Identification and characterization of virulence factors in Enterococcus faecalis

Enterococci are one of the leading causes of nosocomial infections, and Enterococcus faecalis causes the majority of enterococcal infections. However, the mechanisms of enterococcal pathogenesis are still not yet understood. In our initial screening of E. faecalis strain OG1RF genomic libraries, autolysin and a homolog of a protein of Enterococcus faecium previously designated P54 were found to be two major antigens that reacted with human patient sera, and an antigen designated MH-1 antigen that reacted with serum from a endocarditis patient was also identified. To explore a possible role for these antigens in enterococcal infections, the genes encoding these three antigens were disrupted in Enterococcus faecalis OG1RF. ^ To explore a possible role of an E. faecalis gelatinase (encoded by gelE), which belongs to a family of Zn-metalloproteases that have been shown to be virulence factors in other organisms, in enterococcal infections, an insertion mutant was constructed in OG1RF and tested in the mouse peritonitis model. The mice infected with the gelE mutant showed a significantly prolonged survival compared to the wild type strain. To study the expression of gelE, the regions flanking gelE were sequenced. Sequence analysis of the gelE flanking regions revealed three genes (fsrA, fsrB and fsrC) upstream of gelE that show homology to the genes in a locus (agr) that globally regulates the expression of virulence factors in Staphylococcus aureus and one open reading frame (sprE) with homology to bacterial serine protease downstream of gelE. ^ In conclusion, in this study of identification of possible virulence factors in E. faecalis surface and secreted proteins, of three genes encoding antigens detected by human patient sera, none could be shown to effect virulence in the mouse peritonitis model. Inactivation of one of these antigens (autolysin) was shown to slightly increase the tolerance of E. faecalis to penicillin. A serine protease and a locus (fsr) that regulates the expression of gelE and sprE were shown to be important for enterococcal infection in the mouse peritonitis model. (Abstract shortened by UMI.)^

Identification and characterization of antigens and potential virulence factors in Enterococcus

Enterococci are normal flora in the human intestinal tract, and also one of the leading causes of nosocomial infections, with most of the clinical isolates being Enterococcus faecalis and Enterococcus faecium. Despite extensive studies on the antibiotic resistance, the pathogenicity of enterococci is not well understood, especially for E. faecium. To identify potential virulence factors based on their antigenicity during infection, E. faecium genomic libraries were constructed and screened using sera from patients with E. faecium endocarditis. ^ As one of my projects, total polysaccharides were extracted from E. faecalis OG1RF and from two epa mutants constructed previously, TX5179 and TX5180, and western blots with patient sera showed that an immuno-reactive polysaccharide present in wild type OG1RF was not produced by either of the two epa mutants. The epa mutants were more sensitive to ethanol stress, neutrophil killing and neutrophil phagocytosis than the wild type OG1RF. ^ Expression of virulence factors is commonly regulated by two component systems. A BLAST search was performed to identify potential two component systems in the E. faecalis V583 genome database using PhoP/PhoS as query sequences, and 11 gene pairs were identified, seven of which were disrupted in E. faecalis OGIRF. ^ Finally, an in vitro translocation model was established for enterococci. E. faecalis strain OG1RF and E. faecium strain DO were shown to be able to translocate across a T84 monolayer, while E. coli strain DH5α and E. faecalis strain E1 could not. ^ In conclusion, several E. faecium antigens expressed in infection (whose antibodies present in sera from patients with E. faecium endocarditis) were identified, two of which, SagA and GlyA, were characterized and suggested to be involved in cell wall metabolism. E. faecalis epa gene cluster (involving in polysaccharide biosynthesis and known to be involved in virulence of E. faecalis in mice) was shown to be involved in hindering neutrophil killing. Several two-component systems were identified in E. faecalis and two of which, EtaRS and EtbRS, were involved in E. faecalis virulence in a mouse peritonitis model.^

Non-staphylococcal infections of cardiac implantable electronic devices

Background. The number of infections of cardiac implantable electronic devices (CIED) continues to escalate out of proportion to the increase rate of device implantation. Staphylococcal organisms account for 70% to 90% of all CIED infections. However, little is known about non-staphylococcal infections, which have been described only in case reports, small case series or combined in larger studies with staphylococcal CIED infections, thereby diluting their individual impact. ^ Methods. A retrospective review of hospital records of patients admitted with a CIED-related infections were identified within four academic hospitals in Houston, Texas between 2002 and 2009. ^ Results. Of the 504 identified patients with CIED-related infection, 80 (16%) had a non-staphylococcal infection and were the focus of this study. Although the demographics and comorbities of subjects were comparable to other reports, our study illustrates many key points: (a) the microbiologic diversity of non-staphylococcal infections was rather extensive, as it included other Gram-positive bacteria like streptococci and enterococci, a variety of Gram-negative bacteria, atypical bacteria including Nocardia and Mycobacteria, and fungi like Candida and Aspergillus; (b) the duration of CIED insertion prior to non-staphylococcal infection was relatively prolong (mean, 109 ± 27 weeks), of these 44% had their device previously manipulated within a mean of 29.5 ± 6 weeks; (c) non-staphylococcal organisms appear to be less virulent, cause prolonged clinical symptoms prior to admission (mean, 48 ± 12.8 days), and are associated with a lower mortality (4%) than staphylococcal organisms; (d) thirteen patients (16%) presented with CIED-related endocarditis; (e) although not described in prior reports, we identified 3 definite and 2 suspected cases of secondary Gram-negative bacteremia seeding of the CIED; and (f) inappropriate antimicrobial coverage was provided in approximately 50% of patients with non-staphylococcal infections for a mean period of 2.1 days. ^ Conclusions. Non-staphylococcal CIED-related infections are prevalent and diverse with a relatively low virulence and mortality rate. Since non-staphylococcal organisms are capable of secondarily seeding the CIED, a high suspicion for CIED-related infection is warranted in patients with bloodstream infection. Additionally, in patients with suspected CIED infection, adequate Gram positive and -negative antibacterial coverage should be administered until microbiologic data become available.^

The evidence for groundwater as a source of enteric illness in developing countries: A systematic review of the literature

Groundwater constitutes approximately 30% of freshwater globally and serves as a source of drinking water in many regions. Groundwater sources are subject to contamination with human pathogens (viruses, bacteria and protozoa) from a variety of sources that can cause diarrhea and contribute to the devastating global burden of this disease. To attempt to describe the extent of this public health concern in developing countries, a systematic review of the evidence for groundwater microbially-contaminated at its source as risk factor for enteric illness under endemic (non-outbreak) conditions in these countries was conducted. Epidemiologic studies published in English language journals between January 2000 and January 2011, and meeting certain other criteria, were selected, resulting in eleven studies reviewed. Data were extracted on microbes detected (and their concentrations if reported) and on associations measured between microbial quality of, or consumption of, groundwater and enteric illness; other relevant findings are also reported. In groundwater samples, several studies found bacterial indicators of fecal contamination (total coliforms, fecal coliforms, fecal streptococci, enterococci and E. coli), all in a wide range of concentrations. Rotavirus and a number of enteropathogenic bacteria and parasites were found in stool samples from study subjects who had consumed groundwater, but no concentrations were reported. Consumption of groundwater was associated with increased risk of diarrhea, with odds ratios ranging from 1.9 to 6.1. However, limitations of the selected studies, especially potential confounding factors, limited the conclusions that could be drawn from them. These results support the contention that microbial contamination of groundwater reservoirs—including with human enteropathogens and from a variety of sources—is a reality in developing countries. While microbially-contaminated groundwaters pose risk for diarrhea, other factors are also important, including water treatment, water storage practices, consumption of other water sources, water quantity and access to it, sanitation and hygiene, housing conditions, and socio-economic status. Further understanding of the interrelationships between, and the relative contributions to disease risk of, the various sources of microbial contamination of groundwater can guide the allocation of resources to interventions with the greatest public health benefit. Several recommendations for future research, and for practitioners and policymakers, are presented.^

Investigación técnica y económica sobre desinfección de aguas residuales por sistemas de oxidación

Un incremento de la demanda del agua, junto con el aumento de la contaminación, ha provocado que hoy en día la reutilización de las aguas depuradas sea necesaria, pero la reutilización de aguas debe garantizar y minimizar los posibles riesgos sanitarios y medioambientales que su práctica pueda provocar. En España estos parámetros se encuentran regulados por el RD 1620/2007 relativo al régimen jurídico de la reutilización de las aguas depuradas. Las aguas regeneradas son aguas que ya han sido sometidas a un tratamiento de depuración, y a las cuales se aplica un posterior tratamiento adicional o complementario que permita adecuar su calidad al uso al que vaya a destinarse. Siendo requeridos para los distintos reúsos procesos de desinfección, uno de los principales sistemas utilizados es el cloro, debido a su sencilla aplicación y costos bajos, sin tomar en cuenta la posible formación de compuestos organohalogenados potencialmente cancerígenos. Es por esto que surge la necesidad de aplicar distintos sistemas de oxidación objeto de estudio en esta tesis, como el dióxido de cloro estabilizado, ozono y los procesos avanzados de oxidación (Advanced Oxidation Processes, AOP), ozono/peróxido y uv/peróxido. En esta tesis se investiga los rendimientos que pueden alcanzar estos sistemas en la eliminación de los ácidos húmicos y los fenoles, siendo las principales sustancias formadoras de subproductos de la desinfección, así mismo, se considera necesario garantizar la desinfección del agua a través del estudio de tres grupos de microrganismos, los coliformes totales, e. coli y enterococos, siendo un punto importante el posible recrecimiento microbiológico debido a una desinfección escasa, por la permanencia en el agua de los compuestos antes mencionados, o por alguna fuente de alimento que pudieran encontrar en el sistema de distribución. Lo más importante será la calidad que se pueda alcanzar con estos desinfectantes, con el fin de obtener agua para los distintos reúsos que existen en la actualidad. Y así no limitar los alcances que puede tener la reutilización de las aguas residuales. Basándose en lo antes mencionado se procedió a realizar la caracterización del agua del rio Manzanares, con el fin de determinar la cantidad de ácidos húmicos disueltos y fenoles, obteniendo valores bajos, se decidió incorporar a las muestras de rio 5 mg/L de estos compuestos, con el fin de observar de que manera podrían interferir en la desinfección de esta agua. De esta forma se logran obtener resultados óptimos de los sistemas de desinfección estudiados, siendo el Ozono un oxidante eficiente en la desinfección de los microrganismos y en la eliminación de ácidos húmicos y fenoles con tiempos de contacto cortos, mostrando deficiencias al permitir el recrecimiento de los coliformes totales. Del sistema de oxidación avanzada UV/Peróxido se determino como un eficiente desinfectante para garantizar la inexistencia de rebrotes, al paso del tiempo. Así mismo se concluye que tiene buenos rendimientos en la eliminación del ácido húmico y los fenoles. An increase in water demand, coupled with increasing pollution, has caused today reuse of treated water is necessary, but must ensure water reuse and minimize potential health and environmental risks that their practice is cause. In Spain these parameters are regulated by Royal Decree 1620/2007 on the legal regime of the reuse of treated water. The reclaimed water is water that has already been subjected to a depuration treatment, which is applied as a subsequent further treatment that will bring quality to the use to which is to be delivered. As required for various reuses disinfection processes, one of the main systems used is chlorine, due to its simple implementation and low costs, without taking into account the possible formation of potentially carcinogenic halogenated organic compounds. That is why there is a need to apply different oxidation systems studied in this thesis, as stabilized chlorine dioxide, ozone and advanced oxidation processes (AOP), ozone/peroxide and UV/peroxide. This thesis investigates the rates can reach these systems in removing humic acids and phenols, the main substances forming disinfection byproducts, likewise, it is considered necessary to ensure water disinfection through the study of three groups of microorganisms, total coliform, e. coli and enterococci, the important point being a possible regrowth due to microbiological disinfection scarce, the water remaining on the aforementioned compounds, or a food source which may be found in the distribution system. The most important quality is that achievable with these disinfectants, with the water to obtain various reuses that exist today. And thus not limit the scope that can be reuse of wastewater. Based on the above we proceeded to perform characterization Manzanares river water, in order to determine the quantity of dissolved humic acids and phenols, obtaining low values, it was decided to incorporate river samples 5 mg / L of these compounds, in order to observe how they might interfere with the disinfection of the water. Thus optimum results are achieved for disinfection systems studied, being efficient ozone oxidant in the disinfection of microorganisms and the removal of humic acids and phenols with short contact times, showing gaps to allow regrowth total coliforms. Advanced oxidation system UV / peroxide were determined as an efficient disinfectant to ensure the absence of volunteers, the passage of time. Also it is concluded that has good yields in removing humic acid and phenols.

Bacterial resistance to vancomycin: Overproduction, purification, and characterization of VanC2 from Enterococcus casseliflavus as a d-Ala-d-Ser ligase

The VanC phenotype for clinical resistance of enterococci to vancomycin is exhibited by Enterococcus gallinarum and Enterococcus casseliflavus. Based on the detection of the cell precursor UDP-N-acetylmuramic acid pentapeptide intermediate terminating in d-Ala-d-Ser instead of d-Ala-d-Ala, it has been predicted that the VanC ligase would be a d-Ala-d-Ser rather than a d-Ala-d-Ala ligase. Overproduction of the E. casseliflavus ATCC 25788 vanC2 gene in Escherichia coli and its purification to homogeneity allowed demonstration of ATP-dependent d-Ala-d-Ser ligase activity. The kcat/Km2 (Km2 = Km for d-Ser or C-terminal d-Ala) ratio for d-Ala-d-Ser/d-Ala-d-Ala dipeptide formation is 270/0.69 for a 400-fold selection against d-Ala in the C-terminal position. VanC2 also has substantial d-Ala-d-Asn ligase activity (kcat/Km2 = 74 mM−1min−1).

VanX, a bacterial d-alanyl-d-alanine dipeptidase: Resistance, immunity, or survival function?

The zinc-containing d-alanyl-d-alanine (d-Ala-d-Ala) dipeptidase VanX has been detected in both Gram-positive and Gram-negative bacteria, where it appears to have adapted to at least three distinct physiological roles. In pathogenic vancomycin-resistant enterococci, vanX is part of a five-gene cluster that is switched on to reprogram cell-wall biosynthesis to produce peptidoglycan chain precursors terminating in d-alanyl-d-lactate (d-Ala-d-lactate) rather than d-Ala-d-Ala. The modified peptidoglycan exhibits a 1,000-fold decrease in affinity for vancomycin, accounting for the observed phenotypic resistance. In the glycopeptide antibiotic producers Streptomyces toyocaensis and Amylocatopsis orientalis, a vanHAX operon may have coevolved with antibiotic biosynthesis genes to provide immunity by reprogramming cell-wall termini to d-Ala-d-lactate as antibiotic biosynthesis is initiated. In the Gram-negative bacterium Escherichia coli, which is never challenged by the glycopeptide antibiotics because they cannot penetrate the outer membrane permeability barrier, the vanX homologue (ddpX) is cotranscribed with a putative dipeptide transport system (ddpABCDF) in stationary phase by the transcription factor RpoS (σs). The combined action of DdpX and the permease would permit hydrolysis of d-Ala-d-Ala transported back into the cytoplasm from the periplasm as cell-wall crosslinks are refashioned. The d-Ala product could then be oxidized as an energy source for cell survival under starvation conditions.

Detecção de Enterococcus resistentes a vancomicina em criações comerciais de ovinos e caprinos das regiões centro-leste e nordeste do Estado de São Paulo

As exigências das condições higiênico-sanitárias na produção de animais de interesse zootécnico vêm aumentando progressivamente dada à necessidade de aliar-se produtividade a produtos de alta qualidade para atender a mercados consumidores cada vez mais exigentes. Nesse sentido, a utilização de antimicrobianos, tanto na profilaxia como na terapêutica, permanece como estratégia de controle para vários microrganismos patogênicos, de importância não apenas para a produção animal como também para a saúde humana, ainda que restrições ao uso indiscriminado desses produtos têm se intensificado. Não obstante, o uso excessivo desses produtos está associado à seleção de microrganismos resistentes nas áreas de produção. Por outro lado, investigações sobre circulação de cepas resistentes em rebanhos animais, até então restritas a populações humanas, ainda permanecem limitadas no Brasil. Bactérias do gênero Enterococcus, integrantes usuais da microbiota gastrointestinal animal e humana, são indicadoras ambientais de contaminação fecal e tem-se tornado objeto de preocupação em saúde pública e veterinária dada a ocorrência de cepas resistentes à vancomicina (VRE). O presente trabalho teve como objetivo isolar, quantificar e caracterizar VRE presentes em amostras fecais de ovinos oriundos de pequenas propriedades das regiões centro-leste e nordeste do estado de São Paulo. Para tanto, 132 amostras fecais foram coletadas diretamente do reto dos animais ou do piso das instalações. As amostras foram semeadas em ágar m-Enterococcus e subcultivadas em Ágar Bile Esculina acrescido de 6 µg/mL de vancomicina (ABEV), para confirmação de Enterococcus spp e detecção de cepas resistentes. Procedeu-se igualmente a observação da morfologia, características tintoriais, bioquímicas e moleculares. O número máximo de Enterococcus spp. encontrado foi de 2,6 × 105 e 1,70 × 105 UFC/g de fezes do ambiente e dos animais, respectivamente. Na caracterização bioquímica espécies mais prevalentes foram: Enterococcus faecalis e Vagococcus fluvialis. No ABEV, houve crescimento de colônias VRE em 33 das 84 amostras de ovinos-caprinos e em 21 das 48 amostras ambientais, representando, respectivamente 46,7% e 29,3% das amostras analisadas. A análise por multiplex PCR das 54 cepas VRE obtidas indicaram que 23 (43%), 22 (41%), 2 (3,5%) e 2 (3,5%) foram positivas, respectivamente, para os genes vanC2/C3, vanC1, vanA e vanB, sendo que para 5,3% dos isolados nenhum produto foi amplificado, sugerindo a possível ocorrência de genes dos demais grupos van conhecidos entre os isolados. Os resultados obtidos indicam, de forma inédita no país, a circulação de VRE em propriedades produtoras de ovinos e caprinos, sem ocorrência de manifestações clínicas aparentes nos animais, porém com possíveis riscos à saúde dos produtores e profissionais envolvidos, bem como a eventuais consumidores.

An investigation of contact transmission of methicillin resistant-Staphylococcus aureus

Hand hygiene is critical in the healthcare setting and it is believed that methicillin-resistant Staphylococcus aureus (MRSA), for example, is transmitted from patient to patient largely via the hands of health professionals. A study has been carried out at a large teaching hospital to estimate how often the gloves of a healthcare worker are contaminated with MRSA after contact with a colonized patient. The effectiveness of handwashing procedures to decontaminate the health professionals' hands was also investigated, together with how well different healthcare professional groups complied with handwashing procedures. The study showed that about 17% (9-25%) of contacts between a healthcare worker and a MRSA-colonized patient results in transmission of MRSA from a patient to the gloves of a healthcare worker. Different health professional groups have different rates of compliance with infection control procedures. Non-contact staff (cleaners, food services) had the shortest handwashing times. In this study, glove use compliance rates were 75% or above in all healthcare worker groups except doctors whose compliance was only 27%. (C) 2004 The Hospital Infection Society. Published by Elsevier Ltd. All rights reserved.

Bacterial resistance to antibiotics:modified target sites

Alteration in the target sites of antibiotics is a common mechanism of resistance. Examples of clinical strains showing resistance can be found for every class of antibiotic, regardless of the mechanism of action. Target site changes often result from spontaneous mutation of a bacterial gene on the chromosome and selection in the presence of the antibiotic. Examples include mutations in RNA polymerase and DNA gyrase, resulting in resistance to the rifamycins and quinolones, respectively. In other cases, acquisition of resistance may involve transfer of resistance genes from other organisms by some form of genetic exchange (conjugation, transduction, or transformation). Examples of these mechanisms include acquisition of the mecA genes encoding methicillin resistance in Staphylococcus aureus and the various van genes in enterococci encoding resistance to glycopeptides. © 2005 Elsevier B.V. All rights reserved.

Gram-positive infections in patients with end-stage renal disease

Gram-positive microorganisms, specifically coagulase-negative staphylococci are the most common species recovered from clinical culture specimens of patients with end-stage renal disease. The propensity of coagulase-negative staphylococci (CNS) to cause infection in this patient group has been widely debated. However, it is still unclear how this usually avirulent commensal microorganism produces infection that contributes to high rates of morbidity and mortality in patients with end-stage renal disease. The aim of this thesis was to investigate the rate, geographical distribution, molecular and phenotypic mechanisms of Gram-positive microorganisms associated with infection in renal dialysis patients. In addition, it sought to assess the value of early serological diagnosis of dialysis catheter-associated infection and the effect of antimicrobial treatment regimens on the faecal carriage of enteric microorganisms. In this study, the incidence of haemodialysis catheter-associated infection was established with the Meditrend audit tool. This tool was used to assess the infection outcomes of catheter insertion and management procedures until the catheter was explanted. Introduction of a catheter management protocol decreased the incidence of catheter-related infection. Staphylococcal species recovered from episodes of haemodialysis catheter-associated infection and continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis were genotyped by determination of macrorestriction profiles with pulsed-field gel electrophoresis. This highlighted horizontal transfer of microorganisms between different patients and the environment. The phenotypic characteristics of these strains were also investigated to determine characteristics that could be used as markers for dialysis catheter-associated infection. The expression of elastase, lipase and esterase by CNS was significantly associated with infection. A rapid enzyme-linked immunosorbent assay incorporating a novel staphylococcal antigen (lipid S) was used to evaluate the early detection of anti-staphylococcal immunoglobulin gamma in patient sera. The comparison of culture positive and culture negative patients demonstrated a steady state of immune activation in both groups. However anti-lipid S serum antibody titres > 1000 were found to be a predictor of infection. The effect on faecal carriage of vancomycin resistant enterococci (VRE) and Clostridium difficile toxins in patients treated with CAPD when empiric cephalosporin therapy was substituted for piperacillin/tazobactam was investigated. The introduction of piperacillin/tazobactam demonstrated a decrease in the faecal carriage of VRE.

The antimicrobial efficacy of copper alloy furnishing in the clinical environment:a crossover study

Objective. To determine whether copper incorporated into hospital ward furnishings and equipment can reduce their surface microbial load. Design. A crossover study. Setting. Acute care medical ward with 19 beds at a large university hospital. Methods. Fourteen types of frequent-touch items made of copper alloy were installed in various locations on an acute care medical ward. These included door handles and push plates, toilet seats and flush handles, grab rails, light switches and pull cord toggles, sockets, overbed tables, dressing trolleys, commodes, taps, and sink fittings. Their surfaces and those of equivalent standard items on the same ward were sampled once weekly for 24 weeks. The copper and standard items were switched over after 12 weeks of sampling to reduce bias in usage patterns. The total aerobic microbial counts and the presence of indicator microorganisms were determined. Results. Eight of the 14 copper item types had microbial counts on their surfaces that were significantly lower than counts on standard materials. The other 6 copper item types had reduced microbial numbers on their surfaces, compared with microbial counts on standard items, but the reduction did not reach statistical significance. Indicator microorganisms were recovered from both types of surfaces; however, significantly fewer copper surfaces were contaminated with vancomycin-resistant enterococci, methicillin-susceptible Staphylococcus aureus, and coliforms, compared with standard surfaces. Conclusions. Copper alloys (greater than or equal to 58% copper), when incorporated into various hospital furnishings and fittings, reduce the surface microorganisms. The use of copper in combination with optimal infection-prevention strategies may therefore further reduce the risk that patients will acquire infection in healthcare environments.

Faecal bacteria in Perna perna (Linnaeus, 1758) (Mollusca: Bivalvia) for biomonitoring coastal waters and seafood quality

O desenvolvimento urbano em áreas costeiras é intenso e o aumento das descargas de esgoto é uma das conseqüências. Assim, leis específicas que regulem limites para a contaminação microbiológica das águas e de alimentos de origem marinha são necessárias. O objetivo deste estudo é avaliar a utilização de Enterococos e coliformes termotolerantes na carne de mexilhões Perna perna como alternativa para o monitoramento da qualidade microbiológica de águas costeiras. O estudo também considera relações alométricas aplicadas às taxas de clearance para entender a concentração de bactérias pelo molusco. As densidades bacterianas obtidas no molusco foram de 50 a 4300 vezes maiores do que nas amostras de água coletadas próximas dos moluscos e alguns foram considerados impróprios para consumo, mesmo quando as águas não mostraram restrições. A taxa de clearance média obtida para retenção de Enterococos foi 317.7 ml h-1 e essas taxas (CR) puderam ser relacionadas com o tamanho dos mexilhões (L) pela equação CR = 28.3229L1.6421. Os resultados mostram que as densidades bacterianas nos tecidos dos mexilhões podem refletir a contaminação crônica do ambiente e que as taxas de clearance devem ser consideradas para a tomada de decisão em situações onde, por exemplo, se deseja implantar mexilhões para cultivo.

Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.