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.

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

Rapid tests for the detection of the Mycobacterium abscessus subsp. bolletii strain responsible for an epidemic of surgical-site infections in Brazil

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Clonal multidrug-resistant Corynebacterium striatum within a nosocomial environment, Rio de Janeiro, Brazil

Corynebacterium striatum is a potentially pathogenic microorganism with the ability to produce outbreaks of nosocomial infections. Here, we document a nosocomial outbreak caused by multidrug-resistant (MDR) C. striatum in Rio de Janeiro, Brazil. C. striatum identification was confirmed by 16S rRNA and rpoB gene sequencing. Fifteen C. striatum strains were isolated from adults (half of whom were 50 years of age and older). C. striatum was mostly isolated in pure culture from tracheal aspirates of patients undergoing endotracheal intubation procedures. The analysis by pulsed-field gel electrophoresis (PFGE) indicated the presence of four PFGE profiles, including two related clones of MDR strains (PFGE I and II). The data demonstrated the predominance of PFGE type I, comprising 11 MDR isolates that were mostly isolated from intensive care units and surgical wards. A potential causal link between death and MDR C. striatum (PFGE types I and II) infection was observed in five cases.

Outbreak of carbapenem-resistant Klebsiella pneumoniae: two-year epidemiologic follow-up in a tertiary hospital

This study describes a carbapenem-resistant Klebsiella pneumoniae (CRKP) outbreak that occurred from October 2008-December 2010. Polymerase chain reaction assays were performed to detect the blaKPC gene and molecular typing was performed using pulsed-field gel electrophoresis (PFGE). There were 33 CRKP infections; PFGE revealed five genotypes: genotype A in five (15%), B in 18 (55%), C in eight (24%) and two unique profiles. Genotype B was disseminated in all hospital units and belonged to the same clone identified in 11 different hospitals in the state of São Paulo. Sixteen (48%) patients died. Seven isolates (21%) were resistant to polymyxin B and 45% were resistant to tigecycline and amikacin.

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.

The antimicrobial susceptibility, biofilm formation and genotypic profiles of Staphylococcus haemolyticus from bloodstream infections

We analysed the antimicrobial susceptibility, biofilm formation and genotypic profiles of 27 isolates of Staphylococcus haemolyticus obtained from the blood of 19 patients admitted to a hospital in Rio de Janeiro, Brazil. Our analysis revealed a clinical significance of 36.8% and a multi-resistance rate of 92.6% among these isolates. All but one isolate carried the mecA gene. The staphylococcal cassette chromosome mec type I was the most prevalent mec element detected (67%). Nevertheless, the isolates showed clonal diversity based on pulsed-field gel electrophoresis analysis. The ability to form biofilms was detected in 66% of the isolates studied. Surprisingly, no icaAD genes were found among the biofilm-producing isolates.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

The first report of the vanC1 gene in Enterococcus faecium isolated from a human clinical specimen

The vanC1 gene, which is chromosomally located, confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum. Enterococcus faecium TJ4031 was isolated from a blood culture and harbours the vanC1gene. Polymerase chain reaction (PCR) assays were performed to detect vanXYc and vanTc genes. Only the vanXYc gene was found in the E. faecium TJ4031 isolate. The minimum inhibitory concentrations of vancomycin and teicoplanin were 2 µg/mL and 1 µg/mL, respectively. Real-time reverse transcription-PCR results revealed that the vanC1and vanXYc genes were not expressed. Pulsed-field gel electrophoresis and southern hybridisation results showed that the vanC1 gene was encoded in the chromosome. E. faecalis isolated from animals has been reported to harbour vanC1gene. However, this study is the first to report the presence of the vanC1gene in E. faecium of human origin. Additionally, our research showed the vanC1gene cannot serve as a species-specific gene of E. gallinarum and that it is able to be transferred between bacteria. Although the resistance marker is not expressed in the strain, our results showed that E. faecium could acquire the vanC1gene from different species.

Antimicrobial susceptibility patterns, emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.

Biofilm production by multiresistant Corynebacterium striatumassociated with nosocomial outbreak

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatumbound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Isoenzymatic variability in wild potatoes

Two species of wild potato Solanum commersonii, subspecies commersonii and malmeanum, and S. chacoense, subspecies muelleri occur in southern Brazil. Their rusticity and easy adaptation to extreme climatic conditions make them valuable for breeding programs. The objective of this work was to analyze the isoenzymatic variability of 113 clones of wild potato subspecies. They were collected and maintained at Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado, at Pelotas, RS, Brazil. Enzymes involved in energetic (group I) or in peripherical (group II) metabolism constituted the material used. Polyacrylamide horizontal gel electrophoresis was used to analyze peroxidase, aspartate transaminase, phosphoglucomutase and isocitrate dehydrogenase isoenzymes. Solanum spp. has considerable genetic variability for isoenzymatic patterns. Cluster analysis classified the clones into 51 subgroups, based on electrophoretic variants of group I enzymes, and into 89, when group II enzyme variants were added. Genotypic differentiation of S. chacoense muelleri in relation to S. commersonii commersonii and S. commersonii malmeanum is evident when expressed through similarity and cluster analysis.

Characterization of potato genotypes using molecular markers

The objective of this work was to characterize 27 potato genotypes, using molecular markers. Polyacrylamide gel electrophoresis, RAPD techniques and isozymes of esterase, phosphoglucomutase and soluble proteins were analyzed in tubers, and isocitrate dehydrogenase, aspartate transaminase, phosphoglucomutase and peroxidase, in leaves. Eighteen primers were tested and four were chosen, kits OPX (01, 04 and 09) and OPY (07), to analyze RAPD markers in leaf extracts. Similarity and cluster analysis were conducted using Jaccard coefficient and the unweighted pair-group method using arithmetic average. Despite the differences detected in the analysis of proteins and isozymes in the tubers, as well as of isozymes in the leaves, the characterization of all genotypes through gel electrophoresis was not possible, while RAPD markers were efficient to characterize all the 27 genotypes.

Esterase profile in the postembryonic development of Rhipicephalusmicroplus

The objective of this work was to analyze the pattern of esterase activity in the development stages of Rhipicephalus microplus by nondenaturing polyacrylamide gel electrophoresis using specific staining for esterase. The electrophoretical results revealed the presence of nine regions displaying esterase activity, stained with both alpha-naphthyl acetate and beta-naphthyl acetate, and classified as alpha-beta-esterase. Stage-specific esterases were found, with the first nymphal and larval stages showing the greatest esterase activity throughout the development. An esterase called EST-4 was detected only in males and was considered sex-specific. There are differences in the esterase profile among the different postembryonic development stages of R. microplus.