First Detection of oqxAB in Salmonella spp. Isolated from Food

Abstract

Food-borne salmonellosis is an important public health problem worldwide and the second leading cause of food-borne illnesses in Hong Kong. In this study, the prevalence and antimicrobial resistance of Salmonella in meat products in Hong Kong were determined. Interestingly, a plasmid-mediated quinolone resistance (PMQR) gene combination, oqxAB, which mediates resistance to nalidixic acid, chloramphenicol, and olaquindox, was for the first time detectable on the chromosomes of two Salmonella enterica serovar Derby isolates. Further surveillance of oqxAB in Salmonella will be needed.

TEXT

Food-borne salmonellosis is an important public health problem worldwide. More than 1.4 million cases of salmonellosis occur in the United States each year, causing more than 300,000 hospitalization events and around 500 deaths (1). In Hong Kong, Salmonella is the second leading cause of food-borne illnesses (2). There have been over 3,000 Salmonella infection cases reported to the Department of Health within the last several years, but this rate is regarded as underestimated because of the self-limiting nature of the disease. Most human Salmonella infections occur through the consumption of contaminated food of animal origin, such as poultry, beef, pork, eggs, and milk (3). Although antibiotics are not essential for the treatment of most cases of salmonellosis, they can be life-saving in invasive infections, which often occur in children and elderly people (4). Resistance of Salmonella to conventional drugs, including ampicillin, chloramphenicol, and tetracycline, has been found frequently (5). Fortunately, the resistance rates of fluoroquinolones and broad-spectrum cephalosporins, which have been the choices of treatment for multidrug-resistant (MDR) nontyphoidal Salmonella infection in adults and children, respectively (6), remain extremely low.

Nevertheless, cephalosporin-resistant Salmonella food isolates have also been reported recently (7, 8). Fluoroquinolone resistance is relatively rare among Salmonella isolates compared to other food-borne pathogens, such as Escherichia coli and Campylobacter. In recent years, the prevalence of quinolone resistance in Salmonella clinical isolates, especially among the clinically significant serotypes, such as S. enterica serovars Typhimurium and Enteritidis, has been increasing (9). Quinolone resistance in Salmonella may not cause direct treatment failure, yet it may lead to a longer hospital stay, higher treatment cost, and higher treatment failure rate and hence a potential increase in mortality rate (10–12). Quinolone resistance in Salmonella may be attributed to the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants, including derivatives of quinolone resistance proteins (Qnr), aminoglycoside acetyltransferase AAC(6′)-Ib-cr, and quinolone efflux pump QepA (13, 14). This study describes for the first time the prevalence and antimicrobial resistance of Salmonella isolated in retail meats sold in Hong Kong and the spectrum of molecular mechanisms responsible for the phenotypic resistance.

A total of 150 meat samples, which included 80 pork samples (45 from the supermarket and 35 from the wet market/fresh food market) and 70 from chicken samples (36 from the supermarket and 34 from the fresh food market) were collected. Salmonella isolates were obtained from these food samples following the procedures described by the U.S. Food and Drug Administration and confirmed by the API20E kit (bioMérieux) (15). The Salmonella isolation rate for meat samples collected from the supermarket (37%) was similar to that of samples recovered from the wet market (45%). Salmonella isolates were recovered from 45 pork samples and 16 chicken samples at rates of 56% and 23%, respectively, which are recovery rates much higher than those of other countries but similar to those in a recent study from China (16–18). Since some of the samples contained two Salmonella isolates, a total of 112 Salmonella strains were collected for further characterization; among these 112 strains, 86 were isolated from pork and 26 were isolated from chicken.

Antimicrobial susceptibilities to 13 antimicrobials, including ampicillin, cefotaxime, ceftriaxone, sulfamethoxazole, kanamycin, amikacin, gentamicin, tetracycline, chloramphenicol, ciprofloxacin, nalidixic acid, streptomycin, and olaquindox, were determined for these Salmonella isolates by the agar dilution method according to CLSI guidelines (19). Eighty-four (75%) Salmonella strains isolated from retail meats exhibited resistance to at least one antimicrobial. Resistance to tetracycline (55%), sulfamethoxazole (46%), nalidixic acid (35%), and chloramphenicol (30%) was commonly observed. Around 24% of the isolates were resistant to ampicillin, yet among them, only one isolate was also resistant to cefotaxime and ceftriaxone. Various Salmonella isolates also exhibited resistance to kanamycin (4%) and gentamicin (3%). Two isolates were found to exhibit cross-resistance to chloramphenicol, tetracycline, nalidixic acid, and olaquindox (MIC, 256 μg/ml). All Salmonella isolates recovered from retail foods were susceptible to amikacin and ciprofloxacin. The resistance profile of Salmonella isolates is shown in Table 1. Salmonella isolates from Hong Kong showed very low rates of resistance to the antibiotics concerned compared to the isolates from China. However, the antimicrobial resistance rates and patterns in Salmonella food isolates in Hong Kong and mainland China are comparable to those of the clinical isolates in corresponding locations (20–22).

Table 1.

Antimicrobial resistance profiles and resistance genes of Salmonella isolates from food

No. of isolates (n = 84)	Resistance profilea	Resistance gene(s) (n)
14	NAL	qnrS (1)
12	AMP SUL TET CHL NAL	qnrS (1), aac(6′)-Ib-cr (1)
11	SUL TET
9	SUL TET CHL
7	TET
4	AMP SUL TET
3	SUL
2	AMP SUL KAN GEN TET NAL
2	AMP SUL TET CHL
2	AMP SUL TET NAL
2	CHL NAL
2	SUL TET CHL NAL
2	TET CHL NAL OLA	oqxAB (2)
2	TET CHL
1	AMP
1	AMP CTX AXO SUL TET	blaCMY-2 (1)
1	AMP SUL
1	AMP SUL CHL
1	AMP TET
1	GEN NAL
1	KAN TET
1	SUL KAN TET CHL
1	SUL TET NAL

^aAMP, ampicillin; CTX, cefotaxime; CRO, ceftriaxone; SUL, sulfamethoxazole; KAN, kanamycin; GEN, gentamicin; TET, tetracycline; CHL, chloramphenicol; NAL, nalidixic acid; OLA, olaquindox.

β-Lactamase gene screening was performed on the isolate that exhibited resistance to ceftriaxone and cefotaxime, as previously described (23). A PCR product carrying partial bla_CMY-2 sequence was detectable. Insertion sequences (ISs) were frequently detected upstream of sequences of extended-spectrum β-lactamases (ESBLs) and were regarded as being responsible for the capture and mobilization of the antibiotic resistance genes. Forward primers targeting insertion sequences ISCR1 (5′-AGACGCCGTGGAAGCGTGTG), ISEcp1 (5′-CTGCAAACGGTGCTGCGGAA), and IS903 (5′-CGCAGCGTCAGTGAACCCCC) and reverse primer CMY-2R (5′-AGCGGTTATTGCAGCTTTTCAAGAA) were used to amplify the whole length of the CMY variant. An ∼2-kb fragment was amplified by primers targeting ISEcp1 and CMY-2R. DNA sequencing of the whole DNA fragment revealed that the CMY variant was CMY-2. ISEcp1 was 248 bp upstream of CMY-2, and the ∼2-kb fragment was identical to a sequence carried on plasmid pSH696_135 from S. enterica serovar Heidelberg (accession no. JN983048.1). A conjugation experiment was performed for the Salmonella isolate carrying bla_CMY-2, using E. coli J53 as the recipient strain as previously described (24). No transconjugant was obtained, suggesting that bla_CMY-2 transmission was not mediated by a self-transmissible plasmid.

PMQR genes, including qnrA, qnrB, qnrC, qnrD, qnrS, qepA, aac(6′)Ib-cr, and oqxAB, were screened by PCR as described previously (25). Two nalidixic acid-resistant Salmonella isolates with different resistance profiles harbored PMQR genes, one carrying the qnrS gene and the other carrying qnrS and aac(6′)-Ib-cr genes (Table 1). These two PMQR genes have previously been reported in Salmonella (26, 27). Single gyrA mutation has been found to mediate nalidixic acid resistance in isolates without PMQR genes (data not shown). Importantly for the first time, two olaquindox-resistant isolates were found to contain the gene combination oqxAB, which encodes a resistance nodulation division (RND) family efflux pump and confers resistance to olaquindox quinolones and chloramphenicol and reduced susceptibility to other antibiotics (28). Both of these isolates were Salmonella enterica serovar Derby and displayed identical pulsed-field gel electrophoresis (PFGE) profiles. The fact that these two isolates were isolated from pork samples, but at different dates and from different locations (one from the supermarket and one from the wet market), suggests clonal dissemination of oqxAB-positive Salmonella in the local food supply network. The oqxAB genes could not be transferred to E. coli through conjugation. Hybridization of chromosomal and plasmid DNAs from these two isolates with the oqxAB probe confirmed that the oqxAB combination was located on the chromosomal DNA of Salmonella (Fig. 1). The oqxAB genes were first identified on the pOLA52 plasmid, and oqxAB was recently found in environmental and clinical Enterobacteriaceae isolates, including E. coli and Klebsiella pneumoniae (28, 29). The oqxAB genes carried by S. Derby in this study were confirmed to be associated with IS26 by PCR analysis (Fig. 2). The sequence of IS26-flanked oqxAB was found to be identical to that of the same fragment carried on pOLA52, while no other similar sequences of pOLA52 were found in the Salmonella isolates, suggesting that the oqxAB combination originated from pOLA52 and was transmitted to Salmonella through integration of oqxAB into the chromosome of Salmonella mediated by IS26. Further research will be needed to investigate the dissemination features of oqxAB among salmonellae since quinolone resistance in Salmonella will be of huge clinical significance. In other bacteria, like E. coli, fluoroquinolone resistance had been very high already, and the carrying of an additional oqxAB gene combination will not cause a huge change in their resistance profiles. In contrast, fluoroquinolone resistance in Salmonella has been very low, and fluoroquinolones are the choice of treatment for severe Salmonella infections. The prevalence of oqxAB will enable the Salmonella isolates to become resistant to quinolone and show reduced susceptibility to fluoroquinolones. It may also contribute to the further development of resistance to fluoroquinolone in Salmonella under selective pressure.

Fig 1.

Southern hybridization of the oqxAB genes to the chromosomal and plasmid DNA of oqxAB-positive S. Derby (S95 and S163) isolated from food in Hong Kong. Chromosomal (C) and plasmid (P) DNAs from S95 and S163 were denatured by boiling at 100°C for 2 min and placed on ice immediately for 5 min before being loaded onto the nitrocellulose membrane. After cross-linking, the membrane was subjected to Southern hybridization with the digoxigenin (DIG)-labeled oqxAB probe. +ve, PCR product of oqxAB from both isolates; −ve, Tris-EDTA (TE) buffer.

Fig 2.

Genetic environment of oqxAB. The IS26 transposase together with a hypothetical protein (H) were located upstream of the oqxA gene, separated by 188 nucleotides. The sequence showed 100% identity to pOLA52, the plasmid where the oqxAB combination was first discovered.