Association of Transferable Quinolone Resistance Determinant qnrB19 with Extended-Spectrum β-Lactamases in Salmonella Give and Salmonella Heidelberg in Venezuela

Hindawi Publishing Corporation International Journal of Microbiology Volume 2013, Article ID 628185, 6 pages http://dx.doi.org/10.1155/2013/628185 Research Article Association of Transferable Quinolone Resistance Determinant qnrB19 with Extended-Spectrum 𝛽-Lactamases in Salmonella Give and Salmonella Heidelberg in Venezuela Fanny González and María Araque Laboratorio de Microbiologı́a Molecular, Departamento de Microbiologı́a, Facultad de Farmacia y Bioanálisis, Universidad de Los Andes, Mérida 5101, Venezuela Correspondence should be addressed to Marı́a Araque; Received 6 July 2013; Revised 13 August 2013; Accepted 29 August 2013 Academic Editor: Michael McClelland Copyright © 2013 F. González and M. Araque. 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. Four nontyphoidal Salmonella strains with resistance to extended-spectrum cephalosporins and nonclassical quinolone resistance phenotype were studied. Two S. Give were isolated from pediatric patients with acute gastroenteritis, and two S. Heidelberg were recovered from raw chicken meat. Phenotypic characterization included antimicrobial susceptibility testing and detection of extended-spectrum 𝛽-lactamases (ESBLs) by the double-disc synergy method. The detection of quinolone resistance-determining regions (QRDR) of gyrA, gyrB, and gyrC genes, 𝑏𝑙𝑎ESBLs genes, and plasmid-mediated quinolone resistance (PMQR) determinants was carried out by molecular methods. Plasmid analysis included Southern blot and restriction patterns. Transferability of resistance genes was examined by transformation. 𝑏𝑙𝑎TEM-1 + 𝑏𝑙𝑎SHV-12 genes were detected in S. Give SG9611 and 𝑏𝑙𝑎TEM-1 + 𝑏𝑙𝑎CTX-M-2 in the other three strains: S. Give SG9811, S. Heidelberg SH7511, and SH7911. Regardless of origin and serovars, the qnrB19 gene was detected in the 4 strains studied. All determinants of resistance were localized in plasmids and successfully transferred by transformation. This study highlights the circulation of qnrB19 associated with 𝑏𝑙𝑎TEM-1 , 𝑏𝑙𝑎SHV-12 , and 𝑏𝑙𝑎CTX-M-2 in S. Give and S. Heidelberg in Venezuela. The recognition of factors associated with increasing resistance and the study of the molecular mechanisms involved can lead to a more focused use of antimicrobial agents. 1. Introduction Nontyphoidal Salmonella (NTS) are one of the major causes of foodborne infections related to the ingestion of contaminated animal food products in humans [1]. In most cases, these infections are confined to the gastrointestinal tract and are self-limiting. However, for immunocompromised and/or elderly patients, as well as for invasive or prolonged infections, antibiotic treatment is recommended [2]. Fluoroquinolones and extended-spectrum 𝛽-lactams are the firstchoice agents for these cases but the increase of the multidrug resistance (MDR) Salmonella strains reduces the available treatment options [1–5]. The emergence of Salmonella spp. isolates that display resistance to extended-spectrum 𝛽-lactams is mediated by plasmids and is an increasing public health concern [3–5]. The resistance to fluoroquinolones is typically mediated by alterations in the target enzymes DNA gyrase and topoisomerase IV or changes in drug entry and efflux. Also, three plasmid-mediated mechanisms conferring decreased susceptibility to ciprofloxacin have been recently described: QepA efflux, Aac(6󸀠 )-Ib-cr aminoglycoside acetyltransferase, and QNR proteins (qnrA, qnrB, qnrC, qnrD, and qnrS) [6, 7]. The qnr determinants encode pentapeptide repeat proteins, which are thought to bind to DNA gyrase, protecting it from quinolones; aac(6󸀠 )-lb-cr encodes a variant aminoglycoside acetyltransferase that modifies those fluoroquinolones that have a piperazinyl moiety, such as ciprofloxacin and norfloxacin, while qepA encodes a major facilitator efflux pump [6–8]. 2 Several studies have shown the coexistence of plasmidmediated quinolone resistance (PMQR) determinants with extended-spectrum 𝛽-lactamases (ESBL) on the same plasmid or cross-species/genera transferability of the plasmids [6, 9]. Selective pressure exerted by fluoroquinolones may be a driving force leading to the emergence and spread of isolates that carry not only PMQR determinants but also ESBL. Thus, close linkage between different resistance determinants may lead to high prevalence of MDR Salmonella strains under antibiotic-specific selective pressure [4, 5, 8]. In fact, the emergence of the association between PMQR and ESBL is an issue that narrows the usage of valuable antibiotics in managing NTS infections [1, 5, 9]. Epidemiologic studies of more than 2,300 NTS strains from human cases and animals in North America, Europe, and Asia have reported the prevalence of qnrA, qnrB, qnrS, and aac(6󸀠 )-lb-cr genes in 0.2%, 1.0%, 2.4%, and 6.4%, respectively [6]. On the other hand, in a previous study carried out by our team from 2005 to 2007 and in 2008, we found a prevalence of qnrB19 (4.7%) in 117 NTS strains isolated from feces of pediatric patients and raw chicken meat [10]. Nevertheless, molecular and epidemiologic information on mechanisms of resistance in Salmonella enterica in Venezuela is scarce [10, 11]. The aim of this study was to determine the coexistence of PMQR determinant qnrB19 and ESBL in S. Give and S. Heidelberg isolates from clinical sample and raw chicken meat in Mérida, Venezuela. 2. Materials and Methods 2.1. Salmonella Strains. In 2011, four NTS strains expressing a remarkable type of multidrug resistance were studied. These strains were selected from a collection of the Molecular Microbiology Laboratory of the Pharmacy and Bioanalysis Faculty of The Andes University, Mérida, Venezuela, based on resistance to extended-spectrum cephalosporins and an unusual quinolone resistance phenotype with reduced susceptibility to ciprofloxacin and associated susceptibility to nalidixic acid. Two S. Give (SG9611 and SG9811) were isolated from stool specimens obtained from pediatric patients with acute gastroenteritis, and two S. Heidelberg (SH7511 and SH7911) were recovered from raw chicken meat. 2.2. Antimicrobial Susceptibility Testing. The resistance patterns were determined by the standard plate agar dilution method, according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [12]. The antimicrobial agents tested included (Sigma-Aldrich, St Louis, MO): cefoxitin, cefotaxime, cefotaxime-clavulanic acid, ceftazidime, ceftazidime-clavulanic acid, aztreonam, imipenem, meropenem, ertapenem, nalidixic acid, ciprofloxacin, amikacin, gentamicin, and tobramycin. E. coli ATCC 25922 was always used for quality control purposes. The ESBL phenotype was detected by the double-disc synergy (DDS) method [12]. International Journal of Microbiology 2.3. Determination of Antimicrobial Resistance Genes and Sequence Analysis 2.3.1. Detection (...truncated)