. Conclusion: This study has shown that chlorhexidine coating can successfully reduce the number of adhered bacteria and biofilm formation on airways. J Microbiol Infect Dis 2015;5(4: 162-166

Bacterial adhesion and biofilm formation on surfaces of variable roughness and hydrophobicity

DEFF Research Database (Denmark)

Tang, Lone; Pillai, Saju; Iversen, Anders

L.Biofilm formation on surfaces in food production and processing can deteriorate the quality of food products and be a hazard to consumers. The food industry currently uses a number of approaches to either remove biofilm or prevent its formation. Due to the inherent resilience of bacteria...... in biofilm, a particularly attractive approach is the modification of surfaces with the aim to impede the first step in biofilm formation, namely bacterial adhesion. Surface properties such as hydrophobicity, roughness and predisposition for fouling by protein are recognised as important in bacterial...... adhesion. Sol-gel technology and the recent availability of organic modified silicas have lead to development of hybrid organic/inorganic glass ceramic coatings with specialised surface properties. In this study we investigate bacterial adhesion and the subsequent biofilm formation on stainless steel (SS...

Plectranthus amboinicus essential oil and carvacrol bioactive against planktonic and biofilm of oxacillin- and vancomycin-resistant Staphylococcus aureus.

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Vasconcelos, Sara Edwirgens Costa Benício; Melo, Hider Machado; Cavalcante, Theodora Thays Arruda; Júnior, Francisco Eduardo Aragão Catunda; de Carvalho, Mário Geraldo; Menezes, Francisca Gleire Rodrigues; de Sousa, Oscarina Viana; Costa, Renata Albuquerque

2017-09-16

The emergence of multidrug-resistant bacteria is a worldwide concern and in order to find an alternative to this problem, the occurrence of antimicrobial compounds in Plectranthus amboinicus essential oil was investigated. Thus, this study aims to determine susceptibility of Staphylococcus aureus isolated from food to antibiotics, P. amboinicus essential oil (PAEO) and carvacrol. Leaves and stem of P. amboinicus were used for extraction of essential oil (PAEO) by hydrodistillation technique and EO chemical analysis was performed by gas chromatography coupled to a mass spectrometer. S. aureus strains (n = 35) isolated from food and S. aureus ATCC 6538 were used to evaluate the antimicrobial and antibiofilm activity of PAEO and carvacrol. All strains (n = 35) were submitted to antimicrobial susceptibility profile by disk diffusion method. Determination of MIC and MBC was performed by microdilution technique and antibiofilm activity was determined by microtiter-plate technique with crystal violet assay and counting viable cells in Colony Forming Units (CFU). Carvacrol (88.17%) was the major component in the PAEO. Antibiotic resistance was detected in 28 S. aureus strains (80%) and 12 strains (34.3%) were oxacillin and vancomycin-resistant (OVRSA). From the 28 resistant strains, 7 (25%) showed resistance plasmid of 12,000 bp. All strains (n = 35) were sensitive to PAEO and carvacrol, with inhibition zones ranging from 16 to 38 mm and 23 to 42 mm, respectively. The lowest MIC (0.25 mg mL -1 ) and MBC (0.5 mg mL -1 ) values were observed when carvacrol was used against OVRSA. When a 0.5 mg mL -1 concentration of PAEO and carvacrol was used, no viable cells were found on S. aureus biofilm. The antibacterial effect of carvacrol and PAEO proves to be a possible alternative against planktonic forms and staphylococcal biofilm.

Streptococcus suis Serotype 2 Biofilms Inhibit the Formation of Neutrophil Extracellular Traps.

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Ma, Fang; Yi, Li; Yu, Ningwei; Wang, Guangyu; Ma, Zhe; Lin, Huixing; Fan, Hongjie

2017-01-01

Invasive infections caused by Streptococcus suis serotype 2 (SS2) has emerged as a clinical problem in recent years. Neutrophil extracellular traps (NETs) are an important mechanism for the trapping and killing of pathogens that are resistant to phagocytosis. Biofilm formation can protect bacteria from being killed by phagocytes. Until now, there have only been a few studies that focused on the interactions between bacterial biofilms and NETs. SS2 in both a biofilm state and a planktonic cell state were incubated with phagocytes and NETs, and bacterial survival was assessed. DNase I and cytochalasin B were used to degrade NET DNA or suppress phagocytosis, respectively. Extracellular DNA was stained with impermeable fluorescent dye to quantify NET formation. Biofilm formation increased up to 6-fold in the presence of neutrophils, and biofilms were identified in murine tissue. Both planktonic and biofilm cells induced neutrophils chemotaxis to the infection site, with neutrophils increasing by 85.1 and 73.8%, respectively. The bacteria in biofilms were not phagocytized. The bactericidal efficacy of NETs on the biofilms and planktonic cells were equal; however, the biofilm extracellular matrix can inhibit NET release. Although biofilms inhibit NETs release, NETs appear to be an important mechanism to eliminate SS2 biofilms. This knowledge advances the understanding of biofilms and may aid in the development of treatments for persistent infections with a biofilm component.

An Essential Role for Coagulase in Staphylococcus aureus Biofilm Development Reveals New Therapeutic Possibilities for Device-Related Infections.

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Zapotoczna, Marta; McCarthy, Hannah; Rudkin, Justine K; O'Gara, James P; O'Neill, Eoghan

2015-12-15

High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤ 24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin. In contrast, SarA-regulated biofilms formed on uncoated polystyrene in nutrient-rich bacteriological medium were mediated by the previously characterized biofilm factors poly-N-acetyl glucosamine, fibronectin-binding proteins, or autolytic activity and were antibiotic resistant. Coagulase-mediated biofilms exhibited increased antimicrobial resistance over time (>48 hours) but were always susceptible to dispersal by the fibrinolytic enzymes plasmin or nattokinase. Biofilms recovered from infected central venous catheters in a rat model of device-related infection were dispersed by nattokinase, supporting the important role of the biofilm phenotype and identifying a potentially new therapeutic approach with antimicrobials and fibrinolytic drugs, particularly during the early stages of device-related infection. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients

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César de la Fuente-Núñez

2014-10-01

Full Text Available Cystic fibrosis (CF patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1 and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α production by human peripheral blood mononuclear cells (PBMC and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.

Inhibition of biofilm formation in Bacillus subtilis by new halogenated furanones.

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Kayumov, Airat R; Khakimullina, Elvina N; Sharafutdinov, Irshad S; Trizna, Elena Y; Latypova, Lilia Z; Thi Lien, Hoang; Margulis, Anna B; Bogachev, Mikhail I; Kurbangalieva, Almira R

2015-05-01

Gram-positive bacteria can cause various infections including hospital-acquired infections. While in the biofilm, the resistance of bacteria to both antibiotics and the human immune system is increased causing difficulties in the treatment. Bacillus subtilis, a non-pathogenic Gram-positive bacterium, is widely used as a model organism for studying biofilm formation. Here we investigated the effect of novel synthesized chloro- and bromo-containing 2(5H)-furanones on biofilm formation by B. subtilis. Mucobromic acid (3,4-dibromo-5-hydroxy-2(5H)-furanone) and the two derivatives of mucochloric acid (3,4-dichloro-5-hydroxy-2(5H)-furanone)-F8 and F12-were found to inhibit the growth and to efficiently prevent biofilm formation by B. subtilis. Along with the low production of polysaccharide matrix and repression of the eps operon, strong repression of biofilm-related yqxM also occurred in the presence of furanones. Therefore, our data confirm that furanones affect significantly the regulatory pathway(s) leading to biofilm formation. We propose that the global regulator, Spo0A, is one of the potential putative cellular targets for these compounds.

Adhesion, biofilm formation, cell surface hydrophobicity and antifungal planktonic susceptibility: relationship among Candida spp.

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Ana Isabel Silva-Dias

2015-03-01

Full Text Available We have performed the characterization of the adhesion profile, biofilm formation, cell surface hydrophobicity (CSH and antifungal susceptibility of 184 Candida clinical isolates obtained from different human reservoirs. Adhesion was quantified using a flow cytometric assay and biofilm formation was evaluated using two methodologies: XTT and crystal violet assay. CSH was quantified with the microbial adhesion to hydrocarbons test while planktonic susceptibility was assessed accordingly the CLSI protocol for yeast M27-A3 S4.Yeast cells of non-albicans species exhibit increased ability to adhere and form biofilm. However the correlation between adhesion and biofilm formation varied according to species and also with the methodology used for biofilm assessment. No association was found between strain´s site of isolation or planktonic antifungal susceptibility and adhesion or biofilm formation. Finally CSH seemed to be a good predictor for biofilm formation but not for adhesion.Despite the marked variability registered intra and inter species, C. tropicalis and C. parapsilosis were the species exhibiting high adhesion profile. C. tropicalis, C. guilliermondii and C. krusei revealed higher biofilm formation values in terms of biomass. C. parapsilosis was the species with lower biofilm metabolic activity.

Adhesion, biofilm formation, cell surface hydrophobicity, and antifungal planktonic susceptibility: relationship among Candida spp.

Science.gov (United States)

Silva-Dias, Ana; Miranda, Isabel M; Branco, Joana; Monteiro-Soares, Matilde; Pina-Vaz, Cidália; Rodrigues, Acácio G

2015-01-01

We have performed the characterization of the adhesion profile, biofilm formation, cell surface hydrophobicity (CSH) and antifungal susceptibility of 184 Candida clinical isolates obtained from different human reservoirs. Adhesion was quantified using a flow cytometric assay and biofilm formation was evaluated using two methodologies: XTT and crystal violet assay. CSH was quantified with the microbial adhesion to hydrocarbons test while planktonic susceptibility was assessed accordingly the CLSI protocol for yeast M27-A3 S4. Yeast cells of non-albicans species exhibit increased ability to adhere and form biofilm. However, the correlation between adhesion and biofilm formation varied according to species and also with the methodology used for biofilm assessment. No association was found between strain's site of isolation or planktonic antifungal susceptibility and adhesion or biofilm formation. Finally CSH seemed to be a good predictor for biofilm formation but not for adhesion. Despite the marked variability registered intra and inter species, C. tropicalis and C. parapsilosis were the species exhibiting high adhesion profile. C. tropicalis, C. guilliermondii, and C. krusei revealed higher biofilm formation values in terms of biomass. C. parapsilosis was the species with lower biofilm metabolic activity.

Ultrastructural changes in biofilm forms of staphylococci cultivated in a mixed culture with lactobacilli

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G. Lavryk

2017-02-01

Full Text Available The capacity of opportunistic bacteria for biofilm formation plays an important role in the development of chronic inflammatory processes, which are difficult to treat. To improve antimicrobial therapy methods, the influence of lactobacilli on the ultrastructure of biofilm-forming clinical strains of staphylococci when co-cultured was investigated. 5 biofilm-forming clinical strains of S. aureus from the skin of acne vulgaris patients (n = 24 were isolated. Using transmission electron microscopy (TEM the morphological changes of S. aureus cells in the mixed culture with standard strains of Lactobacillus plantarum 8P-A3 and clinical strains of L. fermentum (n = 4 were studied. It was found that in 48 hours after the inoculation on the medium of samples of mixed cultures of L. plantarum 8P-A3 and S. aureus growth of staphylococci was not revealed. Only in some cases of mixed cultures of L. fermentum and biofilm-forming staphylococci was growth of S. aureus obtained. In electron diffraction patterns of control samples of 24-hour staphylococcal monocultures and 48-hour lactobacilli monocultures, natural development of the population at the cellular level was observed. Destructive changes under the influence of lactobacilli (probiotic and clinical strains were detected in all ultrathin sections of the cells of biofilm-forming and planktonic staphylococci. Significant destructive changes in the cell wall of the staphylococci were observed: thickening, obtaining of irregular form, detachment of the cytoplasmic membrane, the complete destruction of the peptidoglycan layer and the emergence of "shadow cells". On all electron diffraction patterns fibrillar-threadlike structures of DNA could not be observed, but in some cases mesosome-like formations were poorly contrasted. It was established that the surface S-layer of lactobacilli was expressed on a significantly larger scale in the mixed culture with staphylococci. In mixed culture of clinical strains

Interference of Pseudomonas aeruginosa signalling and biofilm formation for infection control

DEFF Research Database (Denmark)

Bjarnsholt, Thomas; Tolker-Nielsen, Tim; Høiby, Niels

2010-01-01

Pseudomonas aeruginosa is the best described bacterium with regards to quorum sensing (QS), in vitro biofilm formation and the development of antibiotic tolerance. Biofilms composed of P. aeruginosa are thought to be the underlying cause of many chronic infections, including those in wounds...... and in the lungs of patients with cystic fibrosis. In this review, we provide an overview of the molecular mechanisms involved in QS, QS-enabled virulence, biofilm formation and biofilm-enabled antibiotic tolerance. We now have substantial knowledge of the multicellular behaviour of P. aeruginosa in vitro. A major...

SMU.940 regulates dextran-dependent aggregation and biofilm formation in Streptococcus mutans.

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Senpuku, Hidenobu; Yonezawa, Hideo; Yoneda, Saori; Suzuki, Itaru; Nagasawa, Ryo; Narisawa, Naoki

2018-02-01

The oral bacterium Streptococcus mutans is the principal agent in the development of dental caries. Biofilm formation by S. mutans requires bacterial attachment, aggregation, and glucan formation on the tooth surface under sucrose supplementation conditions. Our previous microarray analysis of clinical strains identified 74 genes in S. mutans that were related to biofilm morphology; however, the roles of almost all of these genes in biofilm formation are poorly understood. We investigated the effects of 21 genes randomly selected from our previous study regarding S. mutans biofilm formation, regulation by the complement pathway, and responses to competence-stimulating peptide. Eight competence-stimulating peptide-dependent genes were identified, and their roles in biofilm formation and aggregation were examined by mutational analyses of the S. mutansUA159 strain. Of these eight genes, the inactivation of the putative hemolysin III family SMU.940 gene of S. mutansUA159 promoted rapid dextran-dependent aggregation and biofilm formation in tryptic soy broth without dextrose (TSB) with 0.25% glucose and slightly reduced biofilm formation in TSB with 0.25% sucrose. The SMU.940 mutant showed higher expression of GbpC and gbpC gene than wild-type. GbpC is known to be involved in the dextran-dependent aggregation of S. mutans. An SMU.940-gbpC double mutant strain was constructed in the SMU.940 mutant background. The gbpC mutation completely abolished the dextran-dependent aggregation of the SMU.940 mutant. In addition, the aggregation of the mutant was abrogated by dextranase. These findings suggest that SMU.940 controls GbpC expression, and contributes to the regulation of dextran-dependent aggregation and biofilm formation. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Copper-Containing Anti-Biofilm Nanofiber Scaffolds as a Wound Dressing Material.

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Jayesh J Ahire

Full Text Available Copper particles were incorporated into nanofibers during the electrospinning of poly-D,L-lactide (PDLLA and poly(ethylene oxide (PEO. The ability of the nanofibers to prevent Pseudomonas aeruginosa PA01 and Staphylococcus aureus (strain Xen 30 to form biofilms was tested. Nanofibers containing copper particles (Cu-F were thinner (326 ± 149 nm in diameter, compared to nanofibers without copper (CF; 445 ± 93 nm in diameter. The crystalline structure of the copper particles in Cu-F was confirmed by X-ray diffraction (XRD. Copper crystals were encapsulated, but also attached to the surface of Cu-F, as shown scanning transmission electron microscopy (STEM and transmission electron microscopy (TEM, respectively. The copper particles had no effect on the thermal degradation and thermal behaviour of Cu-F, as shown by thermogravimetric analysis (TGA and differential scanning calorimeter (DSC. After 48 h in the presence of Cu-F, biofilm formation by P. aeruginosa PA01 and S. aureus Xen 30 was reduced by 41% and 50%, respectively. Reduction in biofilm formation was ascribed to copper released from the nanofibers. Copper-containing nanofibers may be incorporated into wound dressings.

Effects of Myrcia ovata Cambess. essential oil on planktonic growth of gastrointestinal microorganisms and biofilm formation of Enterococcus faecalis

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Cinthya S. Cândido

2010-10-01

Full Text Available The essential oil from the leaves of Myrcia ovata Cambess., commonly used in Brazil for the treatment of gastric illnesses, was screened for antimicrobial activity and action in the formation of microbial biofilms by Enterococcus faecalis. The oil was obtained by hydrodistillation using a clevenger-type system. Its chemical composition was analyzed using GC and GC-MS. Both MIC and MBC of the essential oil were determined by broth microdilution techniques and agar dilution method. The essential oil showed antimicrobial activity against E. faecalis, Escherichia coli, Pseudomonas aeruginosa, Salmonella choleraesuis, Staphylococcus aureus, Streptococcus pneumoniae and Candida parapsilosis. The results showed that the essential oil of M. ovata Cambess. was effective against the formation of biofilm by E. faecalis when compared with the control. Four volatile compounds, representing 92.1 % of the oil, were identified and geranial was the major component (50.4 %. At the best of our knowledge, this is the first report of the chemical composition and antimicrobial activity of the essential oil from leaves of M. ovata.

Novel Treatment of Staphylococcus aureus Device-Related Infections Using Fibrinolytic Agents.

Science.gov (United States)

Hogan, S; O'Gara, J P; O'Neill, E