β-Lactamases, transferable quinolone resistance determinants, and class 1 integron-mediated antimicrobial resistance in human clinical Salmonella enterica isolates of non-Typhimurium serotypes

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Abstract

This work investigates the occurrence and features of class 1 integrons and the presence of transferable quinolone resistance determinants (TQRD) among 382 clinical Salmonella enterica isolates of non-Typhimurium serotypes as well as the β-lactamases produced by amoxicillin-resistant isolates. These isolates were recovered in 2001 and from 2004 to 2009 from patients from the health region of Terres de l’Ebre (Catalonia, Spain) and comprised 41 different serotypes, mostly of serovar Enteritidis (n = 272), being 16.5% multidrug-resistant (MDR). Among the 93 amoxicillin-resistant isolates, 84 produced TEM-1,4 produced an extended-spectrum β-lactamase (CTX-M-9 in one S. Grumpensis and in one S. Virchow, CTX-M-15 in S. Kapemba, and SHV-12 in S. Enteritidis), one produced DHA-1 (S. Newport), and 4 did not present any of the investigated β-lactamases. TQRD were found in 2 isolates (qnrA1 in CTX-M-9-producing S. Grumpensis and qnrB4 in DHA-1-producing S. Newport). Overall, 35 isolates (9.2% of all isolates and 54% of MDR isolates) belonging to 15 different serotypes carried class 1 integrons that were transferred by conjugation in 17 isolates. Eleven distinct cassette arrangements were identified, with dfrA1-aadA1, dfrA17-aadA5, and dfrA12-orfF-aadA2 being the most prevalent and widely distributed ones. Atypical sul3-associated integrons were detected in 5 isolates of serotypes Rissen and Enteritidis. Moreover, the presence of integrons in the serotypes Kapemba, Mikawasima, and [9,12:Iv:i:-], of the estX-psp (linked to sul3) and aadA13-sat cassette arrangements in S. enterica, of extended-spectrum β-lactamases in S. Kapemba and S. Grumpensis, and of TQRD in S. Grumpensis is reported here for the first time.

Introduction

Non-typhoidal Salmonella enterica are one of the leading causes of food-borne diseases related to the ingestion of contaminated food products of animal origin in humans (EFSA, 2009). Although most cases of S. enterica infections are confined to the gastrointestinal tract and are self-limiting, treatment of invasive illness may be hampered by the increase in isolates exhibiting multidrug resistance (MDR) and particularly resistance to fluoroquinolones and to extended-spectrum β-lactams, the first-choice agents for treatment of severe human infections (Soler et al., 2006, EFSA, 2011). Different studies provide increasing evidence of antimicrobial use in food animals as a major factor in the development of decreased susceptibility to antimicrobial agents in non-typhoidal Salmonella (EFSA, 2009, Threlfall, 2002).

The production of plasmid-mediated AmpC or extended-spectrum β-lactamases (ESBL) is responsible for resistance to broad-spectrum cephalosporins in Salmonella (González-Sanz et al., 2009), whilst chromosomal mutations in the quinolone resistance-determining regions of DNA gyrase and topoisomerase IV and mutations leading to reduced drug accumulation are the main mechanisms involved in fluoroquinolone resistance (Giraud et al., 2006). Moreover, the presence of horizontally transferable genes conferring decreased susceptibility to fluoroquinolones as a result of target protection (qnr), drug modification [aac(6′)-Ib-cr], or the action of active efflux pumps (qepA and oqxAB) is an emerging phenomenon in the Enterobacteriaceae that has been observed in Salmonella (Herrera-León et al., 2011, Hopkins et al., 2008, Veldman et al., 2011) and is often linked to coresistance to broad-spectrum cephalosporins and to complex class 1 integrons (Strahilevitz et al., 2009).

Over the past few years, many investigations have highlighted a major role played by integrons in the acquisition and dissemination of MDR in Salmonella and other Gram-negative bacteria, owing to their ability to capture one or more gene cassettes by an integrase-mediated site-specific recombination mechanism and their usual location on mobile genetic elements carrying other resistance determinants. The class 1 integrons, which are the most common in clinical isolates, generally possess the qacEΔ1 and sul1 genes conferring resistance to ammonium quaternary compounds and sulfonamides, respectively, in the 3′-conserved segment (3′-CS) (Fluit and Schmitz, 2004, Mazel, 2006). MDR associated with class 1 integrons is largely widespread among isolates belonging to serotype Typhimurium, which is together with S. Enteritidis the most frequent serotype in human infections (Threlfall, 2002, Weill et al., 2006a, Pérez-Moreno et al., 2009). Additionally, MDR is increasingly found in other less prevalent, but emerging serotypes of Salmonella spp., such as S. Newport, S. Kentucky, or S. Virchow (Hur et al., 2012, Herrera-León et al., 2010), and class 1 integrons, including atypical sul3-associated integrons, have been reported in non-Typhimurium Salmonella isolates of different geographical origins (Antunes et al., 2006, Krauland et al., 2009) in which they contribute to the extent of antimicrobial resistance.

In a previous study, we documented the high prevalence of class 1 integrons in MDR human isolates of S. Typhimurium and its monophasic variant from our geographical setting (Pérez-Moreno et al., 2009). The aim of the present work was to investigate the occurrence and features of class 1 integrons among human antimicrobial drug-resistant clinical S. enterica isolates of non-Typhimurium serotypes from the health region of Terres de l’Ebre (Catalonia, Spain) as well as to search for transferable quinolone resistance determinants (TQRD) and to characterize the β-lactamases produced by amoxicillin-resistant isolates.

Section snippets

Bacterial isolates

All human clinical isolates of S. enterica came from a collection of isolates consecutively recovered at the Microbiology Laboratory of Hospital de Tortosa Verge de la Cinta during the first 10 months of 2001 and from 2004 to 2009. Among a total of 632 S. enterica isolates, the 382 isolates belonging to serotypes other than Typhimurium or its monophasic variant (77 from 2001 and 305 from 2004 to 2009) were included in the study. Only one isolate per patient or per known outbreak was considered.

Antimicrobial susceptibility and resistance genes

The percentages of isolates resistant to the antimicrobial agents studied among the different serotypes identified in this work are shown in Table 1. Sixty-three isolates (16.5%) exhibited resistance to 3 or more antimicrobial families and were considered MDR. The highest rates of resistance were detected to the following antimicrobial agents: nalidixic acid (41.1%), amoxicillin (24.3%), tetracycline (17.5%), streptomycin (14.2%), sulfonamides (11%), and trimethoprim/sulfamethoxazole (9.9%).

Discussion

The results of the present work demonstrate that, although to a lesser extent than in S. Typhimurium (Pérez-Moreno et al., 2009), the MDR phenotype is quite common amongst human S. enterica isolates belonging to some other serotypes recovered in our geographical area. The resistance to individual antimicrobial agents and the MDR phenotype, which was scarce in S. Enteritidis, were comparable to those reported in a global Spanish collection of human S. Enteritidis and S. Hadar isolates (Soler et

Conflict of interests

None to declare.

Ethical approval

Not required.

Acknowledgements

The authors thank M. Carulla Pont, X. Tejedor Ganduxé, M.J. Centelles Serrano, M. Cortell Ortolá, I. Fort Gallifa, M. Pilar Cid Ventura, and Carmen López Escorihuela for excellent technical support in the conduct of this study as well as Donna Pringle for editorial assistance. They are grateful to the Laboratorio Nacional de Referencia de Salmonella y Shigella (Instituto de Salud Carlos III, Majadahonda, Madrid, Spain) for serotyping of Salmonella isolates. They also thank J. Calvo Montes

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