Proteomic Assessment of the Expression of Genes Related to Toluene Catabolism and Porin Synthesis in Pseudomonas stutzeri ST-9.

The organization and expression of Pseudomonas stutzeri ST-9 genes related to toluene catabolism and porin synthesis was investigated. Toluene-degrading genes were found to be localized in the chromosome close to a phage-type integrase. A regulatory gene and 21 genes related to an aromatics degradation pathway are organized as a putative operon. These proteins are upregulated in the presence of toluene. Fourteen outer membrane proteins were identified as porins in the ST-9 genome. The identified porins showed that the main detected porins are related to the OmpA and OprD superfamilies. The percentage of porins in the outer membrane protein fraction, as determined by mass spectrometry, was 73% and 54% when the cells were cultured with toluene and with glucose, respectively. Upregulation of OmpA and downregulation of OprD occurred in the presence of toluene. A porin fraction (90% OprD) from both cultures was isolated and examined as a toluene uptake system using the liposome-swelling assay. Liposomes were prepared with the porin fraction from a culture that was grown on toluene (T-proteoliposome) or glucose (G-proteoliposome). There was no significant difference in the permeability rate of the different solutes through the T-proteoliposome and the G-proteoliposome.

Comparative reduction of Giardia cysts, F+ coliphages, sulphite reducing clostridia and fecal coliforms by wastewater treatment processes.

Advanced wastewater treatment processes are applied to prevent the environmental dissemination of pathogenic microorganisms. Giardia lamblia causes a severe disease called giardiasis, and is highly prevalent in untreated wastewater worldwide. Monitoring the microbial quality of wastewater effluents is usually based on testing for the levels of indicator microorganisms in the effluents. This study was conducted to compare the suitability of fecal coliforms, F+ coliphages and sulfide reducing clostridia (SRC) as indicators for the reduction of Giardia cysts in two full-scale wastewater treatment plants. The treatment process consists of activated sludge, coagulation, high rate filtration and either chlorine or UV disinfection. The results of the study demonstrated that Giardia cysts are highly prevalent in raw wastewater at an average concentration of 3600 cysts/L. Fecal coliforms, F+ coliphages and SRC were also detected at high concentrations in raw wastewater. Giardia cysts were efficiently removed (3.6 log10) by the treatment train. The greatest reduction was observed for fecal coliforms (9.6 log10) whereas the least reduction was observed for F+ coliphages (2.1 log10) following chlorine disinfection. Similar reduction was observed for SRC by filtration and disinfection by either UV (3.6 log10) or chlorine (3.3 log10). Since F+ coliphage and SRC were found to be more resistant than fecal coliforms for the tertiary treatment processes, they may prove to be more suitable as indicators for Giardia. The results of this study demonstrated that advanced wastewater treatment may prove efficient for the removal of Giardia cysts and may prevent its transmission when treated effluents are applied for crop irrigation or streams restoration.

Effect of toluene on Pseudomonas stutzeri ST-9 morphology - plasmolysis, cell size, and formation of outer membrane vesicles.

Isolated toluene-degrading Pseudomonas stutzeri ST-9 bacteria were grown in a minimal medium containing toluene (100 mg·L(-1)) (MMT) or glucose (MMG) as the sole carbon source, with specific growth rates of 0.019 h(-1) and 0.042 h(-1), respectively. Scanning (SEM) as well as transmission (TEM) electron microscope analyses showed that the bacterial cells grown to mid-log phase in the presence of toluene possess a plasmolysis space. TEM analysis revealed that bacterial cells that were grown in MMT were surrounded by an additional "material" with small vesicles in between. Membrane integrity was analyzed by leakage of 260 nm absorbing material and demonstrated only 7% and 8% leakage from cultures grown in MMT compared with MMG. X-ray microanalysis showed a 4.3-fold increase in Mg and a 3-fold increase in P in cells grown in MMT compared with cells grown in MMG. Fluorescence-activated cell sorting (FACS) analysis indicated that the permeability of the membrane to propidium iodide was 12.6% and 19.6% when the cultures were grown in MMG and MMT, respectively. The bacterial cell length increased by 8.5% ± 0.1% and 17% ± 2%, as measured using SEM images and FACS analysis, respectively. The results obtained in this research show that the presence of toluene led to morphology changes, such as plasmolysis, cell size, and formation of outer membrane vesicles. However, it does not cause significant damage to membrane integrity.

Cryptosporidium and Giardia removal by secondary and tertiary wastewater treatment.

Wastewater disposal may be a source of environmental contamination by Cryptosporidium and Giardia. This study was conducted to evaluate the prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated wastewater effluents. A prevalence of 100% was demonstrated for Giardia cysts in raw wastewater, at a concentration range of 10 to 12,225 cysts L(-1), whereas the concentration of Cryptosporidium oocysts in raw wastewater was 4 to 125 oocysts L(-1). The removal of Giardia cysts by secondary and tertiary treatment processes was greater than those observed for Cryptosporidium oocysts and turbidity. Cryptosporidium and Giardia were present in 68.5% and 76% of the tertiary effluent samples, respectively, at an average concentration of 0.93 cysts L(-1) and 9.94 oocysts L(-1). A higher detection limit of Cryptosporidium oocysts in wastewater was observed for nested PCR as compared to immune fluorescent assay (IFA). C. hominis was found to be the dominant genotype in wastewater effluents followed by C. parvum and C. andersoni or C. muris. Giardia was more prevalent than Cryptosporidium in the studied community and treatment processes were more efficient for the removal of Giardia than Cryptosporidium. Zoonotic genotypes of Cryptosporidium were also present in the human community. To assess the public health significance of Cryptosporidium oocysts present in tertiary effluent, viability (infectivity) needs to be assessed.

Antibacterial effects of the tellurium compound OTD on E. coli isolates.

The antibacterial effects of a new organo-tellurium compound [Octa-O-bis-(R,R)-tartarate ditellurane (OTD)] on Escherichia coli isolates as a model are shown. OTD was found to be a bactericidal drug. It exhibits inhibition zones on a protein-rich agar medium but not in a protein-poor medium unless a thiol is added. When applied at the lag phase, OTD inhibits more efficiently than at the log phase. Thiols enhance the efficiency at the log phase. OTD inhibits biofilm formation of E. coli. X-ray microanalysis demonstrated damage caused to the Na⁺/K⁺ pumps and leakage of potassium and phosphorous. Scanning electron microscopy demonstrated an incomplete surface of the bacterial cell wall with a concavity in the center that looks like a hole. Transmission electron microscopy demonstrated severe damage, such as depletion, perforation, and holes in the inner membrane. These results indicate for the first time that the new tellurium compound has antibacterial activities.

Bioconversion of airborne methylamine by immobilized recombinant amine oxidase from the thermotolerant yeast Hansenula polymorpha.

Aliphatic amines, including methylamine, are air-pollutants, due to their intensive use in industry and the natural degradation of proteins, amino acids, and other nitrogen-containing compounds in biological samples. It is necessary to develop systems for removal of methylamine from the air, since airborne methylamine has a negative effect on human health. The primary amine oxidase (primary amine : oxygen oxidoreductase (deaminating) or amine oxidase, AMO; EC 1.4.3.21), a copper-containing enzyme from the thermotolerant yeast Hansenula polymorpha which was overexpressed in baker's yeast Saccharomyces cerevisiae, was tested for its ability to oxidize airborne methylamine. A continuous fluidized bed bioreactor (CFBR) was designed to enable bioconversion of airborne methylamine by AMO immobilized in calcium alginate (CA) beads. The results demonstrated that the bioreactor with immobilized AMO eliminates nearly 97% of the airborne methylamine. However, the enzymatic activity of AMO causes formation of formaldehyde. A two-step bioconversion process was therefore proposed. In the first step, airborne methylamine was fed into a CFBR which contained immobilized AMO. In the second step, the gas flow was passed through another CFBR, with alcohol oxidase from the yeast H. polymorpha immobilized in CA, in order to decompose the formaldehyde formed in the first step. The proposed system provided almost total elimination of the airborne methylamine and the formaldehyde.

Eradication of multi-drug resistant bacteria by a novel Zn-doped CuO nanocomposite.

Zinc-doped copper oxide nanoparticles are synthesized and simultaneously deposited on cotton fabric using ultrasound irradiation. The optimization of the processing conditions, the specific reagent ratio, and the precursor concentration results in the formation of uniform nanoparticles with an average size of ≈30 nm. The antibacterial activity of the Zn-doped CuO Cu0.88Zn0.12O in a colloidal suspension or deposited on the fabric is tested against Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) bacteria. A substantial enhancement of 10,000 times in the antimicrobial activity of the Zn-CuO nanocomposite compared to the pure CuO and ZnO nanoparticles (NPs) is observed after 10 min exposure to the bacteria. Similar activities are observed against multidrug-resistant bacteria (MDR), (i.e., Methicillin-resistant S. aureus and MDR E. coli) further emphasizing the efficacy of this composite. Finally, the mechanism for this enhanced antibacterial activity is presented.

Effect of external voltage on Pseudomonas putida F1 in a bio electrochemical cell using toluene as sole carbon and energy source.

A bio electrochemical cell (BEC) was constructed as a typical two-chamber microbial fuel cell (MFC), except that it was operated under external voltage instead of constant resistance as in an MFC. The anode chamber contained a pure culture of Pseudomonas putida F1 grown in a minimal medium containing toluene as the sole carbon and energy source. Operating the BEC under external voltages of 75, 125, 175, 250 and 500 mV (versus an Ag/AgCl reference electrode) led to increased bacterial cell growth to an OD(600) of 0.62-0.75, while the control BEC, which was not connected to external voltage, reached an OD(600) of only 0.3. Examination of the current generated under external voltages of 75, 125, 175, 250 and 500 mV showed that the maximal currents were 11, 23, 28, 54 and 94 mA m(-2), respectively. Cyclic voltammetry experiments demonstrated an anodic peak at 270 mV, which may imply oxidation of a vital molecule. The average residual toluene concentration after 147 h in the BEC operated under external voltage was 22 %, whereas in the control BEC it was 81 %. Proteome analysis of bacterial cells grown in the BEC (125 mV) revealed two groups of proteins, which are ascribed to charge transfer in the bacterial cells and from the cell to the electrode. In conclusion, operating the BEC at 75-500 mV enabled growth of a pure culture of P. putida F1 and toluene degradation even in an oxygen-limited environment.

Understanding the antibacterial mechanism of CuO nanoparticles: revealing the route of induced oxidative stress.

To date, there is still a lack of definite knowledge regarding the interaction of CuO nanoparticles with bacteria and the possible permeation of the nanoparticles into bacterial cells. This study was aimed at shedding light on the size-dependent (from the microscale down to the small nanoscale) antibacterial activity of CuO. The potent antibacterial activity of CuO nanoparticles was found to be due to ROS-generation by the nanoparticles attached to the bacterial cells, which in turn provoked an enhancement of the intracellular oxidative stress. This paradigm was confirmed by several assays such as lipid peroxidation and reporter strains of oxidative stress. Furthermore, electron microscopy indicated that the small nanoparticles of CuO penetrated the cells. Collectively, the results reported herein may reconcile conflicting concepts in the literature concerning the antibacterial mechanism of CuO nanoparticles, as well as highlight the potential for developing sustainable CuO nanoparticles-based devices for inhibiting bacterial infections.

Bactericidal activity of the organo-tellurium compound AS101 against Enterobacter cloacae.

OBJECTIVES: The antibacterial effect of the organo-tellurium compound AS101 on the Gram-negative bacterium Enterobacter cloacae is shown in this study for the first time. METHODS: The antimicrobial effect of the drug was shown by inhibition of growth, by inhibition of biofilm formation and by its ability to penetrate the bacterial cell and to cause damage and ultrastructural changes. RESULTS: AS101 was found to be a bactericidal drug with MICs and MBCs of 9.4 mg/L. It inhibits bacterial growth and causes a six orders of magnitude decrease in viability in a protein-rich medium, but not in a protein-poorer medium, unless 2-mercaptoethanol is added. Subinhibitory concentrations inhibit motility and biofilm formation. AS101 enters the bacterium through its porins and causes bacterial damage to Na(+)/K(+) pumps and leakage of potassium, phosphorous and sulphur. Ultrastructural changes within the bacterial cell and on its surface demonstrate an incomplete surface with a concavity in the centre that looks like a hole from which aggregates are liberated as well as cell lysis. CONCLUSIONS: AS101 has antibacterial activity, which may be useful against E. cloacae and other species of Enterobacteriaceae as a substitute for current antibiotics that have become ineffective due to increasing bacterial resistance.

Sonochemical coating of cotton and polyester fabrics with "antibacterial" BSA and casein spheres.

A novel antibacterial coating for cotton and polyester fabrics has been developed by using drug-loaded proteinaceous microspheres made of bovine serum albumin and casein proteins. The microbubbles were created and anchored onto the fabrics (see figure) in a one-step reaction that lasts 3 min. The sonochemically produced "antibacterial fabrics" have been characterized. The efficiency of the sonochemical process in converting the native proteins into microspheres, encapsulating the drug, and coating the fabric has also been studied.

Sonochemical coating of paper by microbiocidal silver nanoparticles.

Colloidal silver has gained wide acceptance as an antimicrobial agent, and various substrates coated with nanosilver such as fabrics, plastics, and metal have been shown to develop antimicrobial properties. Here, a simple method to develop coating of colloidal silver on paper using ultrasonic radiation is presented, and the coatings are characterized using X-ray diffraction (XRD), high resolution scanning electron microscope (HRSEM), and thermogravimetry (TGA) measurements. Depending on the variables such as precursor concentrations and ultrasonication time, uniform coatings ranging from 90 to 150 nm in thickness have been achieved. Focused ion beam (FIB) cross section imaging measurements revealed that silver nanoparticles penetrated the paper surface to a depth of more than 1 µm, resulting in highly stable coatings. The coated paper demonstrated antibacterial activity against E. coli and S. aureus, suggesting its potential application as a food packing material for longer shelf life.

Antifungal activity of ZnO nanoparticles--the role of ROS mediated cell injury.

Metal oxide nanoparticles have marked antibacterial activity. The toxic effect of these nanoparticles, such as those comprised of ZnO, has been found to occur due to an interaction of the nanoparticle surface with water, and to increase with a decrease in particle size. In the present study, we tested the ability of ZnO nanoparticles to affect the viability of the pathogenic yeast, Candida albicans (C. albicans). A concentration-dependent effect of ZnO on the viability of C. albicans was observed. The minimal fungicidal concentration of ZnO was found to be 0.1 mg ml(-1) ZnO; this concentration caused an inhibition of over 95% in the growth of C. albicans. ZnO nanoparticles also inhibited the growth of C. albicans when it was added at the logarithmic phase of growth. Addition of histidine (a quencher of hydroxyl radicals and singlet oxygen) caused reduction in the effect of ZnO on C. albicans depending on its concentration. An almost complete elimination of the antimycotic effect was achieved following addition of 5 mM of histidine. Exciting the ZnO by visible light increased the yeast cell death. The effects of histidine suggest the involvement of reactive oxygen species, including hydroxyl radicals and singlet oxygen, in cell death. In light of the above results it appears that metal oxide nanoparticles may provide a novel family of fungicidal compounds.

Enhanced inactivation of bacteria by metal-oxide nanoparticles combined with visible light irradiation.

BACKGROUND: In recent years nano-metaloxides which easily penetrate into the cells with special interest due to their higher chemical reactivity as compared to that of similar materials in the bulk form. Of particular interest are nano-TiO(2) and ZnO, which have been widely used for their bactericidal and anticancerous properties. PURPOSE: The aim of the present study was to examine the bactericidal properties of nano-TiO(2) and ZnO combined with visible light on S. aureus and S. epidermitis, known for their high prevalence in infected wounds. STUDY: Using the technique of electron-spin resonance (ESR) coupled with spin trapping, we examined the ability of TiO(2) and ZnO nanoparticle suspensions in water to produce reactive oxygen species (ROS) with and without visible light irradiation. The possibility of exciting these nanoparticles with visible light in order to enhance their antimicrobial activity was also tested. RESULTS: Electron-spin resonance measurements revealed that ZnO and TiO(2) nanoparticles are able to produce ROS in water suspension. A remarkable enhancement of ROS production was found following illumination with blue light. In addition, illumination significantly enhanced the antibacterial activity of the nanoparticles. CONCLUSION: The results suggest that nanoparticles combined with visible light can be used for sterilization purposes and may be effective for treating infected wounds.

Coliphages as Indicators for the Microbial Quality of Treated Wastewater Effluents.

Wastewater effluents are a reliable water source for non-potable water reuse including unrestricted crop irrigation in arid regions suffering from water scarcity. This study was performed to develop and optimize a procedure to concentrate coliphages from 100 L of treated effluent. Moreover, the reduction of coliphages by filtration and disinfection by either chlorine or UV was compared with that of fecal coliform (FC). The adsorption efficiency of MS2 and Qß coliphages by the NanoCeram filter was similar and reached 99.8%. Elution efficiency of MS2 coliphage from the NanoCeram filters by a solution of 1% NaPP and 0.05 M glycine, pH 9.5, was 74  ±  9.5%. The highest reconcentration efficiency of MS2 and Qß coliphages was obtained with polyethylene glycol (PEG) precipitation and reached 76  ±  28% and 90  ±  11%, respectively. In comparison, the reconcentration efficiency of organic flocculation was 0% and 1.3% for Qß and MS2 coliphages, respectively. The overall recovery efficiency of MS2 coliphages from 100 L tertiary effluent was 57  ±  1.5%. Poor reduction was observed for coliphages compared to FC by filtration and chlorine disinfection although; the reduction of FC, as measured by cultivation, was satisfactory and within the guidelines for unrestricted irrigation. High correlation between the reduction of FC and coliphages was recorded for tertiary effluent disinfected by UV irradiation. Monitoring the microbial quality of tertiary effluent using qPCR for the enumeration of FC was found unsuitable, because DNA levels were unaffected by the treatment processes. The results of this study demonstrated that monitoring the microbial quality of tertiary effluent by FC may not reflect the health risks encountered by the application of these effluents and the addition of coliphages to the monitoring programs may allow for accurate assessment of the health risks introduced by the application of tertiary effluent.

Antibacterial and In Vivo Studies of a Green, One-Pot Preparation of Copper/Zinc Oxide Nanoparticle-Coated Bandages.

Simultaneous water and ethanol-based synthesis and coating of copper and zinc oxide (CuO/ZnO) nanoparticles (NPs) on bandages was carried out by ultrasound irradiation. High resolution-transmission electron microscopy demonstrated the effects of the solvent on the particle size and shape of metal oxide NPs. An antibacterial activity study of metal-oxide-coated bandages was carried out against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative). CuO NP-coated bandages made from both water and ethanol demonstrated complete killing of S. aureus and E. coli bacteria within 30 min., whereas ZnO NP-coated bandages demonstrated five-log reductions in viability for both kinds of bacteria after 60 min of interaction. Further, the antibacterial mechanism of CuO/ZnO NP-coated bandages is proposed here based on electron spin resonance studies. Nanotoxicology investigations were conducted via in vivo examinations of the effect of the metal-oxide bandages on frog embryos (teratogenesis assay-Xenopus). The results show that water-based coatings resulted in lesser impacts on embryo development than the ethanol-based ones. These bandages should therefore be considered safer than the ethanol-based ones. The comparison between the toxicity of the metal oxide NPs prepared in water and ethanol is of great importance, because water will replace ethanol for bulk scale synthesis of metal oxide NPs in commercial companies to avoid further ignition problems. The novelty and importance of this manuscript is avoiding the ethanol in the typical water:ethanol mixture as the solvent for the preparation of metal oxide NPs. Ethanol is ignitable, and commercial companies are trying the evade its use. This is especially important these days, as the face mask produced by sonochemistry (SONOMASK) is being sold all over the world by SONOVIA, and it is coated with ZnO.

Photoinactivation of Candida albicans by its own endogenous porphyrins.

The possibility of photoeradicating the prokaryotic microorganism Candida albicans by enhancing its endogenous porphyrin production and accumulation was investigated in this study. Induction of porphyrin synthesis was performed by the addition of delta-aminolevulinic acid (ALA), or its hydrophobic derivative ALA methyl ester (m-ALA). Photoinactivation of C. albicans was performed under blue light (407-420 nm) illumination. A decrease in viability of about 1.6 or 2.1 orders of magnitudes was obtained with a light dose of 36 J/cm(2) for an initial concentration of 100-mg/ml ALA or m-ALA, respectively. Endogenous porphyrins extracted from the cells showed that cultures incubated with m-ALA accumulated a relatively higher amount of endogenous porphyrins than ALA, indicating better transport through the yeast cell barriers. When a combination of miconazole and ketoconazole (antifungal agents) is given at a sub-inhibitory concentration (0.5 microg/ml each) with an inducer, a 2.1 or 3.2 orders of magnitude decrease in viability is caused with ALA or with m-ALA, respectively, upon illumination. Fluorescence intensities of the accumulated porphyrins as demonstrated by FACS indicate that the combination of the two azole drugs and an inducer cause a relatively high amount of endogenous porphyrins. Although the additive action of both azole drugs allow better penetration of the inducer, especially m-ALA photoeradication remained limited because of an acidic pH generated in the presence of the inducer. The acidic pH is probably the cause for the inefficiency of the photodynamic treatment. More hydrophobic inducers than m-ALA and less acidic must be investigated to improve the photodynamic treatment by endogenous-induced porphyrins.

Isolation, characterization and biological role of camelysin from Bacillus thuringiensis subsp. israelensis.

The present study reports a simple rapid method for isolating the zinc-containing metalloprotease camelysin from Bacillus thuringiensis subsp. israelensis (Bti) by extraction from intact bacterial cells with egg L-alpha-phosphatidylcholine containing monolamellar liposomes, followed by separation on a sucrose gradient. Characterization of the isolated camelysin revealed a molecular weight of 23 kDa and a pI of 6.2. The camelysin exhibited maximal activity against the substrate azocasein at a temperature of 37 degrees C and pH 7.5. However, the enzyme's activity remained high also at basic pH values (8-10). In a rich growth medium (LB), camelysin appeared at the late logarithmic phase of Bti growth and reached its maximum in the stationary phase. Camelysin was shown to activate the protoxins Cyt1Aa and Cyt2Ba produced by Bti. The hemolytic activity of Cyt1Aa increased from 40 to 70% and that of Cyt2Ba from 6 to 50% in the presence of 50% (w/w) camelysin. It is concluded that these protoxins can be activated not only by insect gut proteases, but also by the endogeneous metalloprotease camelysin of the Bti bacterium.

Visible light-induced killing of bacteria as a function of wavelength: implication for wound healing.

BACKGROUND AND OBJECTIVE: Visible light (400-800 nm) at high intensity was previously found to kill bacteria that are frequently found in infected wounds, while low-power white light enhances bacterial proliferation. The phototoxic effect was found to involve induction of reactive oxygen species (ROS) production by the bacteria. The aim of the present study was to identify the most effective wavelengths in the visible range for inducing a bactericidal effect. EXPERIMENTAL: ROS production in Staphylococcus aureus and Escherichia coli as a function of wavelengths in the visible range (400-500, 500-800, 415, and 455 nm) was studied using the electron paramagnetic resonance (EPR) spin trapping technique. The phototoxicity of 415 and 455 nm light at different fluencies on the survival of S. aureus and E. coli was assessed by colony count of the bacteria following irradiation. RESULTS: ROS production following blue (400-500 nm) light illumination was found to be higher than that of red (500-800 nm). Within the blue range, light of 415 nm induced more ROS than 455 nm, which correlated with results obtained for the reduction in colony count of S. aureus and E. coli following illumination using equal intensities of these two wavelengths. At low fluencies, both 415 and 455 nm enhanced proliferation of S. aureus but reduced viability of E. coli. CONCLUSION: Intense blue light, preferably at 415 nm, could be used for bacterial eradication. However, it should be noted that low intensity of visible light can be dangerous since it may promote proliferation of the microorganisms.

Molecular features of heterogeneous vancomycin-intermediate Staphylococcus aureus strains isolated from bacteremic patients.

BACKGROUND: Heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) bacteremia is an emerging infection. Our objective was to determine the molecular features of hVISA strains isolated from bacteremic patients and to compare them to methicillin resistant S. aureus (MRSA) and methicillin sensitive S. aureus (MSSA) blood isolates. RESULTS: We assessed phenotypic and genomic changes of hVISA (n = 24), MRSA (n = 16) and MSSA (n = 17) isolates by PCR to determine staphylococcal chromosomal cassette (SCCmec) types, Panton-Valentine leukocidin (PVL) and the accessory gene regulator (agr) loci. Biofilm formation was quantified. Genetic relatedness was assessed by PFGE. PFGE analysis of isolates was diverse suggesting multiple sources of infection. 50% of hVISA isolates carried SCCmec type I, 21% type II; 25% type V; in 4% the SCCmec type could not be identified. Among MRSA isolates, 44% were SCCmec type I, 12.5% type II, 25% type V, 12.5% were non-typable, and 6% were SCCmec type IVd. Only one hVISA isolate and two MSSA isolates carried the PVL. Biofilm formation and agr patterns were diverse. CONCLUSION: hVISA isolates were diverse in all parameters tested. A considerable number of hVISA and MRSA strains carried the SCCmec type V cassette, which was not related to community acquisition.