Lúcia Martins Teixeira

Phenotypic and genotypic characterization of atypical Lactococcus garvieae strains isolated from water buffalos with subclinical mastitis and confirmation of L. garvieae as a senior subjective synonym of Enterococcus seriolicida.

During a survey of bacterial agents that cause subclinical mastitis in water buffalos, we isolated several strains of gram-positive cocci that appeared to be enterococci except that they grew very slowly at 45 degrees C and grew slowly in broth containing 6.5% NaCl. On the basis of the results of conventional physiologic tests, these strains were identified as Enterococcus durans. However, none of the strains reacted with the AccuProbe Enterococcus genetic probe. The whole-cell protein profiles of these organisms were compared with the profiles of Enterococcus and Lactococcus reference strains. apart from minor quantitative differences, the mastitis isolates had indistinguishable protein profiles that were similar to the profiles of the Lactococcus garvieae and Enterococcus seriolicida type strains. The results of DNA relatedness studies performed by using the hydroxyapatite method at 55 and 70 degrees C indicated that all of the mastitis isolates were related to the type strain of L. garvieae at the species level, despite the fact that they exhibited several uncommon phenotypic characteristics (growth at 45 degrees C, growth in broth containing 6.5% NaCl, and failure to produce acid from mannitol and sucrose). The high levels of DNA relatedness between strains of L. garvieae is a senior synonym of E. seriolicida, L. garvieae should be retained as the species name and strain ATCC 43921 should remain the type strain of this species.

Occurrence of a Multidrug-Resistant Pseudomonas aeruginosa Clone in Different Hospitals in Rio de Janeiro, Brazil

ABSTRACT Multidrug-resistant Pseudomonas aeruginosa nosocomial infections are increasingly recognized worldwide. The existence of metallo-β-lactamase- and extended-spectrum β-lactamase-producing isolates exhibiting resistance to most β-lactam antimicrobial agents greatly complicates the clinical management of patients infected with such isolates. Since 1998, P. aeruginosa isolates resistant to all commercially available antimicrobial agents have been detected at a university-affiliated public hospital in Rio de Janeiro, Brazil. The present study was designed to characterize the antimicrobial resistance profiles and the genetic diversity of the P. aeruginosa strains isolated at this hospital and four private hospitals in Rio de Janeiro. Between April 1999 and March 2000, 200 consecutive isolates were obtained and analyzed for antimicrobial resistance. The genetic diversity of a selected number of them was evaluated by pulsed-field gel electrophoresis and PCR with the ERIC-2 primer. A predominant genotype, designated genotype A, was identified among isolates from four of the five hospitals evaluated. Eighty-four ceftazidime-resistant isolates were evaluated for metallo-β-lactamase production, which was detected in 20 (91%) of 22 genotype A isolates and 11 (18%) of 62 isolates belonging to other genotypes (P < 0.05). Two metallo-β-lactamase-producing genotype A isolates also produced an extended-spectrum β-lactamase. The occurrence of multidrug-resistant P. aeruginosa strains belonging to a unique genotype in different hospitals in Rio de Janeiro underscores the importance of the contribution of a single clone to the increase in the incidence of multidrug-resistant P. aeruginosa nosocomial infections.

Frequency and characteristics of diarrhoeagenic Escherichia coli strains isolated from children with and without diarrhoea in Rio de Janeiro, Brazil

The frequency of diarrhoeagenic Escherichia coli (DEC) strains was investigated in 253 children up to 3 years old, with (patient group, PG, 199 children) and without (control group, CG, 54 children) diarrhoea, living in Rio de Janeiro, Brazil. DEC strains were detected in 70 (27.6%) children, including 54 (27.1%) with diarrhoea and 16 (29.6%) without diarrhoea. Enteroaggregative E. coli (EAEC) was the most frequent DEC category, accounting for 14.6% of the isolates in the PG and for 11.1% in the CG. E. coli strains carrying enteropathogenic E. coli (EPEC) virulence markers showed higher incidence in the CG (12.9%) than in the PG (8.0%). E. coli strains belonging to non-classical EPEC groups that carried eae only or eae and bfpA, designated as attaching-effacing E. coli (AEEC) were the most frequent (79.1%). Simultaneous presence of multiple EPEC virulence factors (EAF/eae/bfpA) were only detected among strains isolated from the PG. Enterotoxigenic E. coli (ETEC) strains were isolated from 5.5% of the children in the CG and from 3.5% of those in the PG. Most of the ETEC isolates were LT-probe positive (70%) and none carried both LT-I and ST-I probe sequences. One enteroinvasive E. coli (EIEC) strain was recovered from a child with diarrhoea. No stx-probe positive E. coli strains were detected. Overall, DEC strains were not found to be significantly associated with diarrhoea (p>0.05). However, the higher incidence of EAEC, the most frequent DEC category, among children with diarrhoea, suggests a potential role of EAEC as an important enteric pathogen in the community investigated.

Epidemic nephritis in Nova Serrana, Brazil

BACKGROUND Outbreaks of nephritis have been rare since the 1970s. From December, 1997, to July, 1998, 253 cases of acute nephritis were identified in Nova Serrana, Brazil. Seven patients required dialysis, and three patients died. We did a case-control study to investigate the cause of the outbreak. METHODS Using a matched cluster design, we examined seven recent patients, their family members (n=23), and members of neighbourhood-matched control households (n=22). We subsequently interviewed 50 patients and 50 matched controls about exposure to various dairy products. We also cultured dairy foods and took udder-swab and milk samples from cows. FINDINGS Throat cultures indicated that nephritis was associated with group C Streptococcus equi subspecies zooepidemicus, a cause of bovine mastitis. S. zooepidemicus was detected in four of seven case households (six of 30 people) and no control households (p=0.09). Patients were more likely than matched controls to have consumed a locally produced cheese called queijo fresco (matched odds ratio 2.1, p=0.05). The nephritis attack rate was 4.5 per 1000 in Nova Serrana but 18 per 1000 in the village Quilombo do Gaia (p=0.003). The largest supplier of unpasteurized queijo fresco was a farm in Quilombo do Gaia. S. zooepidemicus was not detected in food samples or in swabs collected from cows in August, 1998, although mastitis was evident among cows on the suspected farm. Throat cultures of the two women who prepared cheese on this farm yielded the outbreak strain of S. zooepidemicus. After the cheese was removed from the distribution system, no further cases were reported. INTERPRETATION A large outbreak of glomerulonephritis was attributed to S. zooepidemicus in unpasteurised cheese. This outbreak highlights the dangers of consuming unpasteurized dairy products and need for global efforts to promote food safety.

MALDI‐TOF mass spectrometry as a tool for differentiation of invasive and noninvasive Streptococcus pyogenes isolates

A novel mass spectral fingerprinting and proteomics approach using MALDI-TOF MS was applied to detect and identify protein biomarkers of group A Streptococcus (GAS) strains. Streptococcus pyogenes ATCC 700294 genome strain was compared with eight GAS clinical isolates to explore the ability of MALDI-TOF MS to differentiate isolates. Reference strains of other bacterial species were also analyzed and compared with the GAS isolates. MALDI preparations were optimized by varying solvents, matrices, plating techniques, and mass ranges for S. pyogenes ATCC 700294. Spectral variability was tested. A subset of common, characteristic, and reproducible biomarkers in the range of 2000–14 000 Da were detected, and they appeared to be independent of the culture media. Statistical analysis confirmed method reproducibility. Random Forest analysis of all selected GAS isolates revealed differences among most of them, and summed spectra were used for hierarchical cluster analysis. Specific biomarkers were found for each strain, and invasive GAS isolates could be differentiated. GAS isolates from cases of necrotizing fasciitis were clustered together and were distinct from isolates associated with noninvasive infections, despite their sharing the same emm type. Almost 30% of the biomarkers detected were tentatively identified as ribosomal proteins.

DNA typing of methicillin-resistant Staphylococcus aureus : isolates and factors associated with nosocomial acquisition in two Brazilian university hospitals

Control and prevention of methicillin-resistant Staphylococcus aureus (MRSA) infections should include early identification of patients at higher risk of MRSA acquisition and analysis of isolates by discriminatory bacterial DNA typing methods. One hundred and three MRSA isolates cultured between Sept. 1994 and Sept. 1995 from 62 patients in two teaching hospitals (hospital 1, in Rio de Janeiro; hospital 2, in Minas Gerais) were tested for antimicrobial resistance and genomic DNA was analysed by pulsed-field gel electrophoresis (PFGE). Ten profiles were identified: A, B, C, I and J in hospital 1 and A, B, D, E, F, G and H in hospital 2. PFGE patterns A and B were isolated at both hospitals. The majority (80%) of isolates had similar PFGE patterns (type A). Subtype A1 was isolated at both hospitals, but was more frequent in hospital 2 (54%), while subtype A2 predominated in hospital 1 (63%). MRSA isolates were resistant to the majority of antimicrobial agents tested. However, susceptibility to vancomycin alone was found in 32% of the isolates at hospital 1, whereas 48% of isolates from hospital 2 were susceptible to both vancomycin and mupirocin, and 34% demonstrated susceptibility to vancomycin, mupirocin and chloramphenicol. Thirty-nine percent of all isolates were mupirocin-resistant, with 90% of these belonging to PFGE pattern A. Four main risk factors were associated with MRSA infection or colonisation which may be useful in the early identification of patients at risk: >7 days hospitalisation (95%), very dependent patients (84%), invasive procedures (79%) and recent antimicrobial therapy (79%). The data demonstrate that PFGE pattern A is disseminated in both hospitals. However, at both hospitals subtypes of pattern A and the other PFGE types were associated with different antibiotic resistance patterns.

Distribution of Antimicrobial Resistance and Virulence-Related Genes among Brazilian Group B Streptococci Recovered from Bovine and Human Sources

ABSTRACT In the present report we describe the characteristics of 189 antimicrobial-resistant Streptococcus agalactiae isolates from bovine (38 isolates) and human (151 isolates) sources. All the strains were resistant to tetracycline (TET), and 16 (8.5%) were also resistant to erythromycin, corresponding to 23.7% of the TET-resistant bovine isolates and 4.6% of the TET-resistant human isolates. The tet(O), erm(B), and mreA resistance-related genes, as well as the bca and scpB virulence-related genes, were the most frequent among the bovine isolates, while the tet(M), erm(A), mreA, bca, lmb, and scpB genes were the most prevalent among the isolates from humans. Although a few major clusters were observed, pulsed-field gel electrophoresis results revealed a variety of profiles, reflecting the substantial genetic diversity among strains of this species isolated from either humans or bovines.

Enterococcus gilvus sp. nov. and Enterococcus pallens sp. nov. Isolated from Human Clinical Specimens

ABSTRACT Light yellow-pigmented (strain PQ1) and yellow-pigmented (strain PQ2), gram-positive, non-spore-forming, nonmotile bacteria consisting of pairs or chains of cocci were isolated from the bile of a patient with cholecystitis (PQ1) and the peritoneal dialysate of another patient with peritonitis (PQ2). Morphologically and biochemically, the organisms phenotypically belonged to the genus Enterococcus. Whole-cell protein (WCP) analysis and sequence analysis of a segment of the 16S rRNA gene suggested that they are new species within the genus Enterococcus. PQ1 and PQ2 displayed less than 70% identities to other enterococcal species by WCP analysis. Sequence analysis showed that PQ1 shared the highest level of sequence similarity with Enterococcus raffinosus and E. malodoratus (sequence similarities of 99.8% to these two species). Sequence analysis of PQ2 showed that it had the highest degrees of sequence identity with the group I enterococci E. malodoratus (98.7%), E. raffinosus (98.6%), E. avium (98.6%), and E. pseudoavium (98.6%). PQ1 and PQ2 can be differentiated from the other Enterococcus spp. in groups II, III, IV, and V by their phenotypic characteristics: PQ1 and PQ2 produce acid from mannitol and sorbose and do not hydrolyze arginine, placing them in group I. The yellow pigmentation differentiates these strains from the other group I enterococci. PQ1 and PQ2 can be differentiated from each other since PQ1 does not produce acid from arabinose, whereas PQ2 does. Also, PQ1 is Enterococcus Accuprobe assay positive and pyrrolidonyl-β-naphthylamide hydrolysis positive, whereas PQ2 is negative by these assays. The name Enterococcus gilvus sp. nov. is proposed for strain PQ1, and the name Enterococcus pallens sp. nov. is proposed for strain PQ2. Type strains have been deposited in culture collections as E. gilvus ATCC BAA-350 (CCUG 45553) and E. pallens ATCC BAA-351 (CCUG 45554).

Helicobacter pylori Primary Resistance to Metronidazole and Clarithromycin in Brazil

ABSTRACT Helicobacter pylori resistance to metronidazole was detected in 107 (52.97%) of 202 strains. Twenty (9.85%) strains, 18 of them harboring 23S ribosomal DNA mutations, were resistant to clarithromycin. Metronidazole resistance was associated with female gender. Resistance to metronidazole and resistance to clarithromycin were associated. Increasing clarithromycin resistance rates were observed over time.

Analysis of electrophoretic whole-cell protein profiles as a tool for characterization ofEnterococcus species

The whole-cell protein-profiling technique was evaluated for identifyingEnterococcus species. Reference strains, strains from human infections, from animals other than human, and from environmental sources were studied. Whole-cell extracts were obtained by lysozyme treatment and were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and densitometry. EachEnterococcus species had a unique and distinguishable whole-cell protein profile. The major differences among species-specific profiles were found in the positions corresponding to 60-40 and 30-20 kDa. Profiles of the same species did not show qualitative variations. Analysis of whole-cell protein profiles was shown to be a relatively simple, easy, and reproducible procedure for the reliable and fast differentiation and identification of the enterococcal species.