Molecular Characterization of Antibiotic-Resistant Salmonella Isolates from Retail Meat from Markets in Northern Vietnam

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1 1709 Journal of Food Protection, Vol. 75, No. 9, 2012, Pages doi: / x Copyright G, International Association for Food Protection Research Note Molecular Characterization of Antibiotic-Resistant Salmonella Isolates from Retail Meat from Markets in Northern Vietnam TRUONG HA THAI 1,2 AND RYOJI YAMAGUCHI 1 * 1 Department of Veterinary Pathology, Faculty of Agriculture, University of Miyazaki, Miyazaki , Japan; and 2 Department of Microbiology Infectious Diseases Pathology, Faculty of Veterinary Medicine, Hanoi University of Agriculture, Trau Quy, Gia Lam, Ha Noi, Vietnam MS : Received 28 February 2012/Accepted 28 April 2012 ABSTRACT A total of 118 Salmonella isolates were detected from 283 retail meat samples (135 pork and 148 chicken meat) purchased at retail markets in Northern Vietnam. Thirteen serovars, including Infantis, Anatum, Rissen, Reading, Emek, Typhimurium, Blockley, London, Newport, Derby, Weltevreden, Albany, and Hadar, were determined. Resistance to tetracycline (54.2%), sulfonamides (52.5%), streptomycin (41.5%), trimethoprim (36.4%), chloramphenicol (35.6%), and ampicillin (33.1%) was commonly seen in the Salmonella isolates. Fourteen [bla TEM, bla OXA-1, bla PSE-1, aada1, sul1, teta, tetb, tetg, cmla1, flor, dfra1, dfra12, aac(3)-iv, and apha1-1ab] of 17 resistance genes were detected from the isolates demonstrating resistance. Genes for plasmid-mediated quinolone resistance, such as qnra, qnrb, qnrs, qepa, and acc(69)-1b-cr, were not detected in 23 quinoloneresistant isolates. The substitution TCC to TTC at codon 83 of gyra was found in the 18 quinolone-resistant isolates. The data revealed that resistant Salmonella strains were widely distributed in Northern Vietnam via the food chain and that they might contain multiple genes specifying identical resistance phenotypes. Thus, further studies are necessary to clarify the mechanisms of antibiotic resistance in Salmonella strains and their spread in the livestock market. Salmonellosis is one of the most important enteric diseases, affecting many people worldwide. The global human health impact of nontyphoidal Salmonella bacteria is high, with an estimated 93.8 million illnesses annually, of which an estimated 80.3 million are foodborne and associated with 155,000 deaths (18). Pork, beef, and chicken meat have been recognized as significant sources of human salmonellosis. Although more than 2,500 serovars of Salmonella enterica have been identified, most human Salmonella infections are caused by a limited number of serovars, which may vary from country to country and over time (13). The emergence of antibiotic-resistant Salmonella has become a major public health concern. The use of antibiotics in any venue, including disease treatment and growth promotion in domestic livestock, can potentially lead to widespread dissemination of antibiotic-resistant bacteria. In recent years, studies of Salmonella isolates from foodstuffs and animals in Vietnam and other countries have shown that multidrug resistance of Salmonella is increasing (25, 27 29). The increased use of fluoroquinolones has led to increasing resistance to these antibiotics, with rates of resistance that vary by both organism and geographic region (10, 15). The main mechanism of quinolone resistance was believed to arise from chromosomal mutations in genes * Author for correspondence. Tel: ; Fax: ; a0d402u@cc.miyazaki-u.ac.jp. encoding target enzymes, such as DNA gyrase (gyra and gyrb) and/or DNA topoisomerase IV (parc and pare), or affecting drug accumulation (10, 15, 24). Besides mutations in chromosomal genes, plasmid-mediated quinolone resistance (PMQR) has also been reported, including qnr-mediated inhibition of quinolone binding to DNA, a qepa-encoded efflux pump, and the aac(69)-1b-cr-mediated inactivation of fluoroquinolones by an acetyltransferase (21, 22, 24). The aims of this study were to determinate the antibiotic resistance in Salmonella sp. isolates from retail meat from markets in Northern Vietnam and to investigate the genes coding for antibiotic resistance. In addition, the nalidixic acid resistant isolates were further characterized for quinolone resistance mechanisms. MATERIALS AND METHODS Sampling. A total of 283 retail meat samples (135 pork and 148 chicken meat) were collected randomly from the retail markets in three provinces, including Bac Ninh, Ha Noi, and Ha Tay, in Northern Vietnam from July 2008 to June Samples (.100 g) were placed in sterile plastic sampling bags and chilled in an ice box during transport to the laboratory at Hanoi University of Agriculture. All samples were analyzed on the day of arrival. Salmonella isolation and serotyping. Salmonella organisms were isolated according to the International Organization for Standardization standard method ISO 6579 (14), with some modifications. For preenrichment of Salmonella, 25 g of each sample was homogenized in a sterile bag with 225 ml of buffered peptone water and incubated at 37uC for 18 to 24 h. Next, 0.1 ml of

2 1710 THAI AND YAMAGUCHI J. Food Prot., Vol. 75, No. 9 TABLE 1. Prevalence of antibiotic resistance among Salmonella serovars isolated from retail meat No. (%) of isolates resistant to drug Antibiotic a Infantis (n ~ 22) Anatum (n ~ 21) Rissen (n ~ 16) Reading (n ~ 13) Emek (n ~ 9) Typhimurium (n ~ 9) London (n ~ 7) Blockley (n ~ 7) Newport (n ~ 6) Others Total (n ~ 8) b (n ~ 118) A 9 (40.9) 9 (42.9) 7 (43.8) 1 (7.7) 1 (11.1) 6 (66.7) 1 (14.3) 3 (50.0) 2 (25.0) 39 (33.1) Ac 4 (44.4) 4 (3.4) C 7 (31.8) 8 (38.1) 6 (37.5) 3 (23.1) 4 (44.4) 5 (55.6) 2 (28.6) 4 (66.7) 3 (37.5) 42 (35.6) Cf Ci 1 (4.8) 5 (55.6) 1 (12.5) 7 (5.9) G 2 (9.1) 4 (19.0) 4 (25.0) 5 (55.6) 2 (33.3) 1 (12.5) 18 (15.3) K 10 (45.5) 5 (23.8) 5 (31.3) 6 (66.7) 4 (66.7) 1 (12.5) 31 (26.3) Na 4 (18.2) 6 (28.6) 3 (18.8) 6 (66.7) 3 (50.0) 1 (12.5) 23 (19.5) Ne 1 (4.5) 3 (14.3) 3 (18.8) 6 (66.7) 3 (50.0) 1 (12.5) 17 (14.4) No 1 (11.1) 1 (0.8) Su 18 (81.8) 7 (33.3) 11 (68.8) 3 (23.1) 5 (55.6) 6 (66.7) 2 (28.6) 1 (14.3) 4 (66.7) 5 (62.5) 62 (52.5) S 17 (77.3) 7 (33.3) 8 (50.0) 2 (15.4) 1 (11.1) 6 (66.7) 1 (14.3) 1 (14.3) 3 (50.0) 3 (37.5) 49 (41.5) Te 14 (63.6) 12 (57.1) 10 (62.5) 6 (46.2) 2 (22.2) 6 (66.7) 3 (42.9) 2 (28.6) 6 (100) 3 (37.5) 64 (54.2) Tp 12 (54.5) 6 (28.6) 5 (31.3) 2 (15.4) 4 (44.4) 5 (55.6) 1 (14.3) 5 (83.3) 3 (37.5) 43 (36.4) a A, ampicillin; Ac, amoxicillin clavulanic acid; C, chloramphenicol; Cf, ceftazidime; Ci, ciprofloxacin; G, gentamicin; K, kanamycin; Na, nalidixic acid; Ne, neomycin; No, norfloxacin; Su, sulfonamides; S, streptomycin; Te, tetracycline; Tp, trimethoprim;, not found. b Other serovars included Derby (n ~ 4), Weltevreden (n ~ 2), Albany (n ~ 1), and Hadar (n ~ 1).

3 J. Food Prot., Vol. 75, No. 9 ANTIBIOTIC RESISTANCE OF SALMONELLA FROM RETAIL MEAT IN NORTH VIETNAM 1711 TABLE 2. Distribution and prevalence of resistance genes among the Salmonella serovars Drug (no. of isolates tested) and resistance gene(s) No. (%) of isolates positive for gene(s) Serovar (no. of isolates positive for gene[s]) Ampicillin (39) bla TEM 36 (92.3) Anatum (9), Derby (1), Emek (1), Infantis (7), London (1), Newport (3), Reading (1), Rissen (5), Typhimurium (6) bla OXA-1 9 (23.1) Anatum (2), Derby (1), Infantis (1), Rissen (2), Typhimurium (3) bla PSE-1 2 (5.1) Derby (1), Infantis (1) bla TEM zbla OXA-1 5 (12.8) Anatum (2), Typhimurium (3) bla TEM zbla PSE-1 1 (2.6) Derby (1) No. (%) of isolates with at least one gene 39 (100) Chloramphenicol (42) cmla1 24 (57.1) Anatum (6), Derby (1), Emek (1), Infantis (3), London (1), Newport (4), Rissen (4), Typhimurium (4) flor 29 (69.0) Anatum (2), Derby (2), Infantis (6), London (1), Newport (4), Reading (3), Rissen (6), Typhimurium (4), cmla1zflor 24 (51.1) Anatum (1), Derby (1), Infantis (2), Newport (3), Rissen (4), Typhimurium (3) No. (%) of isolates with at least one gene 39 (92.9) Gentamycin (18) aac(3)-iv 17 (94.4) Anatum (4), Derby (1), Infantis (2), Newport (2), Rissen (4), Typhimurium (4) Kanamycin (31) apha1-1ab 31 (100) Anatum (5), Derby (1), Infantis (10), Newport (4), Rissen (5), Typhimurium (6) Streptomycin (49) aada1 41 (83.7) Anatum (7), Derby (3), Emek (1), Infantis (13), Newport (3), Reading (1), Rissen (8), Typhimurium (5) Sulfonamides (62) sul1 47 (75.8) Anatum (7), Derby (3), Emek (2), Infantis (14), London (1), Newport (3), Reading (1), Rissen (9), Typhimurium (6), Tetracycline (64) teta 47 (73.4) Anatum (7), Derby (1), Emek (1), Infantis (10), London (2), Newport (5), Reading (5), Rissen (9), Typhimurium (6), tetb 1 (1.6) Anatum (1) tetg 20 (31.3) Anatum (4), Derby (1), Infantis (2), Newport (4), Reading (1), Rissen (3), Typhimurium (4), tetaztetb 1 (1.6) Anatum (1) tetaztetg 16 (25.0) Anatum (2), Derby (1), Infantis (1), Newport (3), Reading (1), Rissen (3), Typhimurium (4), No. (%) of isolates with at least one gene 52 (81.3) Trimethoprim (43) dfra1 20 (46.5) Anatum (3), Derby (1), Emek (2), Infantis (4), London (1), Reading (2), Rissen (4), Typhimurium (2), dfra12 20 (46.5) Anatum (2), Derby (1), Infantis (6), Newport (3), Reading (1), Rissen (3), Typhimurium (4) dfra1zdfra12 7 (16.3) Anatum (1), Newport (1), Reading (1), Rissen (2), Typhimurium (2) No. (%) of isolates with at least one gene 37 (86.0)

4 1712 THAI AND YAMAGUCHI J. Food Prot., Vol. 75, No. 9 TABLE 3. Resistance profiles and characteristics of quinolone resistance in Salmonella sp. isolates from retail meats a Serovar of isolate Source a of gyra Characterization MIC (mg/ml) of Na b Disk diffusion c Ci No Resistance phenotype d Resistance genes Anatum C Wild type $32 S S A C Te Na blatem, flor Anatum P Wild type $32 S S A S Te Tp Na bla TEM, teta, aada1 Infantis C Wild type $32 S S A C S Su Te K Na blatem, flor, aada1, sul1, teta, apha1-1ab, Infantis P Wild type $64 S S C S Su Te K Ne Na flor, sul1, aada1, teta, apha1-1ab Anatum C Wild type $64 S S A C S Su Te K Tp Na bla TEM, cmla1, aada1, sul1, teta, tetg, apha1-1ab, dfra1 Infantis P Ser83Phe $128 S S C S Su G Tp Na cmla1, flor, aada1, sul1, aac(3)-iv, dfra12 Typhimurium P Ser83Phe $128 S S A S Su Te K Ne Na blatem, aada1, sul1, teta, apha1-1ab Infantis C Ser83Phe $128 S S A C S Su Te K Tp Na bla TEM, cmla1, aada1, sul1, teta, tetg, apha1-1ab, dfra12 Newport P Ser83Phe $128 S S A C S Te K Ne Tp Na bla TEM, cmla1, flor, aada1, teta, apha1-1ab, dfra1 Rissen C Ser83Phe $128 S S A S Su TK Ne Tp Na blatem, aada1, sul1, teta, tetg, apha1-1ab, dfra1 Anatum C Ser83Phe $128 S S A C S Su Te G K Ne Na blatem, blaoxa-1, cmla1, flor, aada1, sul1, tetg, aac(3)-iv, apha1-1ab Anatum C Ser83Phe $128 S S A C S Su Te G K Ne Na blatem, blaoxa-1, cmla1, aada1, sul1, tetg, aac(3)-iv, apha1-1ab Newport C Ser83Phe $256 I S A C S Su Te G K Ne Tp Na blatem, cmla1, flor, aada1, sul1, teta, tetg, aac(3)-iv, apha1-1ab, dfra1, dfra12 Newport C Ser83Phe $256 I S A C S Su Te G K Ne Tp Na blatem, cmla1, flor, aada1, sul1, teta, tetg, aac(3)-iv, apha1-1ab, dfra12 Rissen C Ser83Phe $256 S S A C S Su Te G K Ne Tp Na bla TEM, cmla1, flor, aada1, sul1, teta, tetg, aac(3)-iv, apha1-1ab, dfra1, dfra12 Rissen C Ser83Phe $512 S I A C S Su Te G K Ne Tp Na blatem, cmla1, flor, aada1, sul1, teta, tetg, aac(3)-iv, apha1-1ab, dfra1, dfra12 Anatum C Ser83Phe $512 R S ACSSuTeGKNeTpNaCi bla TEM, cmla1, aada1, sul1, teta, tetb, aac(3)-iv, apha1-1ab, dfra1, dfra12 Derby C Ser83Phe $512 R S ACSSuTeGKNeTpNaCi blatem, cmla1, flor, aada1, sul1, teta, tetg, aac(3)-iv, apha1-1ab, dfra12 Typhimurium C Ser83Phe $512 R I ACSSuTeGKNeTpNaCi blatem, cmla1, aada1, sul1, teta, tetg, aac(3)-iv, apha1-1ab, dfra12 Typhimurium C Ser83Phe $512 R S ACSSuTeGKNeTpNaCiAc blatem, blaoxa-1, cmla1, flor, aada1, sul1, teta, tetg, aac(3)-iv, apha1-1ab, dfra1, dfra12 Typhimurium P Ser83Phe $512 R I ACSSuTeGKNeTpNaCiAc blatem, blaoxa-1, flor, sul1, teta, tetg, apha1-1ab, dfra12 Typhimurium P Ser83Phe $512 R I ACSSuTeGKNeTpNaCiAc blatem, cmla1, flor, aada1, sul1, teta, aac(3)-iv, apha1-1ab, dfra12 Typhimurium C Ser83Phe $512 R R ACSSuTeGKNeTpNaNo blatem, blaoxa-1, cmla1, flor, aada1, sul1, teta, tetb, tetg, Ci Ac aac(3)-iv, apha1-1ab, dfra1, dfra12 a C, chicken meat; P, pork meat. b Na, nalidixic acid. Ci, ciprofloxacin; No, norfloxacin; S, susceptible; I, intermediate; R, resistant. c d A, ampicillin; C, chloramphenicol; Te, tetracycline; Na, nalidixic acid; S, streptomycin; Tp, trimethoprim; Su, sulfonamides; K, kanamycin; Ne, neomycin; G, gentamicin; Ci, ciprofloxacin; Ac, amoxicillin clavulanic acid.

5 J. Food Prot., Vol. 75, No. 9 ANTIBIOTIC RESISTANCE OF SALMONELLA FROM RETAIL MEAT IN NORTH VIETNAM 1713 the preenriched culture in buffered peptone water was added to 10 ml of Rappaport-Vassiliadis soya broth, followed by further incubation at 41.5uC for 24 h. A loopful of culture broth was then sampled from the selective enrichment Rappaport-Vassiliadis soya broth, streaked onto xylose lysine Tergitol 4 agar (Merck), and incubated at 37uC for 24 h. One or two black presumptive colonies were selected from each plate and cultured on nutrient agar slants. The isolates were confirmed to be Salmonella by confirmatory biochemical tests (fermentation of glucose and sucrose, lack of fermentation of lactose, hydrogen sulfide production test, citrate test, lysine decarboxylation, and methyl red and indole tests). Typical Salmonella isolates were serotyped by slide and microtiter agglutination for O and H antigens (Difco Laboratories, Detroit, MI) according to the Kauffmann and White scheme (11) by the Department of Veterinary Hygiene, National Institute of Veterinary Research, Vietnam. Antibiotic susceptibility testing. The antibiotic susceptibilities of isolates were determined according to the guidelines of the Clinical and Laboratory Standards Institute (5). Disk diffusion assays were performed on Mueller-Hinton agar with disks containing 14 different antibiotic agents (Oxoid, Basingstoke, Hampshire, UK). The following antibiotics were tested: ampicillin, 10 mg; amoxicillin clavulanic acid, 20/10 mg; ceftazidime, 30 mg; chloramphenicol, 30 mg; ciprofloxacin, 5 mg; gentamicin, 10 mg; kanamycin, 30 mg; nalidixic acid, 30 mg; neomycin, 10 mg; norfloxacin, 10 mg; streptomycin, 10 mg; tetracycline, 30 mg; trimethoprim, 5 mg; and sulfonamides, 300 mg. The interpretive category susceptible, intermediate, or resistant was determined according to Clinical and Laboratory Standards Institute guidelines (7). An isolate was defined as being multiresistant if it was resistant to three or more antibiotics. Escherichia coli ATCC was used as the control organism. Detection of resistance genes. DNA templates used for PCR were prepared by boiling bacterial cultures (23). The following genes implicated in antibiotic resistance were detected by PCR amplification: bla PSE-1, bla OXA-1, and bla TEM encoding b-lactam resistance; aada1, aada2, aac(3)-iv, apha-1ab, and Kn encoding aminoglycoside resistance; cata1, cmla1, and flor encoding chloramphenicol resistance; sul1 encoding sulfonamide resistance; teta, tetb, and tetg encoding tetracycline resistance; and dfra1 and dfra12 encoding trimethoprim resistance. In the case of an isolate showing resistance to quinolone-based antibiotic compounds, the MIC of nalidixic acid was examined by the broth dilution method (6). The PMQR genes, such as qnra, qnrb, qnrs, qepa, aac(69)- 1b-cr, and quinolone resistance determining regions (QRDRs) of gyra, were detected for quinolone resistance. PCR amplification reactions were performed in a 25-ml reaction mixture containing 12.5 ml of 2 GoTaq Green master mix (Promega, Madison, WI), 1 ml (10 ng/ml) of primers, 4 ml of DNA template, and nuclease-free water. The primer sets and the assay conditions used for amplification were previously described (4, 8, 9, 12, 16, 20). Sequencing of gyra. PCR amplifications of the QRDRs to detect mutations at codons Gly 81, Ser 83, and Asp 87 in gyra were carried out using the primers in a previous report (8). Purified PCR products were sequenced and compared with the wild-type sequences (GenBank accession no. X ). RESULTS AND DISCUSSION Of the 283 retail meat samples, 118 (41.7%) were Salmonella positive, including 63 (42.6%) of the 148 chicken samples and 55 (40.7%) of the 135 pork samples. Thirteen serovars were identified, including Infantis, Anatum, Rissen, Reading, Emek, Typhimurium, Blockley, London, Newport, Derby, Weltevreden, Albany, and Hadar (Table 1). Resistance to tetracycline (54.2%), sulfonamides (52.5%), streptomycin (41.5%), trimethoprim (36.4%), chloramphenicol (35.6%), and ampicillin (33.1%) was commonly seen in the Salmonella isolates (Table 1). These findings were comparable to those in previous reports in Vietnam (25, 27) and other Asian countries (3, 28, 29). These drugs have been commonly used for treatment of Salmonella infection in both humans and animals in these countries because replacing these antibiotics with the new generations of drugs is not easy due to their high cost (26, 28). Fourteen [bla TEM, bla OXA-1, bla PSE-1, aada1, sul1, teta, tetb, tetg, cmla1, flor, dfra1, dfra12, aac(3)-iv, and apha1-1ab] of 17 resistance genes were detected from the resistant isolates (Table 2). In most cases, resistance genes were detected where the corresponding individual resistance phenotypes were observed, suggesting the expression of the genes present. High levels of prevalence of the bla TEM (92.3%), flor (69%), sul1 (75.8%), and dfra1 (46.5%) genes were observed in the antibiotic-resistant isolates, similar to a previous report in Germany (19). In addition, the tetracycline-resistant isolates were mediated mainly by teta (73.4%), consistent with other reports (1, 19). In this study, the mechanism for kanamycin resistance in the Salmonella isolates was mainly encoded by the apha1-iab gene. However, this gene was infrequent in the resistant strains of Salmonella Infantis (23). Resistance of Salmonella isolates to nalidixic acid (19.5%) in this study was consistent with the findings of Van et al. (25). However, this level was lower than that in previous reports in Vietnam (27) and other Asian countries (3, 28). The low levels of resistance to drugs of the fluoroquinolone group (norfloxacin and ciprofloxacin) and third-generation cephalosporins (ceftazidime) were similar to those in previous reports in Vietnam (25, 27) and other countries (3, 29). To date, epidemiological data relating to PMQR genes and QRDRs in Salmonella have been limited in Vietnam. In this study, PMQR genes, such as qnra, qnrb, qnrs, qepa, and acc(69)-1b-cr, were not detected from 23 quinolone-resistant isolates (Table 3), consistent with a previous report from South Korea (17). However, PMQR genes were detected at very low rates from quinoloneresistant Salmonella isolates in France (4), Japan (2), and the United States (10). Substitutions in the codons of gyra, such as Gly 81, Ser 83, and Asp 87, were frequently observed in Salmonella isolates when the MICs of nalidixic acid were $128 ml/ml (8, 17). However, no substitutions at codons Gly 81 and Asp 87 in the QRDR of gyra were identified, but the single mutation at codon Ser 83 (Ser83Phe) was detected from 18 of 23 quinolone-resistant isolates in this study (Table 3). In addition, all of the quinolone-resistant isolates in this study showed multidrug resistance and carried multiple resistance genes. This was also frequently observed in the Salmonella isolates from foodstuffs in Vietnam (27) and other Asian countries (26, 28) and may result in failure in salmonellosis treatment in these countries. Our results showed a high prevalence of antibiotic resistance among Salmonella strains isolated from retail

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