Rapid detection of Van genes in rectal swabs by real time PCR in Southern Brazil

INTRODUCTION: Laboratory-based surveillance is an important component in the control of vancomycin resistant enterococci (VRE). METHODS: The study aimed to evaluate real-time polymerase chain reaction (RT-PCR) (genes vanA-vanB) for VRE detection on 115 swabs from patients included in a surveillance program. RESULTS: Sensitivity of RT-PCR was similar to primary culture (75% and 79.5%, respectively) when compared to broth enriched culture, whereas specificity was 83.1%. CONCLUSIONS: RT-PCR provides same day results, however it showed low sensitivity for VRE detection.

Enterococcus gallinarum carrying the vanA gene cluster: first report in Brazil

In 2000, Enterococcus faecalis resistant to vancomycin was first reported at a tertiary hospital in Porto Alegre, southern Brazil. The resistance spread to other hospitals and surveillance programs were established by hospital infection committees to prevent the spread of vancomycin-resistant enterococci. In February 2002, an isolate initially identified at the genus level as Enterococcus was obtained by surveillance culture (rectal swab) from a patient admitted to a hospital for treatment of septic arthritis in the shoulder. The isolate proved to be resistant to vancomycin by the disc diffusion method and confirmed by an E-test resulting in a minimal inhibitory concentration of > or = 256 µg/ml. This isolate was sent to a reference laboratory (Laboratório Especial de Bacteriologia e Epidemiologia Molecular, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP) for further study and proved to be an E. gallinarum by the polymerase chain reaction (PCR) using specific primers for the species. Due to the phenotype of unusually high vancomycin resistance, the isolate presumably had the resistance genes (vanA and vanB) and this was confirmed by PCR, which indicated the presence of the vanA gene. A 10.8-kb Tn1546-related transposon was also identified by long-PCR. Interspecies transfer of the vancomycin-resistance gene from the donor E. gallinarum was performed in a successful conjugation experiment in vitro, using E. faecium GE-1 and E. faecalis JH22 as receptors. This is the first report of the detection of a vanA determinant naturally acquired by E. gallinarum in Brazil, indicating the importance of characterizing VRE by both phenotype and genotype methods.

Absence of intestinal colonization by vancomycin-resistant enterococci in nonhuman primates

The animal reservoirs of vancomycin-resistant enterococci (VRE) have important role in the epidemiology of the bacteria and resistant genes. The present work searched fecal samples taken off nonhuman primates for the presence of VRE. Resistance profiles, virulence traits, and genetic variability among enterococci isolates were also analyzed. The samples included Capuchin monkeys (Cebus apella, n=28) and Common marmoset (Callithrix penicillata, n=37) housed in the Primate Center of the University of Brasília, Brazil. Most individuals were captive monkeys from the Central-West and South-East regions of Brazil (n=48). We collected rectal swabs and carried out selective isolation followed by multiplex Polymerase Chain Reaction (PCR) to identify species and resistance genes. No vanA or vanB-containing enterococci were found. The carriage rates ranged from 1.5% for the VanC-type E. casseliflavus and E. gallinarum until 12.3% (n=8) for Enterococcus faecalis. All E. faecalis isolates showed susceptibility to vancomycin, teicoplanin, ampicillin, gentamicin, and streptomycin. The virulence genes ace and esp were prevalent (100.0%, 87.5%). Multilocus variable number of tandem repeats (MLVA) revealed diversity in the number of repeats among E. faecalis isolates and targets, which was higher for espC, efa5, and efa6. We identified six different MLVA genotypes that were divergent from those described in human beings. Also, they were clustered into two genogroups that showed host-specificity for the species Cebus apella or Callithrix penicillata. In conclusion, no vanA- or vanB-containing enterococci were found colonizing those primate individuals. This finding suggested that the primate individuals investigated in our study are not directly involved in the epidemiological chain of high-level vancomycin-resistant genes vanA or vanB in Brazil. Our study also showed that E. faecalis isolated from nonhuman primates carry virulence traits and have ability to spread their lineages among different individuals.

Bacteriocinogenic and virulence potential of Enterococcus isolates obtained from raw milk and cheese

Aims To provide molecular and phenotypical characterization of Enterococcus isolates obtained from raw milk and cheese, regarding their bacteriocinogenic and virulence activity. Methods and Results Forty-three bacteriocinogenic enterococci isolates were identified by 16s rDNA, fingerprinted by RAPD-PCR analysis and tested by PCR for the presence of genes for lantibiotics (lanM, lanB and lanC) and enterocins (entA, entB, entP, entL50AB and entAS48) and by phenotypical methods for bacteriocin production and inhibitory spectrum. Also, the virulence of the isolates was evaluated by PCR for genes gelE, hyl, asa1, esp, cylA, efaA, ace, vanA, vanB, hdc1, hdc2, tdc and odc and by phenotypical tests for gelatinase, lipase, DNAse and a- and beta-haemolysis. Most isolates (93.0%) harboured at least one lantibiotic or enterocin gene and were positive for several tested virulence genes, mainly asa1 (100%), gelE (93.0%) and efaA (83.7%). 53.5% of the isolates presented beta-haemolysis. Conclusions Enterococcus spp. isolates presented an interesting potential application for food preservation because of bacteriocin production; however, virulence-related genes were identified in all RAPD profiles. Significance and Impact of the Study The study demonstrated the contradictory characteristics of the tested Enterococcus isolates: they presented a good potential for application in food biopreservation but contained several virulence factors.

Identificação de Enterococcus spp. e avaliação da ocorrência de genes de resistência á vancomicina em amostras de pacientes de hospitais de manaus, AM

Pós-graduação em Fisiopatologia em Clínica Médica - FMB

Diversity of Vancomycin Genes in Sub-tropical Soils

With the increased antibiotic exposure from anthropogenic sources, soil microbes are an ever-increasing ecological pool of resistant bacteria. This is the case with bacterial resistance to vancomycin through transfer of van-resistance genes by transposons. Studies show that bacterial species other than enteroccoci harbor genetic-like elements such as the Tn1546 transposon containing vancomycin-resistant genes. Overuse and misuse of antibiotics in hospital settings and agricultural practices have led to an increase in transferability of vancomycin-resistant genes among microbes. The objective of this project is to analyze the diversity of these genes found in the soil microbes from Miami-Dade County. Bacterial isolates were Gram-stained and the Kirby-Bauer antibiotic disk diffusion test was performed to determine the degree of resistance. Results showed that all bacterial isolates were resistant to penicillin at the 10 µg concentration and most were susceptible to varying vancomycin concentrations (10 µg, 20 µg, and 30 µg). A 1465 bp fragment was amplified from the 16S rDNA gene using 27F and 1492R universal primers from the multi-antibiotic resistant bacteria and sequenced to identify the isolates. Three Gram-negative bacteria genera were identified with the closest phylogenetic match to: Pseudomonas sp., Stenotrophomonas sp., Xanthomonas sp., as well as two Gram-positive bacteria genera: Bacillus sp. and Brevibacillus sp. The isolates’ vanA and vanB genes were amplified using the respective primers. Ongoing work is underway to sequence and compare these known van resistant genes, with the goal of revealing intrinsic vancomycin resistance present in soil bacteria.

The relationship of a clonal outbreak of Enterococcus faecium vanA to methicillin-resistant Staphylococcus aureus incidence in an Australian hospital

Australian isolates of vancomycin-resistant enterococci (VRE) have been widely scattered geographically, predominantly polyclonal and of the VanB phenotype. Forty-nine VRE were isolated from 47 patients in our hospital from October 1996 to December 1999. Forty-four of these VRE were Enterococcus faecium with a vanA glycopeptide resistance genotype. Four isolates were pathogenic. Thirty-five VRE were from an outbreak in the Renal and Infectious Diseases Units over a four-month period. Pulsed-field gel electrophoresis (PFGE) demonstrated that 41 of the 49 VRE were indistinguishable or closely related. Enhanced environmental cleaning, strict contact isolation of colonized patients and reducing inpatient admissions terminated the epidemic. Cohorting of methicillin-resistant Staphylococcus aureus (MRSA)-positive patients was restricted because VPE patients occupied the isolation facilities. This resulted in a statistically significant increase in MRSA infections across the hospital. VRE epidemics have the ability to influence the epidemiology of other nosocomial pathogens when infection control resources are exhausted. (C) 2001 The Hospital Infection Society.

Risk factors for vancomycin-resistant enterococci colonisation in critically ill patients

Vancomycin-resistant enterococci (VRE) are important hospital pathogens and have become increasingly common in patients admitted to the intensive care unit (ICU). To determine the incidence and the risk factors associated with VRE colonisation among ICU patients, active surveillance cultures for VRE faecal carriages were carried out in patients admitted to the ICU of the University Hospital of Uberlândia, Minas Gerais, Brazil. Risk factors were assessed using a case-control study. Seventy-seven patients (23.1%) were found to be colonised with vanC VRE and only one patient (0.3%) was colonised with vanA VRE. Independent risk factors for VRE colonisation included nephropathy [odds ratio (OR) = 13.6, p < 0.001], prior antibiotic use (OR = 5.5, p < 0.03) and carbapenem use (OR = 17.3, p < 0.001). Our results showed a higher frequency (23.1%) of Enterococcus gallinarum and Enterococcus casseliflavus, species that are intrinsically resistant to low levels of vancomycin (vanC), without an associated infection, associated with prior antibiotic use, carbapenem use and nephropathy as comorbidity. This study is the first to demonstrate the risk factors associated with vanC VRE colonisation in ICU hospitalised patients. Although vanA and vanB enterococci are of great importance, the epidemiology of vanC VRE needs to be better understood. Even though the clinical relevance of vanC VRE is uncertain, these species are opportunistic pathogens and vanC VRE-colonised patients are a potential epidemiologic reservoir of resistance genes.

Emergence and characterisation of vanB vancomycin-resistant Enterococcus faecalis in Rio de Janeiro, Brazil

Here we describe the detection and characterisation of three isolates of vancomycin-resistant VanB-type Enterococcus faecalis. Sequence analysis suggested that these isolates harboured the vanB1 gene. The isolates were susceptible to the majority of antimicrobial agents tested, with the exception of chloramphenicol, erythromycin and vancomycin, and showed distinct profiles of high-level resistance to aminoglycosides. Analysis of the clonal relatedness of the vanB E. faecalis isolates showed similar pulsed-field gel electrophoresis profiles. To our knowledge, this is the first report of the occurrence of enterococcal strains carrying vanB genes in Brazil.

Resistência antimicrobiana em Enterococcus faecalis e Enterococcus faecium isolados de carcaças de frango

O objetivo deste trabalho foi realizar o isolamento e analisar o perfil de resistência antimicrobiana de Enterococcus de carcaças de frango resfriadas e congeladas comercializadas no Distrito Federal, detectando genes de resistência antimicrobiana e identificando as espécies Enterococcus faecalis e Enterococcus faecium por reação polimerase em cadeia. Foram analisadas 100 carcaças de frangos, das quais foram isoladas 50 cepas de Enterococcus spp., sendo 42% de E. faecalis e 2% de E. faecium. O teste de susceptibilidade antimicrobiana demonstrou que todas as cepas isoladas apresentaram resistência a pelo menos um antimicrobiano, dos quais 90,47% das cepas de E. faecalis, 100% das cepas de E. Faecium e 82,14% dos Enterococcus spp. apresentaram resistência à Tetraciclina; 80,95% das cepas de E. faecalis e 35,71% das cepas de Enterococcus spp. foram resistentes à Eritromicina; 39,28% dos Enterococcus spp. e 23,80% dos E. faecalis à Ciprofloxacina e 28,57% dos E. faecalis apresentaram resistência ao Cloranfenicol. Foram detectados os genes de resistência antimicrobiana erm(B), vanC-1, aph(3')-llla, ant(6)-la, vanB, vanA, aac(6')-le-aph(2'')-la, erm(A) e tet(M) - este último mais frequente. Estes resultados sugerem sérios problemas para a Saúde Pública, uma vez que esses microrganismos podem possuir a capacidade de transmitir genes de resistência antimicrobiana para outros microrganismos presentes na microbiota intestinal de humanos e animais, podendo inviabilizar o uso destas drogas para tratamentos clínicos.

Vancomycin-dependent Enterococcus faecium vanA: characterization of the first case isolated in a university hospital in Brazil

In this study, we report the characterization of a strain of Enterococcus faecium vanA, which grows only in the presence of vancomycin (VDEfm-UEL). The bacterium was isolated from the feces of a female patient who had undergone surgical treatment of Reinke’s edema and was receiving intravenous vancomycin therapy for infection with methicillin/oxacillin-resistant Staphylococcus aureus, a postoperative complication. Antimicrobial dependence was further confirmed by the vancomycin E-test. VDEfm-UEL was also shown to be resistant to ampicillin, ciprofloxacin, chloramphenicol, erythromycin, levofloxacin, penicillin, rifampicin, and teicoplanin. The putative virulence genes efaA, gelE and esp were detected by PCR. The ddl gene from VDEfm-UEL was cloned and sequenced. Vancomycin dependence seems to be associated with the insertion of a nucleotide in that sequence, which results in a frame-shift mutation, introducing a premature stop codon. This is the first report of vancomycin-dependent E. faecium isolation in a university hospital in Brazil.

Cotransfer of antibiotic resistance genes and a hylEfm-containing virulence plasmid in Enterococcus faecium.

The hyl(Efm) gene (encoding a putative hyaluronidase) has been found almost exclusively in Enterococcus faecium clinical isolates, and recently, it was shown to be on a plasmid which increased the ability of E. faecium strains to colonize the gastrointestinal tract. In this work, the results of mating experiments between hyl(Efm)-containing strains of E. faecium belonging to clonal cluster 17 and isolated in the United States and Colombia indicated that the hyl(Efm) gene of these strains is also carried on large plasmids (>145 kb) which we showed transfer readily from clinical strains to E. faecium hosts. Cotransfer of resistance to vancomycin and high-level resistance (HLR) to aminoglycosides (gentamicin and streptomycin) and erythromycin was also observed. The vanA gene cluster and gentamicin resistance determinants were genetically linked to hyl(Efm), whereas erm(B) and ant(6)-I, conferring macrolide-lincosamide-streptogramin B resistance and HLR to streptomycin, respectively, were not. A hyl(Efm)-positive transconjugant resulting from a mating between a well-characterized endocarditis strain [TX0016 (DO)] and a derivative of a fecal strain of E. faecium from a healthy human volunteer (TX1330RF) exhibited increased virulence in a mouse peritonitis model. These results indicate that E. faecium strains use a strategy which involves the recruitment into the same genetic unit of antibiotic resistance genes and determinants that increase the ability to produce disease. Our findings indicate that the acquisition of the hyl(Efm) plasmids may explain, at least in part, the recent successful emergence of some E. faecium strains as nosocomial pathogens.

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.

VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin

Three Enterococcus faecium strains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P(175)L, which suppressed the activity of the host Ddl D-Ala:D-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRS(B) operon that lowered the free energy of pairing from -13.08 to -6.65 kcal/mol, leading to low-level constitutive expression of the resistance genes from the P(RB) promoter, as indicated by analysis of peptidoglycan precursors and of VanX(B) D,D-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription, initiated from the P(YB) resistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the P(RB) regulatory promoter in strain BM4661, where it was superinducible. Strain BM4661 provides the first example of reversion to vancomycin resistance of a VanB-type dependent strain not due to a compensatory mutation in the ddl or vanS(B) gene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough of the resistance genes from the P(RB) promoter in the absence of vancomycin.

Diet Carotenoid Lutein Modulates The Expression Of Genes Related To Oxygen Transporters And Decreases Dna Damage And Oxidative Stress In Mice.

Lutein (LT) is a carotenoid obtained by diet and despite its antioxidant activity had been biochemically reported, few studies are available concerning its influence on the expression of antioxidant genes. The expression of 84 genes implicated in antioxidant defense was quantified using quantitative reverse transcription polymerase chain reaction array. DNA damage was measured by comet assay and glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were quantified as biochemical parameters of oxidative stress in mouse kidney and liver. cDDP treatment reduced concentration of GSH and increased TBARS, parameters that were ameliorated in treatment associated with LT. cDDP altered the expression of 32 genes, increasing the expression of GPx2, APC, Nqo1 and CCs. LT changed the expression of 37 genes with an induction of 13 mainly oxygen transporters. In treatments associating cDDP and LT, 30 genes had their expression changed with a increase of the same genes of the cDDP treatment alone. These results suggest that LT might act scavenging reactive species and also inducing the expression of genes related to a better antioxidant response, highlighting the improvement of oxygen transport. This improved redox state of the cell through LT treatment could be related to the antigenotoxic and antioxidant effects observed.