Educational Attainment and Staphylococcus aureus Colonization in a Hispanic Border Community: Testing Fundamental Cause Theory.

This study was carried out to evaluate hypotheses generated by fundamental cause theory regarding the socioeconomic status (SES) gradient in colonization with Staphylococcus aureus among Hispanic and non-Hispanic adults living in a border community. Participants (n = 613) recruited in naturally occurring small groups at public and private sites throughout Yuma County, AZ, completed a sociodemographic survey and swabbed their palms, noses, and throats to sample microbial flora. Positive S. aureus colonization among non-Hispanic white participants was nominally higher (39.0%; 95% confidence interval [CI] = 32.4 to 46.1%) than that in Hispanics (31.3%; 95% CI = 26.4 to 36.8%), but there was no education gradient for the sample overall (incidence rate ratio = 1.00; 95% CI = 0.90 to 1.12) or within each ethnic group separately. The education gradient between Hispanic and non-Hispanic whites was statistically equivalent. Results were consistent when home ownership was used as the SES indicator. These data show that S. aureus colonization is not linked to two different SES indicators or Hispanic ethnicity. S. aureus colonization may be considered a less preventable health risk that is outside the influence of SES-based resources.IMPORTANCE Unlike some types of S. aureus infections, S. aureus colonization is not associated with ethnicity or educational attainment and thus may be outside the influence of socioeconomic status-based resources typically mobilized to avoid or mitigate preventable health risks. This assessment of a clinically silent risk that usually precedes infections may illustrate a boundary of fundamental cause theory.

Rapid assessment and prediction of the efficiency of two preservatives against S. aureus in cosmetic products using High Content Screening-Confocal Laser Scanning Microscopy.

Most cosmetic products are susceptible to microbiological spoilage due to contaminations that could happen during fabrication or by consumer's repetitive manipulation. The composition of cosmetic products must guarantee efficient bacterial inactivation all along with the product shelf life, which is usually assessed by challenge-tests. A challenge-test consists in inoculating specific bacteria, i.e. Staphylococcus aureus, in the formula and then investigating the bacterial log reduction over time. The main limitation of this method is relative to the time-consuming protocol, where 30 days are needed to obtain results. In this study, we have proposed a rapid alternative method coupling High Content Screening-Confocal Laser Scanning Microscopy (HCS-CLSM), image analysis and modeling. It consists in acquiring real-time S. aureus inactivation kinetics on short-time periods (typically 4h) and in predicting the efficiency of preservatives on longer scale periods (up to 7 days). The action of two preservatives, chlorphenesin and benzyl alcohol, was evaluated against S. aureus at several concentrations in a cosmetic matrix. From these datasets, we compared two secondary models to determine the logarithm reduction time (Dc) for each preservative concentration. Afterwards, we used two primary inactivation models to predict log reductions for up to 7 days and we compared them to observed log reductions. The IQ model better fits datasets and the Q value gives information about the matrix level of interference.

A simulation study to investigate the added value in using differential somatic cell count as an additional indicator for udder health management in dairy herds.

Mastitis is one of the most costly diseases in dairy herds worldwide. Somatic cell count (SCC) is widely used as an indicator for subclinical intramammary infections (IMI) that may eventually cause mastitis in dairy herds. Differential somatic cell count (DSCC) has recently been introduced as an additional indicator for IMI. The objective of this study was to investigate the value of using DSCC as an additional indicator to select cows for testing and subsequent intervention for subclinical mastitis during the lactation. We parameterized an existing bio-economic simulation model for dairy herds to include DSCC. Then, we simulated three Danish dairy cattle herd situations with different pathogen distributions where the main pathogens were 1) Staphylococcus aureus, 2) Streptococcus agalactiae, and 3) Streptococcus uberis. In these herds, we simulated two different selection strategies for testing (bacterial culture) for subclinical IMI and various intervention strategies for test positive cases. The first selection strategy considered only SCC; cows were selected for testing if they had a low SCC measurement followed by two high SCC measurements. In the second selection strategy, cows additionally had to have a high DSCC measurement. Results showed that both selection strategies led to a similar net income and to a similar number of clinical and subclinical cases for all investigated intervention strategies. However, when using DSCC in the selection of animals, the number of treatment days and the number of cows culled in relation to IMI was reduced: The median annual number of treatment days was reduced by 25-38 days in herd 1, by 25-42 days in herd 2, and by 30-48 days in herd 3, depending on the intervention strategy. The median annual number of cows culled in relation to IMI was reduced by up to 8 cows (10 cows in herd 3) for one of the intervention strategies. Subject to limitations associated with model assumptions, these results suggest that considering DSCC when selecting cows for testing can reduce IMI related culling and the use of antibiotics without changing in-herd prevalence nor resulting in economic loss.

[Criterium pencil mines as an alternative to platinum rods (Inoclic) in achievement of the antibiogram in agar medium for countries with limited resources?] / Les mines de crayon pour critérium comme alternative aux tiges de platine (Inoclic) dans la réalisation de l'antibiogramme en milieu gélosé pour les pays à ressources limitées ?

The realization of the antibiotic susceptibility test in agar is the routine bacteriological examination for the determination and monitoring of bacterial susceptibility to antibiotics. In this study, we report the comparative results between pencil leads for criterium, as an alternative to platinum rods in the realization of the antibiotic susceptibility test. METHODOLOGY: Experimental study evaluating the comparability of the results between Criterium and Inoclic mines (by counting bacterial cells on agar after 5 successive dilutions of reason 10 from a bacterial suspension obtained after piercing through a colony; by measuring the inhibition diameters of 4 ATCC reference bacterial strains on an antibiogram in an agar medium) and evaluating the sterility of the criterium mines by culturing them on enriched broth (heart - brain type). RESULTS: 42 bacterial strains were used for bacterial cell counting. The results were of the same order of magnitude (107 CFU/mL) between Inoclic and criterium mines, for all strains and at all dilutions. The antibiotic susceptibility tests performed for the 4 reference strains by the Inoclics and criterium mines all complied (100%) with the expected limits for determining their sensitivity profile to the antibiotics tested. Compared to the bacterial growth inhibition diameters on antibiotic susceptibility tests, no intra-operator variability was observed, while significant inter-operator variability (both with Inoclic and 0.5 mm criterium mines) was observed with some strains and for inhibition diameters greater than 10 mm. The enriched broth cultures (BCC) and their subculture carried out on 10 criterium mines from 5 different batches were negative. CONCLUSION: Criterium mines seem to be a serious and less expensive alternative to Inoclic for the realization of antibiotic susceptibility testing in our resource-limited countries.

Assessment of predatory bacteria and prey interactions using culture-based methods and EMA-qPCR.

Traditional culture-based enumeration methods were compared with the ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) technique to assess Bdellovibrio-and-like-organisms (BALOs) predator-prey interactions. Gram-negative [Pseudomonas spp. and Klebsiella pneumoniae (K. pneumoniae)] and Gram-positive [Staphylococcus aureus (S. aureus) and Enterococcus faecium (E. faecium)] organisms were employed as prey cells, while a Bdellovibrio bacteriovorus strain (PF13) was used as the predator. The co-culture experiments were also compared in diluted nutrient broth (DNB) and HEPES buffer. In both media, K. pneumoniae (maximum log reduction of 5.13) and Pseudomonas fluorescens (P. fluorescens) (maximum log reduction of 4.21) were sensitive to predation by B. bacteriovorus PF13 as their cell counts and gene copies were reduced during all the co-culture experiments, while the concentration of B. bacteriovorus PF13 increased. The concentration of B. bacteriovorus PF13 also increased in the presence of S. aureus (HEPES buffer) and E. faecium (DNB), indicating that the predator interacted with these Gram-positive prey in order to survive. Moreover, as no predator plaques were produced in the co-culture experiments with P. aeruginosa (DNB and HEPES buffer), S. aureus (DNB and HEPES buffer) and E. faecium (HEPES buffer), EMA-qPCR proved to be beneficial in monitoring the concentration of B. bacteriovorus. In conclusion, the cell counts and/or EMA-qPCR analysis for the HEPES buffer and DNB assays were successfully employed to monitor the predation of P. fluorescens and K. pneumoniae by B. bacteriovorus, while E. faecium was sensitive to predation in DNB and S. aureus was sensitive to predation in HEPES buffer.

Economic and epidemiological impact of different intervention strategies for subclinical and clinical mastitis.

The objective of this study was to evaluate and compare different combinations of intervention strategies for contagious or opportunistic subclinical and clinical intramammary infections (IMI). We simulated two different Danish dairy cattle herds with ten different intervention strategies focusing on cow-specific treatment or culling, including three baseline strategies without subclinical interventions. In one herd, the main causative pathogen of IMI was Staphylococcus (S.) aureus. In the other herd, Streptococcus (St.) agalactiae was the main causative agent. For both herds, we investigated costs and effectiveness of all ten intervention strategies. Intervention strategies consisted of measures against clinical and subclinical IMI, with baselines given by purely clinical intervention strategies. Our results showed that strategies including subclinical interventions were more cost-effective than the respective baseline strategies. Increase in income and reduction of IMI cases came at the cost of increased antibiotic usage and an increased culling rate in relation to IMI. However, there were differences between the herds. In the St. agalactiae herd, the clinical intervention strategy did not seem to have a big impact on income and number of cases. However, intervention strategies which included cow-specific clinical interventions led to a higher income and lower number of cases in the S. aureus herd. The results show that intervention strategies including interventions against contagious or opportunistic clinical and subclinical IMI can be highly cost-effective, but should be herd-specific.

Critical Assessment of Methods to Quantify Biofilm Growth and Evaluate Antibiofilm Activity of Host Defence Peptides.

Biofilms are multicellular communities of bacteria that can adhere to virtually any surface. Bacterial biofilms are clinically relevant, as they are responsible for up to two-thirds of hospital acquired infections and contribute to chronic infections. Troublingly, the bacteria within a biofilm are adaptively resistant to antibiotic treatment and it can take up to 1000 times more antibiotic to kill cells within a biofilm when compared to planktonic bacterial cells. Identifying and optimizing compounds that specifically target bacteria growing in biofilms is required to address this growing concern and the reported antibiofilm activity of natural and synthetic host defence peptides has garnered significant interest. However, a standardized assay to assess the activity of antibiofilm agents has not been established. In the present work, we describe two simple assays that can assess the inhibitory and eradication capacities of peptides towards biofilms that are formed by both Gram-positive and negative bacteria. These assays are suitable for high-throughput workflows in 96-well microplates and they use crystal violet staining to quantify adhered biofilm biomass as well as tetrazolium chloride dye to evaluate the metabolic activity of the biofilms. The effect of media composition on the readouts of these biofilm detection methods was assessed against two strains of Pseudomonas aeruginosa (PAO1 and PA14), as well as a methicillin resistant strain of Staphylococcus aureus. Our results demonstrate that media composition dramatically alters the staining patterns that were obtained with these dye-based methods, highlighting the importance of establishing appropriate biofilm growth conditions for each bacterial species to be evaluated. Confocal microscopy imaging of P. aeruginosa biofilms grown in flow cells revealed that this is likely due to altered biofilm architecture under specific growth conditions. The antibiofilm activity of several antibiotics and synthetic peptides were then evaluated under both inhibition and eradication conditions to illustrate the type of data that can be obtained using this experimental setup.

Epidemiological and partial budget analysis for treatment of subclinical Staphylococcus aureus intramammary infections considering microbiological and cytological scenarios.

An innovative method was investigated to aid in the elimination of Staphylococcus aureus (S. aureus) intramammary infections (IMI) from dairy herds. A stochastic model explore the economic benefit of three-day or eight-day treatment of subclinical IMI in all S. aureus infected cows or in only those with a somatic cell count (SCC) exceeding 200,000 cells/ml. An epidemiological model was developed to run parallel to the economic model that would predict the S. aureus IMI likely to persist, develop new infections and clinical mastitis. In the economic model a first algorithm was used to consider the low prevalence (LP) scenario and made use of S. aureus prevalence information provided by retrospective analysis of microbiological and cytological results in South Africa (2008-2012). The data used considered Staphylococcus aureus prevalence from [1.495; 1.595]95% to [6.72; 6.95]95% for SCC≤200,000 and SCC>200,000 cells/ml respectively. A second algorithm considered the high prevalence (HP) scenario to evaluate a simulated situation with a 5[U1] [R12] to 25% prevalence. Scenarios of low or high transmission ratio (TR) were included in the model according to the hygiene management on the farm. Probabilities and costs were calculated over 255days. The economic models predicted average cost indices for low S. aureus IMI and low TR to vary from -3179 ZAR (South African Rands) when subclinical cases with SCC higher than 200,000 cell/ml were treated for eight days, to -3663 ZAR when all subclinical S. aureus IMI were treated for three days. With a HP and high TR of S. aureus the average cost indices changed from -18,042 ZAR when none to -5433 ZAR per 255days when all S. aureus IMI were treated for eight days. The epidemiological model in this study predicted substantial benefit of treatment mainly in high TR scenarios. New IMI decreased up to77% in the three-day and up to 91% in the eight-day treatment scenarios. In the HP scenarios, persistent IMI were reduced by 94%. The number of clinical cases predicted with no treatment for subclinical infections was higher than the total number of clinical and subclinical cases in scenarios where cows were treated three or eight days. Initial prudent treatment of subclinical IMI resulted in less overall treatments and less new, persistent and clinical cases. Combined results of economic and epidemiological models indicated that the option that cost the least did not always have the best epidemiological outcome. Models may assist in optimising and balancing decisions relating to financial and IMI.

Survivability and fitness cost of heterogeneous vancomycin-intermediate Staphylococcus aureus.

The aim of this study was to observe the survivability and fitness cost of heterogeneous vancomycin-intermediate Staphylococcus aureus(hVISA) isolates. Survivability study was performed on dry cotton swab, and fitness cost was evaluated by estimating growth kinetics and generation time constant in BACTEC automated system. Total mean maximum time of recovery on primary culture was 4.1 and 7.1 weeks (P = 0.0001) for hVISA and vancomycin-sensitive S. aureus (VSSA), respectively, in dry starved condition. No significant difference between the mean value of lag phase duration (P = 0.89) was noted between hVISA and VSSA isolate in growth kinetics. However, we observed lesser generation time of hVISA isolates compared to S. aureus ATCC 29213 (P = 0.0076). This study concluded that a significant difference in generation time between VSSA and hVISA and suggests that hVISA have fitness cost compared to VSSA. However, further studies with more cases are required.

Innovative approaches to treat Staphylococcus aureus biofilm-related infections.

Many bacterial infections in humans and animals are caused by bacteria residing in biofilms, complex communities of attached organisms embedded in an extracellular matrix. One of the key properties of microorganisms residing in a biofilm is decreased susceptibility towards antimicrobial agents. This decreased susceptibility, together with conventional mechanisms leading to antimicrobial resistance, makes biofilm-related infections increasingly difficult to treat and alternative antibiofilm strategies are urgently required. In this review, we present three such strategies to combat biofilm-related infections with the important human pathogen Staphylococcus aureus: (i) targeting the bacterial communication system with quorum sensing (QS) inhibitors, (ii) a 'Trojan Horse' strategy to disturb iron metabolism by using gallium-based therapeutics and (iii) the use of 'non-antibiotics' with antibiofilm activity identified through screening of repurposing libraries.

Assessment of Photodynamic Inactivation against Periodontal Bacteria Mediated by a Chitosan Hydrogel in a 3D Gingival Model.

Chitosan hydrogels containing hydroxypropyl methylcellulose (HPMC) and toluidine blue O were prepared and assessed for their mucoadhesive property and antimicrobial efficacy of photodynamic inactivation (PDI). Increased HPMC content in the hydrogels resulted in increased mucoadhesiveness. Furthermore, we developed a simple In Vitro 3D gingival model resembling the oral periodontal pocket to culture the biofilms of Staphylococcus aureus (S. aureus), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Porphyromonas gingivalis (P. gingivalis). The PDI efficacy of chitosan hydrogel was examined against periodontal biofilms cultured in this 3D gingival model. We found that the PDI effectiveness was limited due to leaving some of the innermost bacteria alive at the non-illuminated site. Using this 3D gingival model, we further optimized PDI procedures with various adjustments of light energy and irradiation sites. The PDI efficacy of the chitosan hydrogel against periodontal biofilms can significantly improve via four sides of irradiation. In conclusion, this study not only showed the clinical applicability of this chitosan hydrogel but also the importance of the light irradiation pattern in performing PDI for periodontal disease.

Can Sophorolipids prevent biofilm formation on silicone catheter tubes?

Given the impact of biofilms in health care environment and the increasing antibiotic resistance and/or tolerance, new strategies for preventing that occurrence in medical devices are obligatory. Thus, biomaterials surface functionalization with active compounds can be a valuable approach. In the present study the ability of the biosurfactants sophorolipids to prevent biofilms formation on silicone rubber aimed for medical catheters was investigated. Sophorolipids produced by Starmerella bombicola, identified by HPLC-MS/MS were used to cover silicone and surface characterization was evaluated through contact angle measurements and FTIR-ATR. Results revealed that sophorolipids presence on silicone surface decreased the hydrophobicity of the material and biofilm formation of Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922. Antibiofilm activity was evaluated through different methods and was more pronounced against S. aureus. Furthermore, biocompatibility of silicone specimens with HaCaT cells was also obtained. From this study it was possible to conclude that sophorolipids seem to be a favourable approach for coating silicone catheters. Such compounds may represent a novel source of antibiofilm agents for technological development passing through strategies of permanent functionalization of surfaces.

Cost-Effectiveness of a National Initiative to Improve Hand Hygiene Compliance Using the Outcome of Healthcare Associated Staphylococcus aureus Bacteraemia.

BACKGROUND: The objective is to estimate the incremental cost-effectiveness of the Australian National Hand Hygiene Inititiave implemented between 2009 and 2012 using healthcare associated Staphylococcus aureus bacteraemia as the outcome. Baseline comparators are the eight existing state and territory hand hygiene programmes. The setting is the Australian public healthcare system and 1,294,656 admissions from the 50 largest Australian hospitals are included. METHODS: The design is a cost-effectiveness modelling study using a before and after quasi-experimental design. The primary outcome is cost per life year saved from reduced cases of healthcare associated Staphylococcus aureus bacteraemia, with cost estimated by the annual on-going maintenance costs less the costs saved from fewer infections. Data were harvested from existing sources or were collected prospectively and the time horizon for the model was 12 months, 2011-2012. FINDINGS: No useable pre-implementation Staphylococcus aureus bacteraemia data were made available from the 11 study hospitals in Victoria or the single hospital in Northern Territory leaving 38 hospitals among six states and territories available for cost-effectiveness analyses. Total annual costs increased by $2,851,475 for a return of 96 years of life giving an incremental cost-effectiveness ratio (ICER) of $29,700 per life year gained. Probabilistic sensitivity analysis revealed a 100% chance the initiative was cost effective in the Australian Capital Territory and Queensland, with ICERs of $1,030 and $8,988 respectively. There was an 81% chance it was cost effective in New South Wales with an ICER of $33,353, a 26% chance for South Australia with an ICER of $64,729 and a 1% chance for Tasmania and Western Australia. The 12 hospitals in Victoria and the Northern Territory incur annual on-going maintenance costs of $1.51M; no information was available to describe cost savings or health benefits. CONCLUSIONS: The Australian National Hand Hygiene Initiative was cost-effective against an Australian threshold of $42,000 per life year gained. The return on investment varied among the states and territories of Australia.

In vitro dose and duration dependent approaches for the assessment of ameliorative effects of nanoconjugated vancomycin against Staphylococcus aureus infection induced oxidative stress in murine peritoneal macrophages.

AIMS: The present study was aimed to evaluate the in vitro ameliorative effect of nanoconjugated vancomycin (NV) against vancomycin sensitive and resistant strains of Staphylococcus aureus infection-induced oxidative stress in murine peritoneal macrophage. METHODS: Peritoneal macrophages from mice were treated with VSSA and VRSA (5 × 10(6) CFU/mL), VSSA + NV (5-250 µg/ml) and VRSA + NV (5-250 µg/ml) for 18 h, having 3 h interval in culture media; and the superoxide anion generation, lipid peroxidation, protein oxidation, antioxidant enzymes status and glutathione enzymes activity were monitored. RESULTS: The significantly increased free radical generation, lipid peroxidation, protein carbonyls and oxidized glutathione levels were observed in VSSA and VRSA treated group as compared to control group; where as reduced glutathione level, antioxidant enzymes status and glutathione dependent enzymes were decreased significantly. All these changes come near to control in NV treated group in a dose and duration dependent fashion. Among the different doses and duration intervals of NV, maximum ameliorative effect was observed by 100 µg/ml for 12 h treatment which does not produce any damage to the cell. CONCLUSIONS: These findings suggest the potential use and beneficial role of nanoconjugated vancomycin as a modulator of S. aureus infection-induced cellular damage in murine peritoneal macrophage.

In vitro assessment of bacterial translocation during needle insertion through inoculated culture media as a model of arthrocentesis through cellulitic tissue.

OBJECTIVE: To determine by use of an in vitro model the potential for translocating sufficient numbers of bacteria into a joint during arthrocentesis through cellulitic tissue to cause sepsis. SAMPLE: Culture media containing 4 concentrations of Staphylococcus aureus and needles of 3 sizes. PROCEDURES: Needles (22, 20, and 19 gauge) were inserted through Mueller-Hinton agar that contained known concentrations of S aureus (10(3),10(4),10(5), and 10(6) CFUs/mL). After a needle exited through the medium, any agar plug within the needle bore was ejected into a sterile syringe and the contaminated portion of the needle was harvested. Sterile saline (0.9% NaCl) solution was used to emulsify the agar plug and wash the contaminated portion of the needle. The resulting solution was cultured to determine the number of bacterial CFUs that could be deposited into a joint during arthrocentesis through contaminated tissue. RESULTS: Needle gauge and bacterial concentration were both associated with the number of bacterial CFUs deposited after insertion through contaminated agar. Although all needle sizes were capable of bacterial translocation sufficient to cause septic arthritis, ORs for 20- and 22-gauge needles translocating > 33 CFUs of S aureus were significantly higher than the OR for a 19-gauge needle. The ORs for 20- or 22-gauge needles translocating > 33 CFUs of S aureus (the minimum population of S aureus known to cause joint sepsis) were 0.22. CONCLUSIONS AND CLINICAL RELEVANCE: Results for this in vitro model indicated that caution should be used when performing arthrocentesis through cellulitic tissue.

Development and application of an indicator assessment tool for measuring health services accreditation programs.

BACKGROUND: Hospital accreditation programs are internationally widespread and consume increasingly scarce health resources. However, we lack tools to consistently identify suitable indicators to assess and monitor accreditation outcomes. We describe the development and validation of such a tool. RESULTS: Using Australian accreditation standards as our reference point we: reviewed the research evidence for potential indicators; looked for links with existing external indicators; and assessed relevant state and federal policies. We allocated provisional scores, on a five point Likert scale, to the five accountability criteria in the tool: research; accuracy; proximity; no adverse effects; and specificity. An expert panel validated the use of the purpose designed indicator assessment tool. The panel identified hand hygiene compliance rates as a suitable process indicator, and hospital acquired Staphylococcus aureus infection (SAB) rates as an outcome indicator, with the hypothesis that improved hand hygiene compliance rates and lower SAB rates would correlate with accreditation performance. CONCLUSIONS: This new tool can be used to identify, analyse, and compare accreditation indicators. Using infection control indicators such as hand hygiene compliance and SAB rates to measure accreditation effectiveness has merit, and their efficacy can be determined by comparing accreditation scores with indicator outcomes. To verify the tool as a robust instrument, testing is needed in other health service domains, both in Australia and internationally. This tool provides health policy makers with an important means for assessing the accreditation programs which form a critical part of the national patient safety and quality framework.

Assessment of adhesion, invasion and cytotoxicity potential of oral Staphylococcus aureus strains.

The oral cavity is regarded as a relevant site for Staphylococcus aureus colonization. However, characterization of virulence mechanisms of oral S. aureus remains to be uncovered. In this study, twenty one S. aureus strains isolated from the oral cavity of Tunisian patients were screened for adherence, invasion and cytotoxicity against HeLa cells. In addition, the presence of adhesins (icaA, icaD, can, fnbA and fnbB) and α-hemolysin (hla) genes in each strain was achieved by polymerase chain reaction (PCR). Our finding revealed that oral S. aureus strains were able to adhere and invade epithelial cells, with variable degrees (P < 0.05). Moreover they exhibited either low (23.8%) or moderate (76.2%) cytotoxic effects. In addition 76.2% of strains were icaA and icaD positive and 90.5% harbor both the fnbA and the fnbB gene. While the cna gene was detected in 12 strains (57.2%). Furthermore, the hla gene encoding the α-toxin was found in 52.4% of the isolates. All these virulence factors give to S. aureus the right qualities to become a redoubtable pathogen associated to oral infections.

Assessment of total bacterial cells in extended aeration activated sludge plants using flow cytometry as a microbial monitoring tool.

The extended aeration activated sludge (EAAS) process is one of the most applied biological processes in small towns. Here, we study the abundance and viability of total bacterial cells in two wastewater treatment plants (WWTPs) operating with an EAAS process. We use flow cytometry (FCM) combined with SYTO13 and propidium iodide (PI) dyes as a rapid, easy, reliable and accurate microbial monitoring tool. A disaggregation procedure with an ultrasonic bath was designed to detach total bacterial cells from activated sludge flocs for subsequent FCM analysis. This procedure permitted the recovery of total bacterial cells from sludge flocs without affecting bacterial viability, as indicated by bacterial strain controls. Since FCM is a multi-parameter technique, it was possible to determine total bacterial abundance and their viability in the activated sludge. As a comparative method, epifluorescence microscopy was also used to quantify total bacterial cells; both methods produced similar results. The FCM analysis revealed relative microbial stability in both the WWTPs. The total bacterial abundance quantified by FCM in the two plants studied was 1.02-6.23 × 10(11) cells L(-1) with 70-72% viability, one logarithm less than that reported in the literature for WWTPs using the conventional activated sludge process. This can be explained by the difference in the operational parameters between the conventional plant and EAAS, mainly the organic loading rate.

Environmental bacteria produce abundant and diverse antibiofilm compounds.

AIMS: The aim of this study was to isolate novel antibiofilm compounds produced by environmental bacteria. METHODS AND RESULTS: Cell-free extracts were prepared from lawns of bacteria cultured on agar. A total of 126 bacteria isolated from soil, cave and river habitats were employed. Extracts were tested for their ability to inhibit Staphylococcus aureus biofilm in a 96-well microtitre plate assay. A total of 55/126 extracts (44%) significantly inhibited Staph. aureus biofilm. Seven extracts were selected for further analysis. The antibiofilm activities in all seven extracts exhibited unique patterns of molecular mass, chemical polarity, heat stability and spectrum of activity against Staph. aureus, Staphylococcus epidermidis and Pseudomonas fluorescens, suggesting that these seven antibiofilm activities were mediated by unique chemical compounds with different mechanisms of action. CONCLUSIONS: Environmental bacteria produce abundant and diverse antibiofilm compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening cell-free extracts is a useful method for identifying secreted compounds that regulate biofilm formation. Such compounds may represent a novel source of antibiofilm agents for technological development.

Assessment of synergistic combination potential of probiotic and bacteriophage against antibiotic-resistant Staphylococcus aureus exposed to simulated intestinal conditions.

This study was designed to evaluate the combined effect of probiotic Lactobacillus rhamnosus and bacteriophage SA11 on the control of antibiotic-sensitive Staphylococcus aureus (ASSA) and antibiotic-resistant S. aureus (ARSA) under the simulated intestinal conditions. The survivability of ASSA and ARSA were determined in the simulated phosphate-buffered saline (PBS)-, trypticase soy broth (TSB)-, and milk-based gastric juices adjusted to pH 2.0, 3.0, and 5.0 at 37 °C for 30 min. The inhibitory effect of bacteriophage SA11 and probiotic on the growth of ASSA and ARSA was evaluated in the simulated intestinal juices at 37 °C for 20 h. The least reductions in the numbers of ASSA and ARSA were observed in the milk-based gastric juices at pH 2.0 (<1 log). No significant changes in the teichoic acid-mediated sliding motility were observed for ASSA and ARSA after 30-min exposure to the simulated gastric juices (pH 2.0, 3.0, and 5.0), responsible for the enhanced bacterial attachment to the epithelial cells. The bacteriophage SA11 was stable down to pH 5.0 and up to 0.06 % bile salts. The bacteriophage SA11 combined with probiotic effectively inhibited the growth of ASSA and ARSA in the simulated intestinal conditions, showing more than 4 log reduction. The relative expression levels of adhesion-related genes (clfA, eno, and fnbA) and efflux-related genes (mdeA, norB, and norC) were less decreased in ARSA than in ASSA after exposure to the simulated gastrointestinal conditions. These results might shed light on the application of bacteriophage to control the ingested antibiotic-resistant foodborne pathogens in the intestinal tract.