Rosa spp. Extracts as a Factor That Limits the Growth of Staphylococcus spp. Bacteria, a Food Contaminant.

Due to their richness of bioactive substances, rose hips are a valuable raw material for obtaining extracts with potential antimicrobial activity. The aim of the study was to determine the antagonistic potential of whole pseudo-fruit and flesh extracts of three Rosa sp. varieties against Staphylococcus spp. bacteria isolated as food contaminants. The biological material in this study consisted of seven strains of bacteria from the genus Staphylococcus. Two strains-Staphylococcus aureus ATCC 25923 and Staphylococcus epidermidis DSMZ 3270-were used as reference strains. The other five strains were food-derived isolates-S. epidermidis A5, S. xylosus M5, S. haemolyticus M6, S. capitis KR6, and S. warneri KR2A. The material was the pseudo-fruits of Rosa canina, Rosa pomifera Karpatia, and Rosa rugosa. The polyphenols were extracted from the fleshy part and the whole pseudo-fruit for all rose varieties. The tested preparations differed significantly in their polyphenol composition. The sum of polyphenols ranged from 28 862 to 35 358 mg/100 g of lyophilisate. The main groups of polyphenols found in the preparations were flavanols and ellagitannins. All of the tested extracts inhibited the growth of staphylococci at a concentration of 500 mg/mL. Rosa rugosa fruit extract showed the strongest antimicrobial properties among the studied extracts. For all the strains, the growth inhibition had a diameter of 20.3-29.0 mm. Moreover, six out of the seven tested strains showed the highest inhibition with the use of this extract. The MIC of rose extracts was in the range of 3.125-500 mg/mL and was strictly dependent on the bacterial species, the species of the rose, and the part of the fruit from which the extract was obtained. Correlations were assessed between the main groups of polyphenols in the extracts and their inhibition of bacterial growth. In the case of pseudo-fruit extracts, the inhibitory effect on bacterial growth positively correlated with the content of ellagitannins, and this effect was observed for almost all the tested strains. The results presented herein follow the current trend of minimising the use of chemical preservatives in food; from this point of view, rose extracts are very promising.

Identification of surface proteins in a clinical Staphylococcus haemolyticus isolate by bacterial surface shaving.

BACKGROUND: The skin commensal Staphylococcus haemolyticus is an emerging nosocomial pathogen. Despite its clinical relevance, published information about S. haemolyticus virulence factors is scarce. In this study, the adhesive and biofilm forming properties of ten clinical and ten commensal S. haemolyticus strains were examined using standard adhesion and biofilm assays. One of the clinical strains was used to identify expressed surface proteins using bacterial surface shaving. Protein abundance was examined by a comparative analysis between bacterial protein expression after human keratinocyte (HaCaT) colonization and growth in cell culture media supplemented with serum. Relative protein quantification was performed by labeling peptides with tandem mass tags (TMT) prior to Mass Spectrometry analysis. Surface proteins can be used as novel targets for antimicrobial treatment and in diagnostics. RESULTS: Adherence to fibronectin, collagen and plastic was low in all tested strains, but with significantly higher adhesion to fibronectin (p = 0.041) and collagen (p = 0.001) in the commensal strains. There was a trend towards higher degree of biofilm formation in the clinical strains (p = 0.059). By using surface shaving, 325 proteins were detected, of which 65 were classified as surface proteins. Analyses showed that the abundance of nineteen (5.8%) proteins were significantly changed following HaCaT colonization. The bacterial Toll/interleukin-1 like (TIRs) domain containing protein (p = 0.04), the transglycosylase SceD (p = 0.01), and the bifunctional autolysin Atl (p = 0.04) showed a 1.4, 1.6- and 1.5-fold increased abundance. The staphylococcal secretory antigen (SsaA) (p = 0.04) was significantly downregulated (- 1.5 fold change) following HaCaT colonization. Among the 65 surface proteins the elastin binding protein (Ebps), LPXAG and LPXSG domain containing proteins and five LPXTG domain containing proteins were identified; three Sdr-like proteins, the extracellular matrix binding protein Embp and a SasH-like protein. CONCLUSIONS: This study has provided novel knowledge about expression of S. haemolyticus surface proteins after direct contact with eukaryotic cells and in media supplemented with serum. We have identified surface proteins and immune evasive proteins previously only functionally described in other staphylococcal species. The identification of expressed proteins after host-microbe interaction offers a tool for the discovery and design of novel targets for antimicrobial treatment.

Influence of antibiotics on biofilm formation by different clones of nosocomial Staphylococcus haemolyticus.

Staphylococcus haemolyticus is the most common organism among clinical isolatesof methicillin-resistant staphylococci. Aim: This study evaluated the ability to produce biofilm with the presence of the antibiotics (1/4 minimum inhibitory concentrations) of S. haemolyticus strains isolated from blood culture. Methods: Clonal distribution was assessed in pulsed-field gel electrophoresis. PCR assays were performed to detect mecA, icaA, aap, atlE, atl, fbp genes. S. haemolyticus strains grown in the presence of the antibiotics were investigated for biofilm formation on glass, polystyrene and catheter surfaces. Results: Biofilm formation was independent of the presence of the icaA and mecA genes, pulsed-field gel electrophoresis type. Vancomycin, oxacillin, moxifloxacin, rifampicin, teicoplanin, tigecycline and linezolid did not inhibit biofilm formation on abiotic surfaces. Conclusion: This study demonstrated that the biofilm formation process is complex and may not be related to ica gene carriage. Furthermore, in this study the biofilm formation was increased in the presence of antimicrobial agents.

Bacterial Coaggregation and Cohesion Among Isolates From Contact Lens Cases.

Purpose: The aim of this study was to examine cohesion, coaggregation, and coculture between bacteria commonly isolated from contact lens cases. Methods: Staphylococcus epidermidis, Staphylococcus haemolyticus, Micrococcus luteus, and Acinetobacter radioresistens (two strains each) isolated from contact lens cases of two asymptomatic wearers were used in this study. In the cohesion assay, bacteria were grown, washed, and examined by incubating lens cases with two different types of bacteria sequentially and assessing the number of adhered cells of each isolate. The ability of isolates to interfere with the growth of other isolates was tested by growing strains in cocultures for 24 hours and determining the numbers of cells of individual strains. For coaggregation, equal proportions of two bacterial suspensions were mixed and allowed to coaggregate for 24 hours. Inhibition of coaggregation was tested by the addition of lactose (0.06 M) or sucrose (0.06 M) or pronase. Results: The initial adhesion of M. luteus or A. radioresistens significantly (P < 0.05) enhanced the subsequent adhesion of the staphylococci. The addition of A. radioresistens in liquid media significantly (P < 0.05) enhanced the growth of staphylococci. S. epidermidis or S. haemolyticus coaggregated with M. luteus or A. radioresistens. The degree of coaggregation varied between 30% and 54%. The highest coaggregation (54% ± 5%) was seen between A. radioresistens 22-1 and S. epidermidis 22-1, isolated from the same lens case. Only lactose or sucrose treatment of staphylococci could partly inhibit coaggregation of some pairs. Conclusions: Coaggregation, cohesion, and growth promotion may facilitate the process of bacterial colonization of contact lens cases.

Activities of Oxadiazole Antibacterials against Staphylococcus aureus and Other Gram-Positive Bacteria.

The activities of four oxadiazoles were investigated with 210 methicillin-resistant Staphylococcus aureus (MRSA) strains. MIC50 and MIC90 values of 1 to 2 and 4 µg/ml, respectively, were observed. We also evaluated the activity of oxadiazole ND-421 against other staphylococci and enterococci and in the presence of oxacillin for selected MRSA strains. The MIC for ND-421 is lowered severalfold in combination with oxacillin, as they synergize. The MIC90 of ND-421 against vancomycin-resistant enterococci is ≤1 µg/ml.

Comparative study on nutrient depletion-induced lipidome adaptations in Staphylococcus haemolyticus and Staphylococcus epidermidis.

Staphylococcus species are emerging opportunistic pathogens that cause outbreaks of hospital and community-acquired infections. Some of these bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) are difficult to treat due to their resistance to multiple antibiotics. We carried out a comparative study on the lipidome adaptations in response to starvation in the two most common coagulase-negative Staphylococcus species: a S. epidermidis strain sensitive to ampicillin and erythromycin and a S. haemolyticus strain resistant to both. The predominant fatty acid composition in glycerolipids was (17:0-15:0) in both bacteria. During the exponential phase, the two bacterial lipidomes were similar. Both were dominated by diacylglycerol (DAG), phosphatidylglycerol (PG), lysyl-phosphatidylglycerol (Lysyl-PG) and Diglucosyl-diacylglycerol (DGDG). Alanyl-PG was detected in small amounts in both bacterial lipids. N-succinyl-lysyl-PG was detected only in S. haemolyticus, while lysyl-DAG only in S. epidermidis. As the two bacteria entered stationary phase, both lipidomes became essentially nitrogen-free. Both bacteria accumulated large amounts of free fatty acids. Strikingly, the lipidome of S. epidermidis became dominated by cardiolipin (CL), while that of S. haemolyticus was simplified to DGDG and PG. The S. epidermidis strain also produced acyl-phosphatidylglycerol (APG) in the stationary phase.

Skin squames contribute to ammonia and volatile fatty acid production from bacteria colonizing in air-cooling units with odor complaints.

One of the most notable Indoor Air Quality problems is odor emission. This study investigated the potential contribution of skin squames to the production of ammonia (NH3 ) and volatile organic acids (VFAs) by 7 bacteria isolated from air-cooling (AC) units with complaints of urine and body odors. Our previous study showed that keratinolytic activity is higher in AC units with odor complaints than those without. In the offices where these units are located, the most likely source of keratins is from human skin squames. Most bacteria can produce NH3 and VFAs in the skin squame culture. Some correlations between the levels of NH3 , NH4+, VFAs, and keratinolytic activity were found. The odor production pathway with skin squames was proposed. Staphylococcus haemolyticus was abundant in the AC units with odor problems and had a high level of keratinolytic activity in addition to odor production. For long-term odor control, it is important to reduce the level of skin squames entering the AC units.

Biofilm formation of methicillin-resistant coagulase negative staphylococci (MR-CoNS) isolated from community and hospital environments.

Methicillin-resistant coagulase negative staphylococci (MR-CoNS) are the major cause of infectious diseases because of their potential ability to form biofilm and colonize the community or hospital environments. This study was designed to investigate the biofilm producing ability, and the presence of mecA, icaAD, bap and fnbA genes in MR-CoNS isolates. The MR-CoNS used in this study were isolated from various samples of community environment and five wards of hospital environments, using mannitol salt agar (MSA) supplemented with 4 µg/ml of oxacillin. The specie level of Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus hominis and Staphylococcus warneri was identified by specific primers of groESL (S. haemolyticus), rdr (S. epidermidis) and nuc (S. hominis and S. warneri). The remainder isolates were identified by tuf gene sequencing. Biofilm production was determined using Congo red agar (CRA) and Microtiter plate (MTP) assay. The mecA and biofilm associated genes (icaAD, fnbA and bap) were detected using PCR method. From the 558 samples from community and hospital environments, 292 MR-CoNS were isolated (41 from community environments, and 251 from hospital environments). S. haemolyticus (41.1%) and S. epidermidis (30.1%) were the predominant species in this study. Biofilm production was detected in 265 (90.7%) isolates by CRA, and 260 (88.6%) isolates were detected by MTP assay. The staphylococci isolates derived from hospital environments were more associated with biofilm production than the community-derived isolates. Overall, the icaAD and bap genes were detected in 74 (29.5%) and 14 (5.6%) of all isolates from hospital environments. When tested by MTP, the icaAD gene from hospital environment isolates was associated with biofilm biomass. No association was found between bap gene and biofilm formation. The MR-CoNS isolates obtained from community environments did not harbor the icaAD and bap genes. Conversely, fnbA gene presented in MR-CoNS isolated from both community and hospital environments. The high prevalence of biofilm producing MR-CoNS strains demonstrated in this study indicates the persisting ability in environments, and is useful in developing prevention strategies countering the spread of MR-CoNS.

Licheniocin 50.2 and Bacteriocins from Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 Inhibit Biofilms of Coagulase Negative Staphylococci and Listeria monocytogenes Clinical Isolates.

BACKGROUND: Coagulase negative staphylococci (CoNS) and Listeria monocytogenes have important roles in pathogenesis of various genital tract infections and fatal foetomaternal infections, respectively. The aim of our study was to investigate the inhibitory effects of two novel bacteriocins on biofilms of CoNS and L. monocytogenes genital isolates. METHODS: The effects of licheniocin 50.2 from Bacillus licheniformis VPS50.2 and crude extract of bacteriocins produced by Lactococcus lactis subsp. lactis biovar. diacetylactis BGBU1-4 (BGBU1-4 crude extract) were evaluated on biofilm formation and formed biofilms of eight CoNS (four S. epidermidis, two S. hominis, one S. lugdunensis and one S. haemolyticus) and 12 L. monocytogenes genital isolates. RESULTS: Licheniocin 50.2 and BGBU1-4 crude extract inhibited the growth of both CoNS and L. monocytogenes isolates, with MIC values in the range between 200-400 AU/ml for licheniocin 50.2 and 400-3200 AU/ml for BGBU1-4 crude extract. Subinhibitory concentrations (1/2 × and 1/4 × MIC) of licheniocin 50.2 inhibited biofilm formation by all CoNS isolates (p < 0.05, respectively), while BGBU1-4 crude extract inhibited biofilm formation by all L. monocytogenes isolates (p < 0.01 and p < 0.05, respectively). Both bacteriocins in concentrations of 100 AU/mL and 200 AU/mL reduced the amount of 24 h old CoNS and L. monocytogenes biofilms (p < 0.05, p < 0.01, p < 0.001). CONCLUSIONS: This study suggests that novel bacteriocins have potential to be used for genital application, to prevent biofilm formation and/or to eradicate formed biofilms, and consequently reduce genital and neonatal infections by CoNS and L. monocytogenes.

[THE MYCOBIOTA OF TUNICA MUCOSA OF MOUTH AND SURFACE OF REMOVABLE ACRYLIC LAMINAR DENTAL PROSTHESES UNDER ORTHOPEDIC REHABILITATION].

The analysis was carried out to detect mycobiota of tunica mucosa of mouth and surface of dental prostheses under orthopedic rehabilitation using removable acrylic laminar dental prostheses. The inoculation of biosamples received from examined patients permitted to isolate Candida albicans. The C. albicans from tunica mucosa of mouth of patients before prosthetics inoculated in low concentration making up 0.33±0.23 CFU/ml in comparison with concentration of 1.92±0.53 CFU/ml after prosthetics. The highest content of C. albicans was marked in biosample from surface of dental prostheses in comparison with biotope of tunica mucosa of mouth of patients. The concentration of microbiota from surface of dental prostheses signicantly surpassed the same on tunica mucosa of mouth of patients prior prosthetics. In patients with removable acrylic laminar dental prostheses under orthopedic rehabilitation various spectrum of representatives of microbiota was detected From biosamples from surface of dentalprostheses of patients the most frequently were inoculated such representatives of gram-positive microbiota as S. aureus, Micrococcus spp., S.haemolyticus, and of gram-negative microbiota Klebsiella pneumonae, Pseudomonas aeruginosa. The cultural analysis of biosamples from patients with removable acrylic laminar dental prostheses detected Candida albicans on tunica mucosa of mouth before and after prosthetics as well as on surfaces of prostheses. The highest concentration of C.albicans is established in case of colonization of removable acrylic laminar dental prostheses. The received data testifies possible involvement of fungi capable of expressed potential ofpathogenicity, in development and maintenance of inflammatory process of tunica mucosa of mouth under orthopedic rehabilitation using removable acrylic laminar dental prostheses.

Isolation and characterization of two novel psychrotrophic decabromodiphenyl ether-degrading bacteria from river sediments.

Decabromodiphenyl ether (BDE-209) is a brominated flame retardant and a priority contaminant. Currently, little information is available about its significance in the environment, specifically about its susceptibility to aerobic biotransformation at low temperature. In this work, five phylogenetically diverse BDE-209-degrading bacterial strains were isolated from river sediments of northern China. These strains were distributed among four different genera-Acinetobacter, Pseudomonas, Bacillus and Staphylococcus. All five isolates were capable of growing on BDE-209, among which two isolates show better growth. By detailed morphological, physiological, and biochemical characteristics and 16S rDNA sequence analysis, the two strains were identified and named as Staphylococcus haemolyticus LY1 and Bacillus pumilus LY2. The two bacteria can grow in mineral salt medium containing BDE-209 substrate across the temperatures ranging from 2.5 to 35 °C, with an optimum temperature of 25 °C which could be considered as psychrotrophs accordingly. The degradation experiment showed that more than 70.6 and 85.5 % of 0.5 mg/L BDE-209 were degraded and the highest mineralization efficiencies of 29.8 and 39.2 % were achieved for 0.5 mg/L BDE-209 by S. haemolyticus LY1 and B. pumilus LY2, respectively. To the best of our knowledge, this is the first demonstration for the biodegradation of BDE-209 by two psychrotrophic bacteria isolated from environment.

Susceptibility Profile of Staphylococcus epidermidis and Staphylococcus haemolyticus Isolated from Blood Cultures to Vancomycin and Novel Antimicrobial Drugs over a Period of 12 Years.

The aim of this study was to evaluate the antimicrobial susceptibility profile of 85 Staphylococcus epidermidis and 84 Staphylococcus haemolyticus strains isolated from blood cultures to oxacillin, vancomycin, tigecycline, linezolid, daptomycin, and quinupristin/dalfopristin over a period of 12 years. S. epidermidis and S. haemolyticus isolated from blood cultures of inpatients, attended at a teaching hospital, were analyzed for the presence of the mecA gene and by SCCmec typing. The minimum inhibitory concentration (MIC) values of tigecycline, linezolid, daptomycin, quinupristin/dalfopristin, and vancomycin were determined. Isolates exhibiting vancomycin MICs of ≥2 µg/ml were typed by pulsed-field gel electrophoresis (PFGE). The rate of mecA positivity was 92.9% and 100% in S. epidermidis and S. haemolyticus, respectively. The most frequent SCCmec types were type III (53.2%) in S. epidermidis and type I (32.1%) in S. haemolyticus. All isolates were susceptible to linezolid and daptomycin, but 7.1% of S. haemolyticus and 2.3% of S. epidermidis isolates were resistant to tigecycline, and 1.2% each of S. haemolyticus and S. epidermidis were resistant and intermediately resistant to quinupristin/dalfopristin, respectively. S. epidermidis exhibited higher vancomycin MICs (40% with MIC of ≥2 µg/ml). Clonal typing of strains with vancomycin MIC of ≥2 µg/ml revealed the presence of different PFGE types of S. epidermidis and S. haemolyticus over a period of up to 4 years (2002-2004, 2005-2008, 2006-2009, 2010-2011). Despite the observation of a high prevalence of mecA, the clinical strains were fully susceptible to vancomycin and to the new drugs linezolid, daptomycin, tigecycline, and quinupristin/dalfopristin. The PFGE types with vancomycin MIC of ≥2 µg/ml exhibited a great diversity of SCCmec cassettes, demonstrating that S. epidermidis and S. haemolyticus may easily acquire these resistance-conferring genetic elements.

Phenotypic and genotypic characterization of biofilm formation in Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most frequently isolated coagulase-negative staphylococci. The ability to produce biofilm has contributed to its emergence as a nosocomial pathogen. In this study, some growth conditions were tested to determine their influence on biofilm formation. Brain-heart infusion (BHI) broth containing glucose was used to screen 64 clinical strains. A strong biofilm producer strain showed cells surrounded by a thick layer of extracellular matrix. The presence of atlE, fbp, bap, and icaA genes was analyzed. We concluded that S. haemolyticus biofilm production can be increased with cells grown in BHI, and highlighted that it could be an ica-independent process.

Virulence and the presence of aminoglycoside resistance genes of Staphylococcus haemolyticus strains isolated from clinical specimens.

We examined thirty methicillin-resistant Staphylococcus haemolyticus isolates cultured from clinical specimens for antibiotic resistance, various important interactions of the bacteria with epithelial cells and putative virulence determinants. All strains were resistant to oxacillin and carried the mecA gene. Aminocyclitol-3'-phosphotransferase (aph(3')-IIIa) gene encoding nucleotidyltransferases was detected in 43 %, aminocyclitol-6'-acetyltransferase-aminocyclitol-2″-phosphotransferase (aac(6')/aph(2″)) gene encoding bifunctional acetyltransferases/phosphotransferases in 33 %, aminocyclitol-4'-adenylyltransferase (ant(4')-Ia) gene encoding phosphotransferases in 20 %. The coexistence of resistance to methicillin and aminoglycosides was investigated in multi-resistant strains. Coexisting (aac(6')/aph(2″)) and (aph(3')-IIIa) genes were detected in 33 % of isolates, whereas 63 % of isolates had at least one of these genes. All strains revealed adherence ability and most of them (63 %) were invasive to epithelial cells. Electron microscopy revealed that the bacteria were found in vacuoles inside the cells. We observed that the contact of the bacteria with host epithelial cells is a prerequisite to their cytotoxicity at 5 h-incubation. Culture supernatant of the strains induced a low effect of cytotoxicity at the same time of incubation. Cell-free supernatant of all isolates expressed cytotoxic activity which caused destruction of HEp-2 cells at 24 h. None of the strains was cytotonic towards CHO cells. Among thirty strains, 27 % revealed lipolytic activity, 43 % produced lecithinase and 20 % were positive for proteinase activity. Analyses of cellular morphology and DNA fragmentation exhibited typical characteristic features of those undergoing apoptosis. The Pearson linear test revealed positive correlations between the apoptotic index at 24 h and percentage of cytotoxicity. Our results provided new insights into the mechanisms contributing to the development of S. haemolyticus-associated infections. The bacteria adhered and invaded to non-professional phagocytes. The invasion of epithelial cells by S. haemolyticus could be similar to phagocytosis that requires polymerization of the actin cytoskeleton. The process is inhibited by cytochalasin D. Moreover, they survived within the cells by residing in membrane bound compartments and induced apoptotic cell death.

Inhibition of exotoxin production by mobile genetic element SCCmec-encoded psm-mec RNA is conserved in staphylococcal species.

Staphylococcal species acquire antibiotic resistance by incorporating the mobile-genetic element SCCmec. We previously found that SCCmec-encoded psm-mec RNA suppresses exotoxin production as a regulatory RNA, and the psm-mec translation product increases biofilm formation in Staphylococcus aureus. Here, we examined whether the regulatory role of psm-mec on host bacterial virulence properties is conserved among other staphylococcal species, S. epidermidis and S. haemolyticus, both of which are important causes of nosocomial infections. In S. epidermidis, introduction of psm-mec decreased the production of cytolytic toxins called phenol-soluble modulins (PSMs) and increased biofilm formation. Introduction of psm-mec with a stop-codon mutation that did not express PSM-mec protein but did express psm-mec RNA also decreased PSM production, but did not increase biofilm formation. Thus, the psm-mec RNA inhibits PSM production, whereas the PSM-mec protein increases biofilm formation in S. epidermidis. In S. haemolyticus, introduction of psm-mec decreased PSM production, but did not affect biofilm formation. The mutated psm-mec with a stop-codon also caused the same effect. Thus, the psm-mec RNA also inhibits PSM production in S. haemolyticus. These findings suggest that the inhibitory role of psm-mec RNA on exotoxin production is conserved among staphylococcal species, although the stimulating effect of the psm-mec gene on biofilm formation is not conserved.

[The formation of bio-films by opportunistic microorganisms isolated from patients with rheumatic diseases].

The study was carried out concerning capability of 194 strains of opportunistic microorganisms to form bio-films. It is established that bacteria ecizing organism of patients with rheumatic diseases have capacity to form microbial bio-films. The formation of bio-films is manifested with the same rate as in agents of inflammatory processes. At that, Escherichia coli, Staphylococcus haemolyticus and bacteria of genus Proteus isolated under rheumatic diseases have significantly higher capability to form biofilms that matters for development of comorbide infections.

Comparative study of two plasticins: specificity, interfacial behavior, and bactericidal activity.

A comparative study was designed to evaluate the staphylococcidal efficiency of two sequence-related plasticins from the dermaseptin superfamily we screened previously. Their bactericidal activities against Staphylococcus aureus as well as their chemotactic potential were investigated. The impact of the GraS/GraR two-component system involved in regulating resistance to cationic antimicrobial peptides (CAMPs) was evaluated. Membrane disturbing activity was quantified by membrane depolarization assays using the diS-C3 probe and by membrane integrity assays measuring beta-galactosidase activity with recombinant strain ST1065 reflecting compromised membranes and cytoplasmic leakage. Interactions of plasticins with membrane models composed of either zwitterionic lipids mimicking the S. aureus membrane of CAMP-resistant strains or anionic lipids mimicking the negative charge-depleted membrane of CAMP-sensitive strains were analyzed by jointed Brewster angle microscopy (BAM), polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and differential scanning calorimetry (DSC) to yield detailed information about the macroscopic interfacial organization, in situ conformation, orientation of the peptides at the lipid-solvent interface, and lipid-phase disturbance. We clearly found evidence of distinct interfacial behaviors of plasticins we linked to the distribution of charges along the peptides and structural interconversion properties at the membrane interface. Our results also suggest that amidation might play a key role in GraS/GraR-mediated CAMP sensing at the bacterial surface.

Effects of growth in the presence of subinhibitory concentrations of dicloxacillin on Staphylococcus epidermidis and Staphylococcus haemolyticus biofilms.

Low concentrations of antibiotics can inhibit microbial adherence to medical device surfaces. However, little is known about the changes that occur in the physiology of bacteria within biofilms formed in the presence of subinhibitory (sub-MIC) concentrations of antibiotics. In this study, the densities and matrix compositions of biofilms formed by two coagulase-negative Staphylococcus species in the absence and in the presence of sub-MIC concentrations of dicloxacillin were evaluated. Biofilms formed in the presence of sub-MIC concentrations of dicloxacillin contained less biomass, and there were notable changes in the composition of the biofilm matrix. Changes in the spatial structure were also verified by confocal scanning laser microscopy, indicating that biofilms grown in the presence of sub-MIC concentrations of dicloxicilln had a lower cell density. Physiological alterations in the bacteria within biofilms grown in the presence of subinhibitory concentrations of the antibiotic were also evaluated. The results showed that there were differences in bacterial surface characteristics when cultures were grown in the presence of sub-MIC concentrations of dicloxacillin, including decreased hydrophobicity and decreased expression of the exopolysaccharide poly-N-acetylglucosamine. The elemental composition of the cell surface was also analyzed, and whereas in Staphylococcus epidermidis there were decreases in the oxygen and nitrogen contents, in Staphylococcus haemolyticus there were increases in these two parameters. Additionally, increases in resistance to several antibiotics were observed for the cells within biofilms formed in the presence of dicloxacillin.

Survey of extreme solvent tolerance in gram-positive cocci: membrane fatty acid changes in Staphylococcus haemolyticus grown in toluene.

We exploited the unique ecological niche of oil fly larval guts to isolate a strain of Staphylococcus haemolyticus which may be the most solvent-tolerant gram-positive bacterium yet described. This organism is able to tolerate 100% toluene, benzene, and p-xylene on plate overlays and saturating levels of these solvents in monophasic liquid cultures. A comparison of membrane fatty acids by gas chromatography after growth in liquid media with and without toluene showed that in cells continuously exposed to solvent the proportion of anteiso fatty acids increased from 25.8 to 33.7% while the proportion of 20:0 straight-chain fatty acids decreased from 19.3 to 10.1%. No changes in the membrane phospholipid composition were noted. Thus, S. haemolyticus alters its membrane fluidity via fatty acid composition to become more fluid when it is exposed to solvent. This response is opposite that commonly found in gram-negative bacteria, which change their fatty acids so that the cytoplasmic membrane is less fluid. Extreme solvent tolerance in S. haemolyticus is not accompanied by abnormal resistance to anionic or cationic detergents. Finally, six strains of Staphylococcus aureus and five strains of Staphylococcus epidermidis, which were not obtained by solvent selection, also exhibited exceptional solvent tolerance.

Comparative assessment of antibiotic susceptibility of coagulase-negative staphylococci in biofilm versus planktonic culture as assessed by bacterial enumeration or rapid XTT colorimetry.

OBJECTIVES: To quantitatively compare the antibiotic susceptibility of biofilms formed by the coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis and Staphylococcus haemolyticus with the susceptibility of planktonic cultures. METHODS: Several CoNS strains were grown planktonically or as biofilms to determine the effect of the mode of growth on the level of susceptibility to antibiotics with different mechanisms of action. The utility of a new, rapid colorimetric method that is based on the reduction of a tetrazolium salt (XTT) to measure cell viability was tested by comparison with standard bacterial enumeration techniques. A 6 h kinetic study was performed using dicloxacillin, cefazolin, vancomycin, tetracycline and rifampicin at the peak serum concentration of each antibiotic. RESULTS: In planktonic cells, inhibitors of cell wall synthesis were highly effective over a 3 h period. Biofilms were much less susceptible than planktonic cultures to all antibiotics tested, particularly inhibitors of cell wall synthesis. The susceptibility to inhibitors of protein and RNA synthesis was affected by the biofilm phenotype to a lesser degree. Standard bacterial enumeration techniques and the XTT method produced equivalent results both in biofilms and planktonic assays. CONCLUSIONS: This study provides a more accurate comparison between the antibiotic susceptibilities of planktonic versus biofilm populations, because the cell densities in the two populations were similar and because we measured the concentration required to inhibit bacterial metabolism rather than to eradicate the entire bacterial population. While the biofilm phenotype is highly resistant to antibiotics that target cell wall synthesis, it is fairly susceptible to antibiotics that target RNA and protein synthesis.