Enterococcus and biocides: mechanisms of tolerance and selection for vancomycin resistance

Dissertation presented to obtain a Doctoral degree in Biology, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa.

Genetic diversity and antimicrobial resistance of enterococcal isolates from Southern region of Brazil

In the present study, a total of 455 enterococcal isolates, recovered from patients living in the city of Porto Alegre, State of Rio Grande do Sul, Brazil, during the period from July 1996 to June 1997, were identified to the species level by conventional biochemical and microbiological tests, and assayed for their susceptibilities to antimicrobial agents. The genetic diversity of antimicrobial resistant strains was evaluated by pulsed-field gel electrophoresis (PFGE) analysis of SmaI restricted chromosomal DNA. The most frequent species was Enterococcus faecalis (92.8%). Other species identified were: E. faecium (2.9%), E. gallinarum (1.5%), E. avium (1.1%), E. hirae (0.7%), E. casseliflavus (0.4%), E. durans (0.4%) and E. raffinosus (0.2%). The overall prevalence of isolates with high-level resistance (HLR) to aminoglycosides was 37.8%. HLR to gentamicin was found in 24.8%. No strains with acquired resistance to vancomycin were found. PFGE analysis showed the predominance of clonal group A, comprising strains isolated from different clinical specimens obtained from patients in three hospitals. These results suggest intra and inter-hospital dissemination of one predominant clonal group of E. faecalis isolates with HLR to gentamicin in the hospitals included in this study.

Enterococcus gallinarum meningitis in an immunocompetent host: a case report

We describe a rare case of a 53-year-old man with a long history of alcohol abuse, with Enterococcus gallinarum meningitis, an organism that rarely causes human infection and is primarily found in the gastrointestinal tract of poultry. The patient improved with high-dose ampicillin and gentamicin therapy. To our knowledge, this is the first Brazilian reported case of E. gallinarum meningitis and probably the first case described in an immunocompetent host.

Precious transition metals: the importance of Zn2+, Mn2+ and Cu2+ in the human pathogen Enterococcus faecalis

Dissertation presented to obtain the Ph.D degree in Biology

Enterococcus faecalis V583 prophages: Dynamic interactions and contribution to bacterial pathogenic traits

Dissertation presented to obtain the Ph.D degree in Biology.

FSR Quorum Sensing: Role in Enterococcus faecalis Biology & Host Infection

Dissertation presented to obtain the Ph.D degree in Biology.

PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

Resistência antimicrobiana associada em isolados clínicos de Enterococcus spp

INTRODUÇÃO: O aumento da prevalência de isolados de enterococos em hospitais, particularmente Enterococcus resistente à vancomicina (VRE), é importante por causa da limitada terapia antimicrobiana efetiva para o tratamento de infecções enterocócicas. MÉTODOS: O presente trabalho apresentou uma investigação retrospectiva de dados de suscetibilidade in vitro quantitativa para uma variedade de antimicrobianos frente aos isolados de Enterococcus spp. e avaliação da associação de resistência entre os agentes antimicrobianos apontados como escolha para o tratamento de infecções causadas por VRE, através do cálculo do risco relativo. RESULTADOS: Dos 156 isolados de enterococos, 40 (25,6%) foram resistentes a três ou mais antimicrobianos, incluindo 7,7% (n = 12/156) resistentes à vancomicina. A associação de resistência elevada foi mais pronunciada entre os isolados de VREs com antimicrobianos alternativos e primários para o tratamento de infecções causadas por estes patógenos, incluindo ampicilina (100%, RR = 7,2), estreptomicina (90,9%, RR = 4,9), rifampicina (91,7%, RR = 3,1) e linezolida (50%, RR = 11,5), apesar da alta taxa de suscetibilidade a esta droga (94,9%). CONCLUSÕES: A resistência associada significativa aos antimicrobianos de primeira escolha e alternativos, usados no tratamento de infecções graves por cepas com o fenótipo VRE e que requerem um regime terapêutico combinado, evidencia alternativas terapêuticas ainda mais limitadas na instituição analisada.

Influence of a subinhibitory concentration of vancomycin on the in vitro expression of virulence-related genes in the vancomycin-resistant Enterococcus faecalis

ABSTRACTINTRODUCTION:Exposure to subinhibitory concentrations (SICs) of antimicrobials may alter the bacterial transcriptome.METHODS: Here, we evaluated the expression of nine virulence-related genes in vancomycin-resistant enterococci (VRE) urinary tract infection isolates grown at SICs of vancomycin.RESULTS:A Subinhibitory concentrations of vancomycin interferes with gene modulation, but does not affect the phenotype of a VRE strain in vitro .CONCLUSIONS:Subinhibitory concentrations of vancomycin may regulate the expression of virulence factors in vivo or contribute to the selection of vancomycin-resistant strains.

Escherichia coli and Enterococcus faecalis are able to incorporate and enhance a pre-formed Gardnerella vaginalis biofilm

Gardnerella vaginalis is the most frequent microorganism found in bacterial vaginosis (BV), while Escherichia coli and Enterococcus faecalis are amongst the most frequent pathogens found in urinary tract infections (UTIs). This study aimed to evaluate possible interactions between UTIs pathogens and G. vaginalis using an in vitro dual-species biofilm model. Our results showed that dual-species biofilms reached significantly higher bacterial concentration than mono-species biofilms. Moreover, visualization of dual-populations species in the biofilms, using the epifluorescence microscopy, revealed that all of the urogenital pathogens co-existed with G. vaginalis. In conclusion, our work demonstrates that uropathogens can incorporate into mature BV biofilms.

Identification and molecular characterization of Van A-type vancomycin-resistant Enterococcus faecalis in Northeast of Brazil

The isolation of vancomycin resistant enterococci (VRE) in Brazil has rapidly increased, following the world wide tendency. We report in the present study the first isolation of vancomycin resistant Enterococcus faecalis (VRE) in the Northeast of Brazil. The four VRE isolates were characterized for antimicrobial susceptibility, genotypic typing by macro restriction of chromosomal DNA followed by pulsed-field gel electrophoresis and for characterization of the Tn1546-like element and plasmid contents. The isolates showed resistance to multiple antibiotics and a single genotype profile, suggesting the dissemination of a single clone among the patients. Tn1546 associated to genetic elements as plasmids shows the importance of infection control measures to avoid the spreading of glycopetide resistance by conjugative transfer of VanA elements.

Antimicrobial resistance profiles of enterococci isolated from poultry meat and pasteurized milk in Rio de Janeiro, Brazil

The enterococci are important nosocomial pathogens with a remarkable capacity of expressing resistance to several antimicrobial agents. Their ubiquitous nature and resistance to adverse environmental conditions take account for their ability to colonize different habitats and for their potential for easy spreading through the food chain. In the present study we evaluated the distribution of species and antimicrobial susceptibility among enterococcal isolates recovered from food obtained in retail stores in Rio de Janeiro, Brazil. The following species were identified among 167 isolates obtained from poultry meat and 127 from pasteurized milk: Enterococcus faecalis (62.6%), E. casseliflavus (17.3%), E. durans (6.5%), E. gallinarum (3.0%), E. gilvus (2.4%), E. faecium (2.0%), E. hirae (1.4%), and E. sulfureus (1.0%). The overall percentages of antimicrobial resistant isolates were: 31.2 % to tetracycline, 23.8% to erythromycin, 11.3% to streptomycin, 4.3% to chloramphenicol, 3.9% to gentamicin, 1.4% to norfloxacin, 1.1% to imipenem, 0.7% to ciprofloxacin, nitrofurantoin, and penicillin, and 0.4% to ampicillin. Intermediate resistance was detected in frequencies varying from 0.5% for linezolid to 58.2% for erythromycin. None of the isolates showed resistance to glycopeptides. High-level resistance to aminoglycosides was observed in 13.1% of the isolates. Multiresistance was observed in E. faecalis, E. casseliflavus, E. faecium, E. gallinarum, E. durans and E. gilvus.

RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene as a tool to identify Enterococcus strains

Restriction fragment length polymorphism (RFLP) analysis of a PCR-amplified fragment of the 16S rRNA gene was performed on reference strains belonging to 21 different enterococcal species and on 75 Enterococcus isolates recovered from poultry meat, pasteurised milk and fresh cheese. PCR amplification generated a 275 bp fragment, which was digested with three restriction endonucleases (DdeI, HaeIII, HinfI). The strains were divided into five groups (groups A-E) on the basis of their restriction patterns. Five biochemical tests (arabinose, arginine, manitol, methyl-β-D-glucopyranoside and raffinose) were then performed in addition to RFLP analysis to narrow the identification of enterococcal strains to the species level. PCR-RFLP, in conjunction with the selected biochemical tests, allowed the precise identification of the 21 species of Enterococcus included in the present study. This proposed method is relatively simple and rapid and can be useful as an adjunct tool for accurate identification of Enterococcus.

In vitro and in vivo effects of Enterococcus faecalis CECT7121 on Toxocara canis

The aim of the present paper was to evaluate the larvicidal effect of Enterococcus faecalis CECT7121 (Ef7121) on the Toxocara canis cycle both in vitro and in vivo. For the in vitro experiments, T. canis larvae were incubated with the supernatants of Ef7121 (EI) and mutant Ef7121 (EIm), in a pre-culture of Ef7121 (EII) and in a fresh culture with Ef7121 (EIII) and the Ef7121 mutant strain (EIIIm). The viability of the larvae was calculated after a 48 h incubation. A significant reduction of the viability of T. canis larvae was observed in EI, EII and EIII. A decrease of this inhibitory effect was observed in EIm and EIIIm (p = 0.008). In the in vivo experiments, mice were orally inoculated with three doses of Ef7121. To study the probiotic persistence in the intestine, the animals were sacrificed every four days and their intestines were dissected. The initial average bacterial levels were 9.7 x 10(4) for Ef7121 (colony forming units/g). At the end of the assay the levels were 1.46 x 10(4). No bacterial translocation was detected in mesenteric lymphatic nodules and spleen. Ef7121 interference with the biological cycle was evaluated in mice challenged with T. canis. The interference was significant when the mice were challenged with probiotic and T. canis simultaneously (p = 0.001), but it was not significant when the challenge was performed 15 days after administration of the bacterial inoculum (p = 0.06). In conclusion, Ef7121 possessed in vitro and in vivo larvicidal activity.

Granular layer in the periplasmic space of gram-positive bacteria and fine structures of Enterococcus gallinarum and Streptococcus gordonii septa revealed by cryo-electron microscopy of vitreous sections.

High-resolution structural information on optimally preserved bacterial cells can be obtained with cryo-electron microscopy of vitreous sections. With the help of this technique, the existence of a periplasmic space between the plasma membrane and the thick peptidoglycan layer of the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus was recently shown. This raises questions about the mode of polymerization of peptidoglycan. In the present study, we report the structure of the cell envelope of three gram-positive bacteria (B. subtilis, Streptococcus gordonii, and Enterococcus gallinarum). In the three cases, a previously undescribed granular layer adjacent to the plasma membrane is found in the periplasmic space. In order to better understand how nascent peptidoglycan is incorporated into the mature peptidoglycan, we investigated cellular regions known to represent the sites of cell wall production. Each of these sites possesses a specific structure. We propose a hypothetic model of peptidoglycan polymerization that accommodates these differences: peptidoglycan precursors could be exported from the cytoplasm to the periplasmic space, where they could diffuse until they would interact with the interface between the granular layer and the thick peptidoglycan layer. They could then polymerize with mature peptidoglycan. We report cytoplasmic structures at the E. gallinarum septum that could be interpreted as cytoskeletal elements driving cell division (FtsZ ring). Although immunoelectron microscopy and fluorescence microscopy studies have demonstrated the septal and cytoplasmic localization of FtsZ, direct visualization of in situ FtsZ filaments has not been obtained in any electron microscopy study of fixed and dehydrated bacteria.