Izabel Cristina Vanzato Palazzo

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.

Prevalence of community-occurring extended spectrum β-lactamase-producing Enterobacteriaceae in Brazil

The occurrence of extended-spectrum-β-lactamase (ESBL)-producing strains in the community was investigated in a private laboratory located in Juiz de Fora, Brazil. All enterobacterial isolates analysed were collected from urine of human patients between the years 2000 and 2002. ESBL production was confirmed by double disk screening, combination disk method, and Etest ESBL strip. The isoelectric point of each β-lactamase was determined in the crude extracts from each isolate. Detection of ESBL genes was performed by polymerase chain reaction and the genetic relatedness of the isolates determined by pulsed-field gel electrophoresis (PFGE). Of the 1,481 isolates, 22 (12 Klebsiella pneumoniae, 7 Escherichia coli, 1 Providencia stuartii, 1 Citrobacter freundii, and 1 Serratia marcescens) were identified as ESBL producers. The frequency of ESBL producers in the community was 1.48%. TEM-type enzymes were identified in 95.4% of the isolates, followed by the SHV type. Seven strains produced CTX-M–type enzymes. This study showed that strains producing multiple β-lactamases are also present in community-acquired bacterial isolates. Multiple strains exhibiting identical PFGE genotypes were found in individual patients indicating a common source of acquisition.

Antimicrobial resistance of Enterococcus sp. isolated from the intestinal tract of patients from a university hospital in Brazil

This study reports the results about antimicrobial resistance of Enterococcus spp. isolated from intestinal tract of patients from a university hospital in Brazil. The identification of strains at species level was performed by conventional biochemical tests, API 20 Strep (bioMérieux), and polymerase chain reaction assay. The species distribution was E. faecium (34%), followed by E. faecalis (33%), E. gallinarum (23.7%), E. casseliflavus (5.2%), E. avium (1%), and E. hirae (1%). Intrinsic resistance to vancomycin characterized by presence of vanC genes was found in E. gallinarum and E. casseliflavus. The high prevalence of VanC phenotype enterococci is very important because these species have been reported as causing a wide variety of infections. Vancomycin-resistant E. faecium or E. faecalis were not found and no one isolate of these species was a beta-lactamase producer. Thirteen clinical isolates of enterococci (13.4%) showed multiresistance patterns, which were defined by resistance to three classes of antibiotics plus resistance to at least one aminoglycoside (gentamicin and/or streptomycin). The resistance to several antimicrobials shown by enterococcal strains obtained in this study is of concern because of the decrease in the therapeutic options for treatment of infections caused by enterococci.

Clonal Transmission of ESBL-Producing Klebsiella spp. at a University Hospital in Brazil

The present study was designed to determine the prevalence and extended-spectrum β-lactamase (ESBL) types in clinical isolates of Klebsiella spp. at a university hospital located in the Brazilian southern region (Ribeirão Preto, São Paulo) as well as their antibiotic susceptibility and genetic profiles. This study included 147 non-repeat Klebsiella spp. isolates collected from January to June 2000, of which 23 K. pneumoniae and 8 K. oxytoca were selected as ESBL producers by using the Oxoid combination disk method and Etest ESBL strip. β-lactamases were characterized by IEF, PCR and sequencing assays using primers for ESBL genes. Antibiotic susceptibility was evaluated by MicroScan system. Dissemination of two major clones of ESBL-producing Klebsiella spp. occurred in the hospital. According to the results obtained in this study there was a clonal spread of CTX-M-producing K. oxytoca in five clinics and dissemination of ESBL-producing K. pneumoniae in the nursery and pediatrics wards.

Staphylococcus hominis subsp. novobiosepticus strains causing nosocomial bloodstream infection in Brazil

OBJECTIVES To report the isolation of six Staphylococcus hominis subsp. novobiosepticus (SHN) strains from hospitalized patients with bloodstream infections in two Brazilian hospitals and to characterize their susceptibility profile to several antimicrobials. METHODS Species identification was performed by biochemical methods and sodA gene sequencing. The MICs of antimicrobials were determined by broth and agar dilution methods and by Etest. Isolates were typed by PFGE and PCR amplification was used to detect the ccr gene complex and the mec class. Morphometric evaluation of cell wall was performed by transmission electron microscopy (TEM). RESULTS Susceptibility profiles indicated that the majority of isolates (five) were multidrug-resistant. Overlapping and multiplex PCR showed that five out of the six strains harboured SCCmec type III with class A mec and type 3 ccr. The initial vancomycin MIC value of 4 mg/L for these strains increased to 16-32 mg/L after growth for 10 days in BHI broth supplemented with this antimicrobial. TEM indicated that vancomycin resistance was associated with cell wall thickening and to another mechanism not fully elucidated. Only one SHN strain was oxacillin- and vancomycin-susceptible. The nosocomial infections in at least five of the patients from both hospitals were caused by a single clone of SHN. CONCLUSIONS It is very important to consider SHN strains as the cause of nosocomial infections. The clinical implications resulting from the pattern of multidrug resistance in these strains may be complicated by the emergence of vancomycin resistance.

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 glycopeptide resistance by conjugative transfer of VanA elements.

Emergence of VanB phenotype-vanA genotype in vancomycin-resistant enterococci in Brazilian hospital

In Brazil, vancomycin-resistant enterococci (VRE) have been reported as nosocomial pathogens since 1998. Recently, in a VRE surveillance in a hospital, we detected two Enterococcus faecalis isolates with vanA genotype and susceptible to teicoplanin. This is the first report of VanB phenotype-vanA genotype enterococci isolated from humans in Brazil.

Molecular characterization of enterococci harboring genotype and phenotype incongruence related to glycopeptide resistance isolated in Brazilian hospitals

Three Enterococcus faecalis and one Enterococcus faecium strains were characterized by plasmid profile, pulsed-field gel electrophoresis (PFGE) and determination of antimicrobial minimal inhibitory concentrations. VanA elements were characterized by Long PCR, overlapping PCR and DNA sequencing. Enterococcal strains showed resistance to vancomycin and harbored the vanA gene, and three these were teicoplanin susceptible while one showed intermediate resistance to teicoplanin. Two E. faecalis strains showed indistinguishable PFGE profile while the third was unrelated. E. faecalis strains showed a deletion in the right terminal region of the Tn1546-like element. The E. faecium strain showed an insertion element in the vanXY intergenic region. Mutations in VanA elements were not found. Rearrangements in the VanA element could be responsible for incongruities in genotype and phenotype in these strains.

Cefoxitin Does Not Induce Production of Penicillin Binding Protein 2a in Methicillin-Susceptible Staphylococcus aureus Strains

Beta-lactam resistance in methicillin-resistant staphylococci (MRS) is caused by the expression of penicillin-binding protein 2a (PBP2a), encoded by the mecA gene, which has low binding affinities to practically all beta-lactam antibiotics so far introduced into clinical use ([7][1]). Detection of

First report of vancomycin-resistant Enterococcus faecalis in Uberaba, Minas Gerais state

In this study we report the first isolation of VanA-type vancomycin-resistant Enterococcus faecalis strains from two different patients hospitalized in the same intensive care unit at the hospital of Universidade Federal do Triângulo Mineiro (UFTM), Uberaba, Minas Gerais, Brazil.