Carvalhais, Virgínia Maria Dinis

Cerca, Nuno

França, Ângela Maria Oliveira Sousa

Pier, Gerald B.

Vilanova, M.

Universidade do Minho: RepositoriUM

1076th ASM Conference on Biofilmsn 81ALOW LEVELS Of β-LAcTAM ANTIBIOTIcS INducE ExTRAcELLuLAR dNA RELEASE ANd BIOfILM fORMATION IN STAPHYLOcOccuS AuREuSJ. B. Kaplan1, E. Izano1, P. Gopal1, M. T. Kar-wacki1, S. Kim1, J. L. Bose2, K. W. Bayles2, A. R. Horswill3; 1Univ. of Med. and Dentistry of New Jersey, Newark, NJ, 2University of Nebraska Medical Center, Omaha, NE, 3University of Iowa, Iowa City, IA.Subminimal inhibitory concentrations of an-tibiotics have been shown to induce bacterial biofilm formation. Few studies have investi-gated antibiotic-induced biofilm formation in Staphylococcus aureus, an important human pathogen. Our goal was to measure S. aureus biofilm formation in the presence of low levels of β-lactam antibiotics. Fifteen phylogeneti-cally diverse MRSA and MSSA strains were employed. Methicillin, ampicillin, amoxicillin or cloxicillin were added to cultures at con-centrations ranging from 0-1 MIC. Biofilm formation was measured in 96-well microtiter plates using a crystal violet binding assay. Autoaggregation was measured using a visual test tube settling assay. Extracellular DNA was quantitated using agarose gel electrophoresis. All four antibiotics induced biofilm formation in some strains. The amount of biofilm induc-tion was as high as 10-fold and was inversely proportional to the amount of biofilm produced by the strain in the absence of antibiotics. MRSA strains of lineages USA300, USA400 and USA500 exhibited the highest levels of methicillin-induced biofilm induction. Biofilm formation induced by low-level methicillin was inhibited by DNase. Low-level methicillin also induced DNase-sensitive autoaggregation and extracellular DNA release. The biofilm induction phenotype was absent in an autolysin (atl) deficient strain. Our findings demonstrate that subminimal inhibitory concentrations of β-lactam antibiotics significantly induce autolysin-dependent extracellular DNA release and biofilm formation in some strains of S. aureus. More studies are needed to determine the mechanism and potential clinical relevance of this process.n 82AstaPhylococcus EPidErmidis BIOfILM-dETAcHEd cELLS: dIffERENTIAL GENE ExPRESSION ANd ANTIBIOTIc RESISTANcEÂ. França1, V. Carvalhais2, M. Vilanova3, G. Pier4, N. Cerca1; 1University of Minho, Braga, PORTUGAL, 2University of Porto and University of Minho, Porto, Braga, PORTUGAL, 3University of Porto (ICBAS), Porto, PORTUGAL, 4Harvard Medical School, Boston, MA.Staphylococcus epidermidis has emerged over the last 3 decades as a leading cause of nosocomial infections due to its ability to form biofilms, particularly, on the surface of indwelling medical devices. The detachment of cells from the biofilm by both active and pas-sive mechanisms is believed to be crucial for the dissemination of infection, often leading to serious complications such as embolic events, endocarditis, sepsis and pneumonia. Hence, a better characterization of biofilm-detached cells may help to employ more effective strate-gies against biofilm-related infections. In order to characterize the phenotype of S. epidermidis biofilm-detached cells, we assessed cell prop-erties by measuring growth curves, expres-sion profiles of key genes involved in initial adhesion, biofilm regulation, detachment and immune evasion (atlE, agrB, psmβ1, rsbU and icaA), and the susceptibility to vancomycin, a cell wall synthesis inhibitor, and tetracycline, a protein synthesis inhibitor, comparing plank-tonic, biofilm-detached and biofilm-derived cell populations. Despite their planktonic state, biofilm-detached cells had some specific fea-tures of biofilm cells, including low expression levels of agrB and higher expression of rsbU and ica transcripts. In contrast, the biofilm-detached cells had higher expression of psmβ1 transcripts, a class of surfactant peptides that Poster AbstractsASM Conferences108has been related to dispersal mechanisms, with the biofilm-detached cells resembling the psmβ1 expression profile of planktonic cells. Similar to biofilm-derived cells, the antibi-otic susceptibility properties of the biofilm-detached cells showed more resistance to tetracycline than stationary planktonic cultures. For vancomycin susceptibility, no differences were found among the 3 populations. Finally, biofilm-detached cells mostly followed the growth kinetics of planktonic cells with only small differences found, and had a higher growth rate compared to biofilm cells during the first 5 h of culture. The results suggest that biofilm-detached S. epidermidis cells may constitute a distinct phenotype, present-ing some features of biofilm-derived cells and other features associated with the planktonic cell phenotype. Targeting the properties of the biofilm-detached cells could present opportuni-ties to more effectively treat these infections and prevent the pathologic events associated with dissemination of cells from a biofilm to more distant sites.n 83AA MOLEcuLAR BASIS fOR NO/H-NOx REGuLATION Of BAcTERIAL BIOfILMSE. M. Boon; Stony Brook University, Stony Brook, NY.Although several reports have documented nitric oxide (NO) regulation of biofilm forma-tion, the molecular basis of this phenomenon is unknown. In many bacteria, a H-NOX (heme-nitric oxide/oxygen binding) gene is found near a di-guanylate cyclase (DGC) gene. H-NOX domains are conserved hemoproteins that are known NO sensors. It is widely recognized that cyclic-di-GMP is a ubiquitous bacterial signaling molecule that regulates the transi-tion between motility and biofilm. Therefore, NO may influence biofilm formation through H-NOX regulation of DGC, thus providing a molecular-level explanation for NO regulation of biofilm formation. We have demonstrated that, indeed, NO/H-NOX negatively affects biofilm formation by directly regulating c-di-GMP turnover in several different bacterial species. Evidence from biochemical charac-terization of proteins in the H-NOX signaling pathway, genetic, and growth studies will be presented to support our conclusions.n 84AMORPHOLOGIcAL cHANGE Of cLOSTRIdIAL BIOfILMS IN RESPONSE TO TEMPERATuREN. Obana, K. Nakamura, N. Nomura; University of Tsukuba, Tsukuba, JAPAN.Clostridium perfringens is a gram-positive anaerobic spore-forming bacterium that is widespread in environmental soil and sewage, as well as in the gastrointestinal tract of hu-mans and animals. It produces numerous extra-cellular enzymes and toxins, and is a causative agent of gas angrene and food poisoning. Recently, it have been reported that C. perfrin-gens also formed biofilms providing increased resistance to oxygen and antibiotics, suggest-ing that the biofilm formation is involved in the pathogenesis. However, environmental signals and regulatory genes involved in the biofilm formation in Clostridium remained unclear. C. perfringens strain 13 formed biofilms as-sociated with the substrate surface when the cells were cultured in flat-bottom wells at 37°C (henceforth called “adhered biofilms”). On the other hand, at 25°C, C. perfringens formed pellicle-like viscous biofilms on the bottom of the culture well but not adhered to the substrate surface (henceforth called “pellicle biofilms”). Thus, the morphology of C. perfringens bio-films changes in response to temperature. Con-focal laser scanning microscopy revealed that the cells were densely packed in the adhered biofilms, but in the pellicle biofilms, the dis-tance between each cell was large and the cell density was low, suggesting that the pellicle biofilms predominantly contain the extracel-lular matrix. We previously identified a protein Poster Abstracts6th ASM Conference onBiofilms September 29 – October 4, 2012 Miami, Florida Final Program and Abstracts© 2012, American Society for Microbiology1752 N Street, N.W.Washington, DC  20036-2904Phone: 202-737-3600World Wide Web: www.asm.orgAll Rights ReservedPrinted in the United States of AmericaISBN: 978-1-55581-876-0

Staphylococcus epidermidis biofilm-detached cells: differential gene expression and antibiotic resistance

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