High frequency of aac(6')-Ib-cr gene associated with double mutations in gyrA and parC in Escherichia coli isolates from patients with urinary tract infections.

OBJECTIVES: The aims of this study were (i) to determine the frequency of plasmid-mediated resistance to fluoroquinolones (FQs) in Escherichia coli isolated from patients with urinary tract infections (UTIs) of nosocomial and community origin and (ii) to determine the relationships between the presence of extended-spectrum ß-lactamases (ESBLs), mutations in the gyrA and parC genes, and resistance to FQs. METHODS: A total of 71 E. coli isolates, including 38 ESBL-producers and 33 non-ESBL-producers, were analysed. The aac(6')-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6')-Ib-cr, a variant associated with FQ resistance. Detection of qnr genes was performed by multiplex PCR. In isolates that tested positive for these genes, the gyrA and parC genes were sequenced and the modulation factor of an efflux pump inhibitor was determined on the minimum inhibitory concentration (MIC) of norfloxacin. RESULTS: The frequencies of qnrS, qnrB and qnrA were 4.2%, 2.8% and 0%, respectively. The frequency of aac(6')-Ib-cr was 40.8% and this variant was associated with double mutations in gyrA and parC as well as resistance to FQs and ESBL production. Modulation of efflux pump activity was more frequent in resistant isolates that had a wild-type parC gene. CONCLUSION: An interplay of resistance mechanisms increased the level of resistance to FQs, and the high frequency of putative plasmid-mediated quinolone resistance genes associated with ESBL-producing isolates reduced therapeutic options to treat UTIs in the affected population.

Extended-spectrum ß-lactamases in Enterobacteriaceae isolated in Brazil carry distinct types of plasmid-mediated quinolone resistance genes.

One hundred and six nalidixic acid-resistant Enterobacteriaceae isolates from two Brazilian hospitals isolated from June to October 2010 were evaluated to characterize the co-existence of plasmid-mediated quinolone resistant (PMQR) and extended-spectrum ß-lactamase (ESBL) determinants. The qnr genetic environment was determined by PCR and sequencing. Conjugation and hybridization experiments determined whether qnr-carrying plasmids were self-transferable. The aac(6')-Ib-cr and qepA genes were also screened. Thirteen qnr-like genes (12.3 %) were identified, with qnrB1 the most common, followed by qnrS1, qnrB2 and qnrB19. No qnrA, qnrC, qnrD or qepA determinant was detected. All qnr-positive strains possessed chromosomal substitutions in gyrase- and topoisomerase-encoding genes and four harboured a aac(6')-Ib-cr gene. The co-production of blaCTX-M was observed in ten qnr-positive strains. These results indicate the dissemination of PMQR genes shown in clinical isolates from Brazil, and their co-existence with ESBL genes emphasizes the complexity of plasmid-mediated resistance determinants among Enterobacteriaceae.

Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil.

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patients in the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate. Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

CTX-M-producing Klebsiella spp. in a Brazilian hospital: what has changed in 6 years?

CTX-M-encoding genes from Klebsiella spp. strains isolated in 2000 and 2006 were characterized as well as their genetic environment. CTX-M-2 variants were predominant in Klebsiella pneumoniae strains, which showed a greater variability in bla(CTX-M) genes, integrons, and plasmids in 2006 when compared to strains collected in 2000. CTX-M-9-producing Klebsiella oxytoca was identified in 2000 as clonal dissemination.

Clonal relationships determined by multilocus sequence typing among enteropathogenic Escherichia coli isolated in Brazil.

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infantile diarrhea in developing nations. Multilocus sequence typing (MLST) characterizes bacterial strains based on the sequences of internal fragments in housekeeping genes. Little is known about strains of EPEC analyzed by MLST from Brazil. In this study, a diverse collection of 29 EPEC strains isolated from patients with diarrhea, admitted to the University Hospital of Ribeirao Preto, was characterized by MLST. Strain analysis demonstrated 22 different sequence types (STs), of which almost half (48%) were new, indicating a high genotype diversity. The 22 STs were divided by eBURST into 12 clonal complexes. It was not possible to correlate typical and atypical EPEC with other strains in the MLST database. This is the first study that analyzed EPEC strains from South America that are included in the E. coli MLST database. Nine (31%) out of 29 strains are part of the CC10 clonal complex, the major clonal complex in the database, which comprises 174 strains and 86 different STs, suggesting that these strains might be the most important intestinal pathogenic E. coli worldwide. Genetic relationships between typical and atypical EPEC, enterohemorrhagic E. coli, and enteroaggregative E. coli strains were not established by MLST.

Predominance of CTX-M-type extended-spectrum beta-lactamase genes among enterobacterial isolates from outpatients in Brazil.

Two hundred fifty-seven nalidixic acid-resistant enterobacterial isolates were collected in a Brazilian community from January 2000 to May 2005 to determine the prevalence of plasmid-encoded extended-spectrum beta-lactamases. The bla(CTX-M) genetic environment was determined by polymerase chain reaction and sequencing. Eleven isolates (4.2%) harbored a bla(CTX-M-2) gene, 3 isolates bla(CTX-M-9), 2 isolates bla(CTX-M-8), and 6 isolates bla(SHV-5). Two novel bla(CTX-M-2) variants, namely, bla(CTX-M-74) and bla(CTX-M-75), were identified.

Molecular and biochemical characterization of SHV-56, a novel inhibitor-resistant beta-lactamase from Klebsiella pneumoniae.

A clinical strain of Klebsiella pneumoniae was found to possess the chromosomal gene bla(SHV-56), encoding a new inhibitor-resistant beta-lactamase with a pI of 7.6. SHV-56 is derived from SHV-11 by the single substitution K234R. This mutation therefore evidences a new critical site for inhibitor resistance among SHV enzymes.

Plasmid-mediated quinolone resistance determinants among enterobacterial isolates from outpatients in Brazil.

OBJECTIVES: The aim of this study was to determine the spread of plasmid-mediated quinolone resistance determinants [qnr-like, aac(6')-Ib-cr and qepA genes] among nalidixic acid-resistant enterobacterial strains isolated from outpatients from Southeast Brazil, their transferability and the genetic structures associated with the qnr genes. METHODS: The qnrA, qnrB and qnrS genes were screened by a multiplex PCR-based technique from 257 non-repetitive nalidixic acid-resistant enterobacterial isolates collected from January 2000 to May 2005. Conjugation experiments were performed to determine whether the qnr-carrying plasmids were self-transferable. Genetic structures surrounding the qnr genes were analysed by PCR and cloning. The aac(6')-Ib-cr and qepA genes were screened among qnr-positive strains. RESULTS: Six qnrB-like-positive isolates (2.3%) were detected, whereas no qnrA- or qnrS-positive isolates were detected. Three Escherichia coli and two Klebsiella pneumoniae isolates harboured a qnrB2 gene and a single Citrobacter freundii isolate had the qnrB8 gene. One qnrB2-positive isolate also had the extended-spectrum beta-lactamase bla(CTX-M-2) gene. All these isolates also possessed chromosomal substitutions in gyrase- and topoisomerase-encoding genes, explaining their high-level resistance to quinolones. CONCLUSIONS: This study constitutes the first epidemiological survey of the three known Qnr determinants among Brazilian isolates and shows their low prevalence in that country, with the qnrB2 gene being mostly identified.

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 beta-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. beta-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.

Multilocus sequence typing of uropathogenic ESBL-producing Escherichia coli isolated in a Brazilian community.

In the present study, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction detection of three resistance genes were combined to characterize seven uropathogenic E. coli isolated from outpatients. Selected portions of seven housekeeping and three antibiotic-resistance genes of the isolates were sequenced. The seven isolates were classified into four different sequence types (STs) by MLST and five PGFE types. Three isolates had a novel allelic profile representing a new ST designated as ST528 and showed the same PFGE and resistance genes. Two isolates, both characterized as ST359, were differentiated by PFGE and shared only one of the antibiotic-resistance genes studied. Comparison of MLST results with those of PFGE and resistance genes demonstrated that Escherichia coli had acquired different antibiotic-resistance genes and DNA rearrangements, causing alterations in PFGE patterns but maintaining the same ST. Furthermore, this article also reports the first detection of a CTX-M-2 ESBL E. coli and SHV-5 in a Brazilian community.

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

The occurrence of extended-spectrum-beta-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 beta-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 beta-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.