Enterococcus faecalis are Gram-positive bacteria that colonize the gastrointestinal tracts of mammals, birds and invertebrates and are also found in sewage, soil, food and water. In addition to being commensal organisms, Enterococci can also cause nosocomial infections in humans including urinary tract infections, septicemia and endocarditis. Hospital-acquired infections often present a challenge in treatment due to the emergence of multi-drug resistant strains. Enterococcal plasmids may act as extremely stable reservoirs for resistance genes and other virulence factors. Pheromone responsive plasmids such as pCF10 mediate efficient transfer of genetic material within the species E. faecalis but may also be capable of transferring resistance genes across species and genus boundaries. Polymicrobial environments often found in nosocomial infections may expose plasmid-harboring enterococci to pathogenic species, poising cells for this type of promiscuous horizontal gene transfer of resistance determinants. Previous studies showed that prgW, which encodes the pCF10 replication initiation protein PrgW, is the minimal origin of replication for this plasmid. The replicon, which is usually limited to Enterococcal spp., can replicate in Lactococcus lactis if it is engineered to produce pre-cCF10. Three conserved cysteines (C78/C275/C307) are important for plasmid stability and allow for replication of the pCF10 replicon in L. lactis in the absence of pre-cCF10. PrgW has a predicted molecular weight of 38,635. Four polyclonal antibodies targeting PrgW at the N-terminus (aa 1-20), C-terminus (aa 314-333) and two internal regions (aa 64-80 and aa 250-271) were used in current experiments and retrospective studies. When PrgW was overexpressed in E. faecalis, four different apparent approximate molecular weights were detected by Western blotting (p40*, p36*, p24* and p18*), suggestive of processing. In Enterococci where the replicon is active, p36* was consistently detected by all four antisera; when PrgW was overexpressed in Streptococcus mutans where the replicon is non-functional, p49* and p40* were detected but p36* was not observed. PrgW p24* was detected by a mixture of the internally targeting antibodies as well as the C-terminal targeting antibody, but not the N-terminal targeting antibody, suggesting that the N-terminal domain of PrgW has been cleaved off in p24*. The p24* form may play a role in pCF10 stability. Mutations to three cysteines in PrgW (C78/C275/C307), which reduce the stability of pCF10, result in the loss of p24*. Enterococcal conjugative plasmids have been previously implicated in the transfer of antibiotic resistance genes. The pCF10 plasmid contains the conjugative transposon Tn925, which possesses the tetM tetracycline resistance gene. Proximity of donor and recipient cells is a key part of pheromone-responsive conjugation. Aggregation substance allows for formation of clumps of E. faecalis in liquid mating experiments. E. faecalis forms biofilms; in contrast to filter mating experiments, polymicrobial biofilms provide an in vitro model of a natural scenario during which horizontal gene transfer may occur. Rates of cross-genus genetic transfer of tetM between E. faecalis OG1RF(pCF10) donor cells and Staphylococcus aureus recipient cells growing on glass coverslips as mixed-species biofilm populations were determined to be 10-8 after pheromone induction of pCF10 conjugation. This biofilm transfer model also holds potential to test the efficacy of synthetic peptides in the reduction or even prevention of pCF10 transfer, and the consequential dissemination of antibiotic resistance determinants throughout the genus Enterococcus and beyond. / Microbiology and Immunology

The microbial source tracking (MST) methods of antibiotic resistance analysis (ARA) and fluorometry (to detect optical brighteners in detergents) were used in the summers of 2004 and 2005 to determine the origins of fecal pollution at beaches with a past history of, or the potential for, high enterococci counts and posted advisories. At Hilton and Anderson beaches, ARA and fluorometry in the summer of 2004 detected substantial human-origin pollution in locations producing consistently high counts of Enterococcus spp. Investigations by municipal officials led to the fluorometric detection and subsequent repair of sewage infrastructure problems at both beaches. The success of these mitigation efforts was confirmed during the summer of 2005 using ARA and fluorometry, with the results cross-validated by pulsed-field gel electrophoresis (PFGE). Results at other beaches indicated that birds and/or wildlife were largely responsible for elevated enterococci levels during 2004 and 2005. The application of fluorometry proved difficult in opens waters due to high levels of dilution, but showed potential for use in storm drains. An additional study developed and tested a new library-based MST approach based on the pattern of DNA band lengths produced by the amplification of the 16S-23S rDNA intergenic spacer region, and subsequent digestion using the restriction endonuclease MboI. Initial results from small known-source libraries yielded high average rates of correct classification (ARCC). However, an increase in the library size was accompanied by a reduction in the ARCC of the library and the method was deemed unsuccessful, and unsuitable for field application. A final study focused on the potential for classification bias with disproportionate source category sizes using discriminant analysis (DA), logistic regression (LR), and k-nearest neighbor (K-NN) statistical classification algorithms. Findings indicated that DA was the most robust algorithm for use with source category imbalance when measuring both correct and incorrect classification rates. Conversely k-NN was identified as the most sensitive algorithm to imbalances with the greatest levels of distortion obtained from the highest k values. Conclusions of this project include: 1) application of a validation set, as well as a minimum detectable percentage to known-source libraries aids in accurately assessing the classification power of the library and reducing the false positive identification of contributing fecal sources; 2) the validation of MST results using multiple methods is recommended for field applications; 3) fluorometry displayed potential for detecting optical brighteners as indicators of sewage leaks in storm drains; 4) the digestion of the 16S-23S rDNA intergenic spacer region of Enterococcus spp. using MboI does not provided suitable discriminatory power for use as an MST method; and 5) DA was the least, and k-NN the most, sensitive algorithm to imbalances in the size of source categories in a known-source library. / Ph. D.

Doctor of Philosophy / Department of Entomology / Ludek Zurek / House fly (Musca domestica L.) larvae develop in decaying organic substrates such as animal manure and adult flies likely play an important role in the ecology of fecal bacteria, including potentially virulent strains. House fly larval development strictly depends on an active bacterial community in the habitat. Although the principle of this symbiosis is not well understood, this association plays a fundamental role in transmission of microbes by this insect. In this study, enterococci were chosen as a model organism to assess the role of house flies in dissemination of multi-drug resistant bacteria in the agricultural environment. House flies (FF) and cattle manure (FM) from a cattle feedlot (frequent use of antibiotics) and house flies (BF) and manure of the American bison (BM) from the Konza Prairie Nature Preserve (no antibiotic use) were collected and analyzed. Results showed a significantly higher prevalence of enterococci resistant to tetracycline and erythromycin in FM and FF compared to that of BF and BM. Enterococcal diversity did not indicate the house fly development in manure in the corresponding habitats but the antibiotic resistance data showed very similar profiles among isolates from flies and corresponding locations. Resistance genes (tetM, tetS, tetO, ermB) and the conjugative transposon Tn916 were the most commonly detected determinants from resistant isolates from both environments. The house fly digestive tract was evaluated for the potential for horizontal transfer of antibiotic resistance genes among Enterococcus faecalis. Horizontal transfer of the pCF10 plasmid with the tetracycline resistance gene (tetM) occurred in the fly digestive tract with a transfer rate up to 101 T/D. In addition, eight enterococcal species were selected to evaluate their role and survival during house fly development. Overall, the survival rate (egg to adult) was significantly higher with E. hirae, E. durans and E. avium compared to other strains. These results indicate: a) house flies play an important role in the ecology of antibiotic resistant enterococci; b) the house fly digestive tract provides conditions for horizontal gene transfer among enterococci, and c) enterococci support the house fly development and can colonize the gut of newly emerging adult flies.

Doctor of Philosophy / Department of Entomology / Ludek Zurek / Enterococci are a group of commensal bacteria that are important nosocomial pathogens. They are abundant in human sewage and wastewater treatment facilities (WWTF). This study focused on the role of house flies, Musca domestica, in the ecology of enterococci at WWTF in both field and laboratory experiments. The first study objective focused on sampling and characterizing enterococci from house flies and wastewater sludge from four WWTF in northeastern Kansas. Enterococci were quantified, identified, and screened for antibiotic resistance and virulence traits, and genotyped. The profiles of enterococci (spp. diversity, antibiotic resistance and virulence) from WWTF sludge and the house flies were similar, indicating that the flies successfully acquired the bacteria from the WWTF substrate. Enterococci with the greatest amount of antibiotic resistant and virulence traits originated from the WWTF that processed meat waste from a commercial sausage plant. Genotyping of E. faecalis revealed clonal matches from sludge and house flies. The second study objective involved tracking the fate of E. faecalis in the digestive tract of house flies in laboratory assays. Colony forming unit (CFU) counts were highest in the midgut at 1 h and declined during the first 24 h. In the labellum, foregut and hindgut, E. faecalis concentrations were more variable but were overall higher after 24 h. Observations from CFU counts and visualizations under a dissecting microscope revealed that E. faecalis peaked in the crop after 48 h suggesting active proliferation in this region. The third objective of the study involved tracking the emergence of calyptrate muscoid flies from stockpiled biosolid cake at one of the four WWTF. Traps were employed at the site for a total of 47 weeks, totaling 386 trap-weeks. A total of 11,349 calyptrate muscoid flies were identified with the two most common species being stable flies (Stomoxys calcitrans) (9,016, 80.2%) and house flies (2022, 18.0%). Numbers of stable flies and house flies peaked around mid-July each year and a second, smaller peak was observed for stable flies 5-8 weeks later. Estimated annual emergence of stable flies was 551,404 and for house flies 109,188.

Doctor of Philosophy / Department of Grain Science and Industry / Subramanyam Bhadriraju / Ludek Zurek / Feed samples and live stored-product insects from feed mills and swine farms were collected and cultured for Enterococcus spp. The mean concentration of enterococci in insect and feed were 2.7 ± 0.5 × 101 cfu/insect and 6.3 ± 0.7 × 103 cfu/g respectively. A total of 362 isolates of enterococci collected from 89 feed samples and 228 stored-product insects were identified to the species level using PCR. These isolates were represented by Enterococcus casseliflavus (53.0%), E. gallinarum (20.4%), E. faecium (16.2%), E. hirae (5.2%), and E. faecalis (5.0%). Enterococci were phenotypically resistant to tetracycline (48.0%), erythromycin (14.3%), streptomycin (16.8%), kanamycin (12.1%), ciprofloxacin (11.0%), ampicillin (3.3%), and chloramphenicol (1.1%). All isolates were susceptible to vancomycin and gentamicin. Tetracycline resistance was encoded by tetM (50.0%), tetO (15.1%), tetK (0.5%), tetS (0.2%) and other unknown tetracycline determinants. Enterococci carried virulence genes including gelatinase (gelE; 21.5%), an enterococcus surface protein (esp; 1.9%), and cytolysin (cylA; 2.2%). An aggregation substance (asa1) gene was detected in 61.0% of E. faecalis isolates. Fifty perncet of E. faecalis isolates were phenotipically tested positive for aggregation substances. Enterococci with cylA genes were hemolytic (52.0%) and with gelE genes were gelatinolytic (18.5%). The ermB gene, encoding erythromycin resistance was detected in 8.8% of the total isolates. The Tn916/1545 family of conjugative transposons was detected in 10.7% of the isolates. Laboratory experiments showed that adults of the red flour beetle, Tribolium castaneum (Herbst), fed on poultry and cattle feeds inoculated with E. faecalis OG1RF:pCF10, were able to successfully acquire enterococci and contaminate sterile poultry and cattle feeds. To assess the potential of horizontal gene transfer, conjugation assays were carried out with E. faecalis using a donor (wild strains) and recipient (E. faecalis OG1SSP) in ratio of 1:10. Only one isolate (1 out of 18 E. faecalis) could transfer tetM to a recipient using broth mating. However, filter mating assay, followed by PCR confirmation revealed that 89.0% (16 out of 18 E. faecalis) of isolates could transfer tetM to E. faecalis. Transfer ratios of transconjugant per recipients ranged from 2.6 × 10-4 to 1 × 10-9. In summary, feed (52.0%) and stored-product insects (41.6%) collected from feed mills and swine farms carried antibiotic-resistant and potentially virulent enterococci. Our study showed that T. castaneum, a pest commonly associated with feed, served as a potential vector for enterococci in the feed environment. Conjugation assays followed by PCR confirmed presence of the tetM gene on a mobile genetic element(s) such as Tn916 and may be horizontally transferred to other Enterococcus species and to other bacteria of clinical significance.

Made available in DSpace on 2016-12-23T14:04:26Z (GMT). No. of bitstreams: 1 Susanne Mariani Loss.pdf: 2049956 bytes, checksum: 69145238536f7bbbe30c59f18c5e2885 (MD5) Previous issue date: 2012-12-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A Ilha das Caieiras está localizada em uma das regiões mais carentes no município de Vitória/ES, a Grande São Pedro. Esta região é densamente povoada e é conhecida como um bolsão de pobreza da capital, com a população sobrevivendo, principalmente, da coleta e comercialização de frutos do mar do manguezal localizado em seu entorno. Estudos realizados anteriormente na região mostraram que a água deste estuário está contaminada com microrganismos patogênicos provenientes, principalmente, de esgotos domésticos lançados sem prévio tratamento. O objetivo deste trabalho foi avaliar a presença de vírus entéricos (adenovírus AdV, rotavírus RV e norovírus GII NoV) e indicadores bacterianos de poluição fecal (coliformes termotolerantes e enterococos) em amostras de água de quatro pontos deste importante estuário da Ilha das Caieiras. O monitoramento ocorreu de janeiro de 2011 a julho de 2012 (19 meses) e utilizou-se as técnicas moleculares PCR Qualitativa e PCR em Tempo Real (qPCR), para detecção de vírus entéricos, e membrana filtrante, para análise de bactérias, além de avaliação de parâmetros físico-químicos da água. Os resultados das análises microbiológicas da água nos pontos estudados (P1, P2, P3 e P4) demonstraram a presença de coliformes termotolerantes, com médias geométricas de 1,66x102, 1,31x102, 2,29x103 e 3,13x102 UFC / 100 mL de água, respectivamente. Para enterococos, as médias encontradas foram 6,30x101 UFC / 100 mL em P1, 5,56x101 UFC / 100 mL em P2, 1,89x103 UFC / 100 mL em P3 e 1,62x103 UFC / 100 mL em P4. Os vírus entéricos foram detectados nos quatro pontos de monitoramento pelas duas técnicas moleculares utilizadas, com valores máximos de 2,00x103, 5,24x104 e 1,50x104 CG / 100 mL para AdV, RV e NoV, respectivamente, quantificados por qPCR. A frequências de detecção de vírus nas amostras de água do estuário variou de 21 27% para AdV, 42 53% para RV e 10 42% para NoV GII. Neste estudo foi possível verificar que o estuário da Ilha das Caieiras apresenta-se contaminado devido, possivelmente, aos lançamentos de esgoto in natura e/ou de lixiviados de áreas rurais. A ação antropogênica sobre este manguezal é evidente e preocupante, pois se reflete na qualidade ambiental deste meio, na saúde da população e na economia da região / The Ilha das Caieiras is located in one of the poorest regions in Vitória / ES, the Grande São Pedro. This region is densely populated and is known as a pocket of poverty in the capital, with a population surviving mainly on the collection and marketing of seafood mangrove located in their surroundings. Previous studies have shown that the region of this estuary water is contaminated with pathogenic microorganisms from mainly domestic sewage released without prior treatment. The aim of this study was to evaluate the presence of enteric viruses (adenovirus - AdV, rotavirus - RV and norovirus GII - NoV) and bacterial indicators of fecal pollution (fecal coliform and enterococci) in water samples from four sites of this important Ilha das Caieiras estuary. Monitoring occurred from January 2011 to July 2012 (19 months) and used molecular techniques Qualitative PCR and Real Time PCR (qPCR) for detection of enteric viruses, and membrane filter for analysis of bacteria, and assessment of physico-chemical parameters of the water. The microbiological analysis of water studied sites (P1, P2, P3 and P4) showed the presence of fecal coliform, with geometric means of 1.66 x102, 1.31 x102, 2.29 x103 and 3.13 x102 CFU / 100 mL water, respectively. For enterococci, the averages were 6.30 x101 CFU / 100 mL in P1, 5.56 x101 CFU / 100 mL at P2, 1.89 x103 CFU / 100 mL at P3 and 1.62 x103 CFU / 100 mL at P4. Enteric viruses were detected in the four monitoring sites by both molecular techniques used, with maximum values of 2,00x103, 5,24x104 and 1,50x104 GC / 100 mL for AdV, NoV and RV, respectively, quantified by qPCR. The frequencies of virus detection in samples of estuarine water ranged from 21 - 27% for AdV, 42 - 53% for RV and 10 - 42% for NoV GII. In this study we found that the estuary of the Ilha das Caieiras presents contaminated, possibly due to sewage releases fresh and / or leachate from rural areas. The anthropogenic mangrove on this is clear and worrisome because environmental quality is reflected in this environment, the health of the population and the economy of the region

The quality of marine bathing water is currently assessed by monitoring the levels of faecal indicator bacteria. Among other drawbacks, results are retrospective using the traditional culture based methods. A rapid method is thus needed as an early warning to bathers for bacterial contamination in marine bathing waters. Total lipopolysaccharide (LPS) was chosen here as a potential general biomarker for bacterial contamination. Levels of total LPS, measured using a Kinetic QCL™ Limulus Amebocyte Lysate (LAL) assay, highly correlated with enumerated Escherichia coli and Bacteroides species. Levels of LPS in excess of 50 EU mL-1 were found to equate with water that was unsuitable for bathing under the current European Union regulations. Results showed that monitoring the levels of total LPS has a potential applicability as a rapid method for screening the quality of marine bathing water. More importantly, the LAL assay overcome the retrospective results when using culture based assessment since the LAL assay takes less than 30 minutes. Although false positive events were not detected, the occurrence of a false positive has been hypothesised, hence a more specific faecal biomarker was also investigated. LPS of five Bacteroides species (B. fragilis, B. caccae, B. ovatus, B. xylanisolvens and B. finegoldii) isolated from marine bathing waters samples were successfully profiled and showed high similarity between isolates in LPS gel electrophoresis banding pattern. Similar results were shown when investigating the endotoxic activity of Bacteroides species with the Kinetic QCL™ LAL assay. The potential biological relevance of Bacteroides LPS was also investigated in cell culture models indicating that Bacteroides showed similar induction of proinflammatory cytokines (TNF-α, IL-6 and IL-1α) and generally the biological activity was approximately 100 fold less than E. coli LPS. In addition, an ELISA assay was designed for the detection of Bacteroides LPS. Results showed that the Bacteroides LPS has a high potential to be used as a faecal biomarker, however, further work is required to develop a fully functional assay. The potential biological relevance of LPS present in contaminated bathing waters was also investigated in cell culture models. Results showed that there is a significant difference in the production of proinflammatory cytokines in comparison to “clean” bathing waters. Thus, results suggest that the European Directive regulations should be extended to cover the levels of total LPS in bathing waters to assure safety to the users of marine recreational water.

In this diploma thesis, I deal with the influence of the sampling site on water quality in natural bathing lakes (Hostivar reservoir, Seberak pond and Vyzlovka pond) in this. I have established that hygienically significant kinds of phytoplankton may differ in terms of water quality especially where there are cyanobacteria constituting water bloom. Surface water bloom may be moved by wind to the lee side of the water body (Microcystis sp.). Aphanizomenon flos-aquae water bloom floating in water column can be affected by wind and by water flow (which may be partly affected by wind as well). With the prevailing fibrous Planktothrix agardhii which does not connstitute water bloom the concentration of chlorophyll-a and cyanobacterial cell abundance in individual sampling sites are similar although the concentration of chlorophyll-a a is about 200 g.l-1 . Microcystis sp. and Scenedesmus sp. survive under eutrophic conditions in competition. From the microbiological perspective, water quality can differ within a single sampling site in places at a distance of less than 100 m. Microbial contamination can be caused by bathers, water birds, farm animals and probably by the removing of microorganisms from sand, mud and sediments on the beach or shore. Rain episodes probably increase the abundance of E.coli...

Introdução: Enterococcus resistente à vancomicina (VRE do inglês Vancomycin Resistant Enterococcus) é uma importante causa de infecção relacionada a assistência à saúde com alta morbidade e mortalidade principalmente nos pacientes imunocomprometidos sendo a segunda causa mais comum de infecções hospitalares nessa população de pacientes em alguns centros. O uso prolongado da terapia antimicrobiana com vancomicina e outras drogas, são fatores de risco associados com a disseminação desse patógeno. Além disso, espécies de enterococos podem apresentar fatores de virulência tais como: substância de agregação (asa1), gelatinase (gelE), citolisina (cylA), proteína de superfície enterococo (esp) e hidrolase glicosil (hylefm). Entretanto, o papel da virulência na colonização e infecção por VRE é controverso. Objetivos: Avaliar o perfil clonal, virulência e resistência de 86 isolados de VRE (80 E. faecium e 06 E. faecalis) de infecção e colonização de 76 pacientes com doenças hematológicas e/ou submetidos a transplante de Medula Óssea (TMO) internados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) no período de 10 anos (2005-2014). Material e Métodos: Foram realizadas concentração inibitória mínima para: vancomicina, teicoplanina, linezolida, gentamicina e estreptomicina em alta concentração (HLAR); Avaliação da clonalidade por eletroforese em campo pulsado (PFGE); Detecção dos mecanismos de resistência (vanA e vanB) e de virulência (esp, asa1, gelE, cylA e hylefm) pela técnica de PCR e sequenciamento total do genoma de dezoito isolados selecionados de acordo com a clonalidade. Foi criado um banco de dados no programa Epiinfo (CDC) com variáveis clinicas e demográficas dos pacientes. A proporção de isolados de colonização portadores de genes de virulência foi comparada com os isolados de infecção assim como a proporção de isolados de infecção portadores de genes de virulência que evoluíram para óbito. O valor de p < 0,005 foi considerado significativo. Resultados: Trinta e quatro pacientes eram colonizados e 42 infectados por VRE (28 eram infecção de corrente sanguínea), 48 pacientes eram transplantados dos quais 32 eram alogênicos. A mortalidade em 14 dias foi de 21.0% e durante a hospitalização de 53.9%. Todos os isolados foram resistentes à vancomicina e 87,3% à teicoplanina. Resistência a gentamicina e estreptomicina em alta concentração (HLAR) foi observada em 08 e 59 isolados respectivamente. Um isolado apresentou resistência a linezolida identificado pela primeira vez na unidade. O gene vanA foi detectado em todos os isolados. Quanto aos genes de virulência, 96,5% de isolados foram positivos para o gene esp e 69,8% para os genes gelE e asa1. Isolados de infecção de E. faecium carrearam mais genes de virulência: esp (100%), gelE (80,0%) e asa1 (75,5%) e o gene gelE foi significativamente mais frequente entre isolados de infecção que de colonização (p=0,008). Infecções causadas por isolados de E. faecium positivos para o gene asa1 foram significantemente associadas com maior mortalidade (p < 0,05). Quinze diferentes Pulsed field type (PFT) foram observados entre os isolados de E. faecium e 06 entre os isolados de E. faecalis. Dezessete E. faecium foram sequenciados e diferentes sequencias tipo (ST) foram observadas (ST412, ST478, ST78 e ST896) já descritas em outros estudos no Brasil. O isolado resistente a linezolida apresentou mutação no domínio V do gene 23S rRNA com um perfil alélico diferente, caracterizando um novo ST (ST987) descrito pela primeira vez no Brasil. Todos os ST observados nos isolados de E. faecium pertencem ao complexo clonal 17, dos quais dois STs (ST963, ST792) foram descritos pela primeira vez no país. O isolado de E. faecalis sequenciado pertencia ao ST9 e ao complexo clonal 9 já descrito por outros autores. Conclusão: Nosso estudo observou que E. faecium foi predominante em nosso hospital e os ST circulantes pertenciam ao CC17. Os isolados de infecção foram mais virulentos que os isolados de colonização e o gene gelE foi significativamente mais frequente nos isolados de infecção. Infecções causadas por isolados de E. faecium positivos para o gene asa1 foram associadas com a alta mortalidade. Este achado pode ser útil para controlar a disseminação de E. faecium no ambiente hospitalar e em pacientes hematológicos / Introduction: Vancomycin Resistant Enterococcus (VRE) is a important cause of health care-associated infection with high morbidity and mortality, mainly in immunocompromised patients, being the second most common cause of hospital infections in this population of patients in some centers. Prolonged use of antimicrobial therapy with vancomycin and other drugs are risk factors associated with the spread of this pathogen. In addition, enterococcal species may present virulence factors such as: aggregation substance (asa1), gelatinase (gelE), cytolysin (cylA), enterococcal surface protein (esp) and glycosyl hydrolase (hylefm). However, the role of virulence on VRE colonization and infection is controversial. Objectives: To evaluate the clonal profile, virulence and resistance of 86 isolates of VRE (80 E. faecium and 06 E. faecalis) from infection and colonization of 76 patients with hematological diseases and / or submitted to bone marrow transplantation (BMT) at Hospital das Clinics of the Medical School of the University of São Paulo (HC-FMUSP) in the period of 10 years (2005-2014). Material and Methods: Minimum inhibitory concentration to vancomycin, teicoplanin, linezolid, gentamicin and streptomycin in high concentration (HLAR) was performed; Clonality of isolates by pulsed field electrophoresis (PFGE) was evaluated; Detection of resistance (vanA and vanB) and virulence (esp, asa1, gelE, cylA and hylefm) genes by PCR technique and whole genome sequencing of eighteen isolates selected based on clonality. Results: All isolates were resistant to vancomycin and 87.3% to teicoplanin. Resistance to gentamicin and streptomycin in high concentration (HLAR) was observed in 08 and 59 isolates respectively. One isolate presented resistance to linezolid observed for the first time in the unit. The vanA gene was detected in all isolates. Regarding virulence genes, 96.5% of isolates were positive for the esp gene and 69.8% for the gelE and asa1 genes. Isolates of E. faecium infection carried more virulence genes: esp (100%), gelE (80.0%) and asa1 (75.5%) and gelE gene was significantly more frequent among infection isolates than colonization (p = 0.008). Infections caused by E. faecium isolates carrying the asa1 gene were significantly associated with higher mortality (p < 0.05). Fifteen different Pulsed field type (PFT) were observed among the isolates of E. faecium and 06 among E. faecalis isolates. Seventeen E. faecium were sequenced and the following sequences type (ST) were observed (ST412, ST478, ST78 and ST896) all of them already described in Brazil. The linezolid-resistant isolate showed the 23S gene Vdomain mutation with a different allelic profile, characterizing a new ST (ST987) described for the first time in our study. All STs observed in E. faecium isolates belong to clonal complex 17. Two other STs (ST963, ST792) were identified for the first time in the country. The E. faecalis isolate belong to ST9 and clonal complex 9 already described by other authors in Brazil. Conclusion: Our study observed that E. faecium was predominant in our hospital and the circulating STs belonged to CC17 a virulent lineage. E. faecium infection isolates were more virulent than colonization isolates and harbored significantly more gelE gene. It appears that infections caused by E. faecium isolates carrying asa1 gene evolved more frequently to death. This finding may be useful to control the spread of E. faecium in the hospital environment and in haematological patients

Introdução: Enterococcus resistente à vancomicina (VRE do inglês Vancomycin Resistant Enterococcus) é uma importante causa de infecção relacionada a assistência à saúde com alta morbidade e mortalidade principalmente nos pacientes imunocomprometidos sendo a segunda causa mais comum de infecções hospitalares nessa população de pacientes em alguns centros. O uso prolongado da terapia antimicrobiana com vancomicina e outras drogas, são fatores de risco associados com a disseminação desse patógeno. Além disso, espécies de enterococos podem apresentar fatores de virulência tais como: substância de agregação (asa1), gelatinase (gelE), citolisina (cylA), proteína de superfície enterococo (esp) e hidrolase glicosil (hylefm). Entretanto, o papel da virulência na colonização e infecção por VRE é controverso. Objetivos: Avaliar o perfil clonal, virulência e resistência de 86 isolados de VRE (80 E. faecium e 06 E. faecalis) de infecção e colonização de 76 pacientes com doenças hematológicas e/ou submetidos a transplante de Medula Óssea (TMO) internados no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) no período de 10 anos (2005-2014). Material e Métodos: Foram realizadas concentração inibitória mínima para: vancomicina, teicoplanina, linezolida, gentamicina e estreptomicina em alta concentração (HLAR); Avaliação da clonalidade por eletroforese em campo pulsado (PFGE); Detecção dos mecanismos de resistência (vanA e vanB) e de virulência (esp, asa1, gelE, cylA e hylefm) pela técnica de PCR e sequenciamento total do genoma de dezoito isolados selecionados de acordo com a clonalidade. Foi criado um banco de dados no programa Epiinfo (CDC) com variáveis clinicas e demográficas dos pacientes. A proporção de isolados de colonização portadores de genes de virulência foi comparada com os isolados de infecção assim como a proporção de isolados de infecção portadores de genes de virulência que evoluíram para óbito. O valor de p < 0,005 foi considerado significativo. Resultados: Trinta e quatro pacientes eram colonizados e 42 infectados por VRE (28 eram infecção de corrente sanguínea), 48 pacientes eram transplantados dos quais 32 eram alogênicos. A mortalidade em 14 dias foi de 21.0% e durante a hospitalização de 53.9%. Todos os isolados foram resistentes à vancomicina e 87,3% à teicoplanina. Resistência a gentamicina e estreptomicina em alta concentração (HLAR) foi observada em 08 e 59 isolados respectivamente. Um isolado apresentou resistência a linezolida identificado pela primeira vez na unidade. O gene vanA foi detectado em todos os isolados. Quanto aos genes de virulência, 96,5% de isolados foram positivos para o gene esp e 69,8% para os genes gelE e asa1. Isolados de infecção de E. faecium carrearam mais genes de virulência: esp (100%), gelE (80,0%) e asa1 (75,5%) e o gene gelE foi significativamente mais frequente entre isolados de infecção que de colonização (p=0,008). Infecções causadas por isolados de E. faecium positivos para o gene asa1 foram significantemente associadas com maior mortalidade (p < 0,05). Quinze diferentes Pulsed field type (PFT) foram observados entre os isolados de E. faecium e 06 entre os isolados de E. faecalis. Dezessete E. faecium foram sequenciados e diferentes sequencias tipo (ST) foram observadas (ST412, ST478, ST78 e ST896) já descritas em outros estudos no Brasil. O isolado resistente a linezolida apresentou mutação no domínio V do gene 23S rRNA com um perfil alélico diferente, caracterizando um novo ST (ST987) descrito pela primeira vez no Brasil. Todos os ST observados nos isolados de E. faecium pertencem ao complexo clonal 17, dos quais dois STs (ST963, ST792) foram descritos pela primeira vez no país. O isolado de E. faecalis sequenciado pertencia ao ST9 e ao complexo clonal 9 já descrito por outros autores. Conclusão: Nosso estudo observou que E. faecium foi predominante em nosso hospital e os ST circulantes pertenciam ao CC17. Os isolados de infecção foram mais virulentos que os isolados de colonização e o gene gelE foi significativamente mais frequente nos isolados de infecção. Infecções causadas por isolados de E. faecium positivos para o gene asa1 foram associadas com a alta mortalidade. Este achado pode ser útil para controlar a disseminação de E. faecium no ambiente hospitalar e em pacientes hematológicos / Introduction: Vancomycin Resistant Enterococcus (VRE) is a important cause of health care-associated infection with high morbidity and mortality, mainly in immunocompromised patients, being the second most common cause of hospital infections in this population of patients in some centers. Prolonged use of antimicrobial therapy with vancomycin and other drugs are risk factors associated with the spread of this pathogen. In addition, enterococcal species may present virulence factors such as: aggregation substance (asa1), gelatinase (gelE), cytolysin (cylA), enterococcal surface protein (esp) and glycosyl hydrolase (hylefm). However, the role of virulence on VRE colonization and infection is controversial. Objectives: To evaluate the clonal profile, virulence and resistance of 86 isolates of VRE (80 E. faecium and 06 E. faecalis) from infection and colonization of 76 patients with hematological diseases and / or submitted to bone marrow transplantation (BMT) at Hospital das Clinics of the Medical School of the University of São Paulo (HC-FMUSP) in the period of 10 years (2005-2014). Material and Methods: Minimum inhibitory concentration to vancomycin, teicoplanin, linezolid, gentamicin and streptomycin in high concentration (HLAR) was performed; Clonality of isolates by pulsed field electrophoresis (PFGE) was evaluated; Detection of resistance (vanA and vanB) and virulence (esp, asa1, gelE, cylA and hylefm) genes by PCR technique and whole genome sequencing of eighteen isolates selected based on clonality. Results: All isolates were resistant to vancomycin and 87.3% to teicoplanin. Resistance to gentamicin and streptomycin in high concentration (HLAR) was observed in 08 and 59 isolates respectively. One isolate presented resistance to linezolid observed for the first time in the unit. The vanA gene was detected in all isolates. Regarding virulence genes, 96.5% of isolates were positive for the esp gene and 69.8% for the gelE and asa1 genes. Isolates of E. faecium infection carried more virulence genes: esp (100%), gelE (80.0%) and asa1 (75.5%) and gelE gene was significantly more frequent among infection isolates than colonization (p = 0.008). Infections caused by E. faecium isolates carrying the asa1 gene were significantly associated with higher mortality (p < 0.05). Fifteen different Pulsed field type (PFT) were observed among the isolates of E. faecium and 06 among E. faecalis isolates. Seventeen E. faecium were sequenced and the following sequences type (ST) were observed (ST412, ST478, ST78 and ST896) all of them already described in Brazil. The linezolid-resistant isolate showed the 23S gene Vdomain mutation with a different allelic profile, characterizing a new ST (ST987) described for the first time in our study. All STs observed in E. faecium isolates belong to clonal complex 17. Two other STs (ST963, ST792) were identified for the first time in the country. The E. faecalis isolate belong to ST9 and clonal complex 9 already described by other authors in Brazil. Conclusion: Our study observed that E. faecium was predominant in our hospital and the circulating STs belonged to CC17 a virulent lineage. E. faecium infection isolates were more virulent than colonization isolates and harbored significantly more gelE gene. It appears that infections caused by E. faecium isolates carrying asa1 gene evolved more frequently to death. This finding may be useful to control the spread of E. faecium in the hospital environment and in haematological patients