Plasmid-mediated Quinolone Resistance Determinant Qnrb19 in Non- Typhoidal Salmonella Enterica Strains Isolated in Venezuela

Copyright © 2012 Gonzalez et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited Quinolone resistance in Enterobacteriaceae is typically mediated by chromosomal mutations leading to alterations in the target enzymes DNA gyrase and topoisomerase IV, or changes in drug entry and efflux. However, three plasmid-mediated quinolone resistance (PMQR) mechanisms conferring decreased susceptibility to quinolones (including some fluoroquinolones) have been recently described: QepA and OqxAB effluxes, Aac(6´)-Ib-cr aminoglycoside acetyltransferase and Qnr proteins (qnrA, qnrB, qnrC, qnrD and qnrS) [1,2]. Although plasmid-mediated quinolone resistance of Qnr type has been identified in Enterobacteriaceae from the United States, Europe, and Asia [1,2], little is known about the diversity, type or range of qnr genes in Latin America [3-6], especially in Venezuela. Therefore, the objective of this study was to screen for the presence of PMQR genes in non-typhoidal Salmonella enterica (NTS) strains with reduced susceptibility to fluoroquinolones, from clinical samples and chicken meat in Venezuela. A total of 127 NTS strains belonging to the collection of the Molecular Microbiology Laboratory of the Pharmacy Faculty of University of the Andes (Mérida, Venezuela), were enrolled in this study. These included 117 strains from stool specimens of pediatric patients collected from 2005 to 2007 and 10 isolates recovered from raw chicken meat in 2008. Six (4.7%) of these isolates showed a typical phenotype with reduced resistance to ciprofloxacin (MICs: 0.5 – 1 g/mL) and with affiliated susceptibility to nalidixic acid (MICs: 4-8 g/mL). These isolates were screened for extended-spectrum beta-lactamase (ESBL) phenotype, using cefotaxime and ceftazidime with and without clavulanic acid per Clinical and Laboratory Standards Institute (CLSI) guidelines [7] and confirmed by polymerase chain reaction (PCR) using specific primers for bla TEM , bla SHV and group bla CTX-M, [8]. Presence of qnrA, qnrB, qnrS, qnrD, aac(6´)-Ib and qepA genes was screened by multiplex and simplex PCR amplifications, using primers and conditions previously described [9,10]. In addition, mutations in quinolone resistance-determining regions (QRDR) of the gyrA, gyrB and parC genes were also determined [11]. Amplicons were sequenced to determine the gene variants and mutations. Regardless of origin and serovar, the qnrB gene was detected in six Salmonella strains. Sequence analysis of the amplification product revealed the qnrB19 variant (Table). No mutations were identified in the QRDR of the gyrA, gyrB and parC genes [11]. …

Quinolone resistance in Enterobacteriaceae is typically mediated by chromosomal mutations leading to alterations in the target enzymes DNA gyrase and topoisomerase IV, or changes in drug entry and efflux.However, three plasmid-mediated quinolone resistance (PMQR) mechanisms conferring decreased susceptibility to quinolones (including some fluoroquinolones) have been recently described: QepA and OqxAB effluxes, Aac(6´)-Ib-cr aminoglycoside acetyltransferase and Qnr proteins (qnrA, qnrB, qnrC, qnrD and qnrS) [1,2].Although plasmid-mediated quinolone resistance of Qnr type has been identified in Enterobacteriaceae from the United States, Europe, and Asia [1,2], little is known about the diversity, type or range of qnr genes in Latin America [3][4][5][6], especially in Venezuela.Therefore, the objective of this study was to screen for the presence of PMQR genes in non-typhoidal Salmonella enterica (NTS) strains with reduced susceptibility to fluoroquinolones, from clinical samples and chicken meat in Venezuela.
A total of 127 NTS strains belonging to the collection of the Molecular Microbiology Laboratory of the Pharmacy Faculty of University of the Andes (Mérida, Venezuela), were enrolled in this study.These included 117 strains from stool specimens of pediatric patients collected from 2005 to 2007 and 10 isolates recovered from raw chicken meat in 2008.Six (4.7%) of these isolates showed a typical phenotype with reduced resistance to ciprofloxacin (MICs: 0.5 -1 g/mL) and with affiliated susceptibility to nalidixic acid (MICs: 4 -8 g/mL).These isolates were screened for extended-spectrum beta-lactamase (ESBL) phenotype, using cefotaxime and ceftazidime with and without clavulanic acid per Clinical and Laboratory Standards Institute (CLSI) guidelines [7] and confirmed by polymerase chain reaction (PCR) using specific primers for bla TEM , bla SHV and group bla CTX-M, [8].Presence of qnrA, qnrB, qnrS, qnrD, aac(6´)-Ib and qepA genes was screened by multiplex and simplex PCR amplifications, using primers and conditions previously described [9,10].In addition, mutations in quinolone resistance-determining regions (QRDR) of the gyrA, gyrB and parC genes were also determined [11].Amplicons were sequenced to determine the gene variants and mutations.
Regardless of origin and serovar, the qnrB gene was detected in six Salmonella strains.Sequence analysis of the amplification product revealed the qnrB19 variant (Table ).No mutations were identified in the QRDR of the gyrA, gyrB and parC genes [11].In four of these strains the presence of ESBLs was suspected from ceftazidime or cefotaxime resistance (MICs: 64 -256 g/mL) and by reestablishing the susceptibility in the presence of clavulanic acid (4 µg/mL) [7].PCR amplification, using specific primers for bla TEM , bla SHV and group bla CTX-M [11], followed by sequencing analysis, allowed us to identify bla TEM-1 + bla SHV-12 in S. Give LMM96 and bla TEM-1 + bla CTX-M-2 in the other three strains: S. Give LMM183, S. Heidelberg LMM175 and S. Heidelberg LMM179 (Table).Association between QnrB-like determinants and ESBLs has been previously reported [2][3][4]6].
Qnr-positive plasmids were successfully transferred by transformation from serovars Salmonella to E. coli HB101.Transformants designated as LMM46-T, LMM96-T, LMM183-T, LMM175-T, LMM179-T and LMM300-T, could be selected on Mueller Hinton Agar plates supplemented with 0.06 µg/ml ciprofloxacin.PCR and sequencing confirmed the presence of qnrB19 in the transformants obtained.Susceptibility testing showed that the MICs of ciprofloxacin and nalidixic acid for all transformants were similar to corresponding host strains.Plasmid DNA was purified from transformants by an alkaline lysis method [12].Electrophoresis showed the presence of a 17 kb plasmid with a similar restriction pattern, using the PstI enzyme (Promega, Madison, WI, USA) in all the transformants obtained.All qnr-positive isolates were typed using PFGE [13].In total, four PFGE clusters were identified (A-D) and isolates of different serotypes were clustered separately.Similar distribution patterns have also been observed in NTS isolates with reduced susceptibility to ciprofloxacin from infants in Wuhan, China [14].This result indicates that the horizontal transfer PMQR occurs, since the same plasmid profile was observed in strains from different origins, times, and serovars.Fluoroquinolones are widely used in veterinary medicine as well as in poultry production, and qnr-positive NTS isolates could be selected and transmitted to humans through the food chain.
As far as the authors know, this is the first description of the occurrence of the qnrB19 gene in NTS isolates from pediatric patients and chicken meat in Venezuela.Isolates similar to those described in this study may be hard to identify in clinical laboratories since this phenotype is difficult to recognize by conventional methods.In addition, the qnrB19 gene found in enteropathogens, classified as susceptible to fluoroquinolones, may promote further selection from low-to high-level resistance when fluoroquinolones are used.Hence it is necessary to increase the sensitivity and optimize the screening procedures when strains that might contain such resistance determinants are studied.