Isolation and Characterization of Antimicrobial-Resistant Nontyphoidal

1348 Journal of Food Protection, Vol. 79, No. 8, 2016, Pages 1348–1354 doi:10.4315/0362-028X.JFP-15-564

Isolation and Characterization of Antimicrobial-Resistant Nontyphoidal Salmonella enterica Serovars from Imported Food Products DONGRYEOUL BAE, OHGEW KWEON,

AND

ASHRAF A. KHAN*

U.S. Food and Drug Administration, National Center for Toxicological Research, Division of Microbiology, Jefferson, Arkansas 72079, USA MS 15-564: Received 7 December 2015/Accepted 22 March 2016

ABSTRACT The objective of this study was to determine antimicrobial resistance and elucidate the resistance mechanism in nontyphoidal Salmonella enterica serovars isolated from food products imported into the United States from 2011 to 2013. Food products contaminated with antimicrobial-resistant nontyphoidal S. enterica were mainly imported from Taiwan, Indonesia, Vietnam, and China. PCR, DNA sequencing, and plasmid analyses were used to characterize antimicrobial resistance determinants. Twentythree of 110 S. enterica isolates were resistant to various antimicrobial classes, including b-lactam, aminoglycoside, phenicol, glycopeptide, sulfonamide, trimethoprim, and/or fluoroquinolone antimicrobial agents. Twelve of the isolates were multidrug resistant strains. Antimicrobial resistance determinants blaTEM-1, blaCTX-M-9, blaOXA-1, tetA, tetB, tetD, dfrA1, dfrV, dhfrI, dhfrXII, drf17, aadA1, aadA2, aadA5, orfC, qnrS, and mutations of gyrA and parC were detected in one or more antimicrobial-resistant nontyphoidal S. enterica strains. Plasmid profiles revealed that 12 of the 23 antimicrobial-resistant strains harbored plasmids with incompatibility groups IncFIB, IncHI1, IncI1, IncN, IncW, and IncX. Epidemiologic and antimicrobial resistance monitoring data combined with molecular characterization of antimicrobial resistance determinants in Salmonella strains isolated from imported food products may provide information that can be used to establish or implement food safety programs to improve public health. Key words: Antimicrobial resistance; Food; Plasmid; Salmonella enterica; Type IIA topoisomerase

Enhanced surveillance and investigation to determine the diversity and antimicrobial resistance of pathogens isolated from food-related environments is now required according to the U.S. Food and Drug Administration (FDA) Food Safety Modernization Act (33), which includes a rule for preventive controls for food facilities. The rule is to ensure food safety and security in the food supply. In spite of efforts to protect consumers from food products contaminated with pathogens, few studies have been conducted in the United States examining the detection rate, antimicrobial resistance, and molecular characterization of Salmonella enterica isolated from imported food products (2–5, 21, 22, 34, 35, 39). The importance of monitoring and detection of foodborne pathogens from imported food products has increased with rapidly expanding international trade in foods, including red meat, fish, shellfish, fruits, nuts, and vegetables (www.ers.usda.gov/media/563776/import_1. xls). According to the U.S. Department of Agriculture, approximately 16.8% of foods consumed in the United States during 2009 were imported. Between 2002 and 2005, imports constituted up to 79.1% of fish and shellfish products, 36.3% of fruits and nuts, 9.6% of red meat, and 9.3% of vegetables consumed in the United States (15). * Author for correspondence. Tel: 870-543-7601; Fax: 870-5437307; E-mail: [email protected].

Canada, Mexico, China, Chile, Thailand, Australia, Brazil, Indonesia, India, Vietnam, Costa Rica, Ecuador, Malaysia, Guatemala, the Philippines, and Bangladesh have been large-scale seafood, fruit, nut, and/or vegetable exporters to the United States (8). More than 2,500 S. enterica serovars have been reported (29). Nontyphoidal S. enterica (NTS) serovars mostly cause gastrointestinal infections via consumption of contaminated foods (10, 31, 37). Antimicrobial-resistant S. enterica strains have been isolated from various imported food products, mostly seafood and vegetables (38, 39). The FDA maintains a zero tolerance policy for Listeria and Salmonella in ready-to-eat foods. The prevalence (Supplementary Table 1 [S1]; all supplementary materials are available at https://app.box.com/s/ fm4uty7v5ur2mei6ziw2ymzu0lr16kax) and antimicrobial resistance (Table 1) of pathogens in imported food products were much higher than those in domestically produced food products (37). The Food Safety Modernization Act, which was signed into law by the U.S. Congress in 2011, has fundamentally changed the way food is regulated in the United States and abroad, and this law affects the entire supply chain from farm to fork. The law also provides the FDA with new enforcement authorities and new tools to hold imported foods to the same standards as required for domestic foods. Persistent

Senftenberg (E4) Virchow (C1) Senftenberg (E4) Senftenberg (E4) Newport (C2)

Senftenberg (E4) Frozen albacore, ground Senftenberg (E4) Frozen albacore steak Typhimurium (B) Frozen whole tilapia

Virchow (C1) Enteritidis (D1)

Litchfield (C2)

Give (E1)

Enteritidis (D1)

Newport (C2) Panama (D1) Enteritidis (D1) Infantis (C1) Bareilly (C1)

PSS_940 PSS_942 PSS_945 PSS_946 PSS_948

PSS_950 PSS_954 PSS_955

PSS_957 PSS_959

PSS_970

PSS_984

PSS_988

PSS_1006 PSS_1009 PSS_1011 PSS_1013 PSS_1018

Frozen raw barramundi fillets Frozen black pomfret Cumin powder Dried parsley Frozen broadhead fish

White pepper powder

Ginger powder

Frozen farm-raised perch

Frozen largemouth bass Szechuan pepper

2013 2013 2013 2013 2013

2013

2012

2012

2012 2012

2012 2012 2012

Frozen yellowfin steak 2012 Crocodile striploin meat and tail fillet 2012 Frozen albacore 2012 Frozen albacore 2012 Frozen whole snakehead 2012

2012

2011 2011 2011 2011

Year

Taiwan Vietnam Pakistan China Vietnam

Taiwan

Vietnam

Taiwan

Taiwan China

Indonesia Indonesia Taiwan

Indonesia Australia Indonesia Indonesia Vietnam

Vietnam

Indonesia Taiwan Thailand China

Country

50, ,8 76, 60, 38

þ þ

blaTEM-1, tetA

ND ND ND þ þ

ND ND ND 9, ,8 10, ,8

90

ND þ ND

ND ND 55, 35, 13, 11, 9, ,8 10, 9, ,8 50, 38

þ þ þ ND ND blaTEM-1, blaOXA-1, tetD, qnrS tetA blaTEM-1, dfr17, aadA5 blaTEM-1

ND ND ND ND 24

110, 95

þ ND þ þ þ þ

55, 30 ND ND 150, 85

NDb þ ND þ

blaTEM-1 tetD, dhfrXII, aadA2 ND blaTEM-1, blaCTX-M-9, tetA blaTEM-1, blaCTX-M-9, tetA, dhfrI, aadA1 ND tetA, dfrV dfrA1, orfC ND blaTEM-1, tetA, qnrS

Plasmid size (kb)

Class I integrase

Antimicrobial resistance determinants

AMP, CHL, GEN, KAN, NAL, SXT, blaTEM-1, dhfrXII, TET aadA2 TET tetB TET tetB STR, TET tetA GEN, NAL, SXT, TET tetA, aadA1 CHL, STR, SXT, TET tetB, dfrA1, orfC

AMP, CHL, GEN, KAN, NAL, SXT AMP, CHL, GEN, NAL, SXT, TET

SXT SXT, TET SXT SXT AMP, CHL, CIP, KAN, NAL, STR, SXT, TET SXT SXT AMP, CHL, CIP, GEN, KAN, NAL, SXT, TET STR, TET AMP, NAL, SXT

AMP, NAL, SXT, TET CHL, SXT, TET NAL AMP, CHL, CIP, GEN, KAN, NAL, STR, SXT, TET AMP, NAL, SXT, TET

Antimicrobial resistancea

NA NA NA IncHI1 IncHI1

IncHI1, X IncI1, N

IncI1

NA NA

NA NA IncI1

NA IncW, N IncW IncFIIB IncN

NA

NAc NA NA NA

Replicon type

AMP, ampicillin; NAL, nalidixic acid; SXT, trimethoprim-sulfamethoxazole; TET, tetracycline; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; STR, streptomycin. Resistance was evaluated by both antibiotic diffusion disk and broth microdilution assays. b ND, not detected. c NA, replicon was not amplified with the primers used in this study.

a

Weltevreden (E1) Frozen whole blue anchovy

PSS_928

octopus farm-raised tilapia yellowstripe trevally tilapia

PSS_903 PSS_909 PSS_912 PSS_913

Frozen Frozen Frozen Frozen

Enteritidis (D1) London (E1) Braenderup (C1) Indiana (A)

ID

Food product

Salmonella serotype (serogroup)

TABLE 1. Description and molecular characterization of nontyphoidal Salmonella enterica strains from imported food products

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monitoring and characterization of pathogens in imported food products are necessary to enhance food safety and public health. Monitoring and characterization of antimicrobial resistance in enteric bacteria from humans, animals, and foods in the United States have been conducted through the National Antimicrobial Resistance Monitoring System (NARMS) since 1996 (11). Among enteric bacteria, NTS strains have been considered the most common foodborne causes of gastroenteritis, enteric fever, diarrhea, and bacteremia, resulting in more than 23,000 hospitalizations and 450 deaths per year in the United States (10, 13). Serotyping of NTS isolates is important for monitoring and tracking of these foodborne pathogens because only some NTS serovars have been commonly associated with outbreaks in the United States (10). According to the Centers for Disease Control and Prevention (9), the most prevalent Salmonella serovars associated with the 45,828 laboratory-confirmed NTS infection cases recorded in 2011 were Enteritidis (17%), Typhimurium (13%), Newport (11%), and Javiana (6%). The most common serovar, Salmonella Enteritidis, has been dominant in recent cases of human salmonellosis in the United States (10). Although most NTS infections cause mild symptoms, severe systemic infections, such as bacteremia or meningitis, can occur in immunocompromised persons (17). Fluoroquinolones (FQs) (e.g., ciprofloxacin) and expanded spectrum cephalosporins (e.g., ceftriaxone) are currently used to treat severe systemic infections caused by NTS strains (18). NTS strains resistant to three or more classes of antibiotics have been reported (9), and the prevalence of multiple antimicrobial-resistant (MAR) NTS strains increased between 2001 and 2011. The common MAR Salmonella strains included I,4,[5],12:i: (27%, 22 of 82 strains), Typhimurium (26%, 85 of 323), Heidelberg (30%, 21 of 70), Newport (3.9%, 11 of 285), Enteritidis (2.3%, 9 of 391), and Dublin (60%, 6 of 10) (9). Most MAR NTS strains that are highly resistant to various classes of antibiotics, especially extended-spectrum b-lactams, have become more prevalent in the United States (27). As a follow-up to our previous study (7), we report here epidemiologic information (source, detection year, and country) for 110 foodborne Salmonella isolates and molecular characterization data (serotype, antimicrobial resistance phenotype, antimicrobial resistance determinant, plasmid size, and replicon type) for 23 NTS strains resistant to antimicrobial agents.

MATERIALS AND METHODS Salmonella isolates. From 1 October 2011 through 31 July 2013, 110 NTS strains were isolated from a variety of imported food products (n ¼ 3,840) that included vegetables, fruits, meats, and seafood (Supplementary Table 1 [S1]). These strains were then characterized and serotyped. Table 1 shows the origin and serotypes of 23 NTS strains resistant to antimicrobial agents. The isolates were grown in Luria-Bertani (LB) broth (BD, Franklin Lakes, NJ). The Salmonella strains evaluated in this study were isolated, identified, and serotyped in the FDA Pacific Regional Laboratory–Southwest (Irvine, CA) according to the FDA Bacteriological Analytical Manual procedure (6).

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Antimicrobial susceptibility test. Antimicrobial susceptibility testing of the 110 isolates was performed using Mueller-Hinton agar plates and antibiotic diffusion assay disks (BD) containing ampicillin (10 lg/ml), amoxicillin with clavulanic acid (20 and 10 lg/ml), gentamicin (10 lg/ml), kanamycin (30 lg/ml), streptomycin (100 lg/ml), chloramphenicol (30 lg/ml), tetracycline (30 lg/ ml), nalidixic acid (30 lg/ml), ciprofloxacin (5 lg/ml), and trimethoprim-sulfamethoxazole (1.25 and 23.75 lg/ml) according to the procedures specified by the Clinical and Laboratory Standards Institute (12). The broth microdilution assay also was used to confirm resistance of NTS strains to antimicrobial agents. One hundred microliters of bacterial inoculum (about 1.0 3 106 CFU/ml) was added to 96-well microtiter plates containing 100 ll of LB broth with various concentrations of antimicrobial agents. The plates were incubated at 378C for 18 h. Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212 were used as quality control strains. Detection of plasmids and antimicrobial resistance genes and replicon typing. Twenty-three NTS food isolates resistant to antimicrobial agents were cultured overnight in LB broth. Genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA) following the manufacturer’s procedure, and plasmids were extracted (23). One and 5 ml of bacterial culture were used for genomic DNA and plasmid extraction, respectively. The plasmids of the 23 isolates were loaded on 0.8% agarose gels were run using a CHEF Mapper XA (Bio-Rad Laboratories, Hercules, CA) at 6 V/cm for 7 h. A supercoiled DNA ladder (BAC-Tracker, Epicentre Biotechnologies, Madison, WI) was used as a molecular marker. To determine genes conferring antimicrobial resistance, a PCR assay was performed with the genomic DNA and plasmids using primers specific to extended-spectrum or AmpC b-lactamase (ESBL), aminoglycosides, phenicols, trimethoprim, sulfonamides, tetracycline, and quinolones. The primer sequences are given in Supplementary Table 2 (S2). PCRamplified integrons and the type IIA topoisomerase (DNA gyrase and topoisomerase IV) genes also were sequenced. The antimicrobial resistance determinants from class I integrons and mutations in the quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC, and parE involved in quinolone resistance were identified by sequence analysis. Plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS, and aac(6 0 )Ib-cr were also identified. Replicon typing was conducted to classify plasmids (26). Primers specific to replicon types are also given in Supplementary Table 2 (S2).

RESULTS Distribution of NTS foodborne isolates resistant to antimicrobial agents. A total of 23 antimicrobial-resistant Salmonella strains were identified among the 110 NTS isolates recovered from various imported food products, including frozen seafood, freshwater fish, and dried herbs (Table 1). The Salmonella serovars identified were Senftenberg, Enteritidis, Newport, Virchow, London, Braenderup, Indiana, Weltevreden, Typhimurium, Litchfield, Give, Panama, Infantis, and Bareilly. Serogroups C and E were most common in this study (Supplementary Table 1 [S1]). Salmonella Senftenberg was the most frequently recovered of the 23 antimicrobial-resistant NTS strains, followed by Salmonella Enteritidis and Salmonella Newport (Table 1). The contaminated food products were mostly processed and imported into the United States from Southeast and East


ANTIMICROBIAL-RESISTANT S. ENTERICA FROM IMPORTED FOODS

Asian countries (Fig. 1A). As revealed in the tree map (Fig. 1B), antimicrobial-resistant NTS strains were predominantly found in products imported from Taiwan, Indonesia, Vietnam, and China (Table 1).

Antimicrobial susceptibility. The 23 NTS strains were resistant to a variety of antimicrobial agents, including ampicillin, gentamicin, kanamycin, streptomycin, chloramphenicol, tetracycline, ciprofloxacin, nalidixic acid, and trimethoprim-sulfamethoxazole (Table 1). According to the antimicrobial susceptibility data from both the disk diffusion agar and microdilution assays, resistance was most common to trimethoprim-sulfamethoxazole (18 of 110 NTS strains, 16.4%), tetracycline (13.6%), nalidixic acid (10.0%), ampicillin (8.2%), chloramphenicol (7.3%), gentamicin (5.5%), kanamycin (4.5%), streptomycin (4.5%), and ciprofloxacin (2.7%) (Table 1), but all strains were susceptible to amoxicillin with clavulanic acid (data not shown). Twelve (10.9%) of the antimicrobial-resistant strains were resistant to more than three classes of antimicrobial agents (Table 1). Strain PSS_913 was resistant to all antimicrobial agents tested except amoxicillin with clavulanic acid (Table 1). All isolates from foods imported from Indonesia and Vietnam were resistant to trimethoprimsulfamethoxazole and tetracycline, respectively (Table 1). In comparison, NTS strains isolated from foods from Thailand were less resistant to antimicrobial agents. Only one strain from Thailand, PSS_912, was resistant to nalidixic acid. Antimicrobial resistance determinants. As found in a previous study (7), blaTEM-1, blaCTX-M-9, and blaOXA-1, which confer b-lactam resistance, were detected from ampicillin-resistant strains. The aadA1, aadA2, and aadA5 genes involved in aminoglycoside (gentamicin, kanamycin, and streptomycin) resistance were detected in NTS strains PSS_909, PSS_928, PSS_959, PSS_988, and PSS_1013 (Table 1). Strains resistant to chloramphenicol were negative for catI, cmlA, and floR genes. NTS strains PSS_909, PSS_928, PSS_942, PSS_945, PSS_959, and PSS_988, which were resistant to trimethoprim-sulfamethoxazole, were positive for dfrA1, dfrV, dfr17, dhfrI, and dhfrXII genes (Table 1). Gene tetA, tetB, or tetD was detected in all tetracycline-resistant NTS strains (Table 1). For quinolone resistance, mutations of the type IIA topoisomerase genes (gyrA, gyrB, parC, and parE) were determined by DNA sequencing, and PMQR genes were identified by PCR. Among the 23 antimicrobial-resistant NTS strains, PSS_913 (one of the quinolone-resistant strains) had high levels of resistance to ciprofloxacin (MIC . 64) and nalidixic acid (MIC . 128). Sequence analysis of gyrA, gyrB, parC, and parE revealed that strain PSS_913 had a double mutation in gyrA (S83F and D87N) and parC (S80R and T57S) but no amino acid substitution in GyrB or ParE. In the primary and secondary structures, all the mutations were located in the QRDRs of gyrA (data not shown) and parC (Supplementary Fig. 1 [S3]), which are highly conserved mutational spots responsible for high levels of FQ resistance. As revealed in the tertiary and quaternary structures, the three residues, Ser-83 and Asp-87

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in GyrA and Ser-80 in ParC, are located within the a helices in the DNA-FQ–binding sites of the complexes (Supplementary Fig. 1 [S3] for ParCE), whereas the residue Thr-57 on a loop in ParC is positioned far away from the bound ˚ DNA and FQ with an approximate distance of ~30 A (Supplementary Figure 1 [S3]). However, no PMQR genes (qnrA, qnrB, qnrS, and aac(6 0 )Ib-cr) were detected in PSS_913. In contrast, two FQ-sensitive strains, PSS_948 and PSS_955, harbored qnrS. In strain PSS_948, substitution of Thr-57 to Ser in ParC was also identified. Fifteen of the 23 antimicrobial-resistant strains harbored class I integrase (Table 1). Aminoglycoside resistance genes (aadA1, aadA2, and aadA5) and trimethoprim resistance genes (dfrA1, dfrV, dfr17, dhfrI, and dhfrXII) were identified in class I integrons in NTS strains harboring the class I integrase gene. Plasmid profiles revealed that the plasmid band patterns of 12 isolates containing plasmids were different (Table 1). The isolates had different sizes of plasmids from ,8 to 150 kb. When the plasmids were classified using the PCR-based replicon assay, 10 isolates with IncI1, IncFIIB, IncHI1, IncN, IncW, and/or IncX replicon types were identified (Table 1).

DISCUSSION The detection rate of antimicrobial-resistant NTS strains revealed that Salmonella Senftenberg was the most commonly isolated (5 of 23 NTS strains) followed by Salmonella Enteritidis (4 of 23). In a recent study (10), Salmonella Enteritidis was the most common serovar among food isolates. In the present study, all Salmonella Senftenberg strains, which were recovered from tuna products with high frequency (77.8%), were found among products exported from Indonesia and were resistant to trimethoprim-sulfamethoxazole (Supplementary Table 1 [S1]). Recently, frozen raw tuna contaminated with Salmonella Paratyphi B was linked to multistate outbreaks in the United States (32), and the FDA determined that the contaminated tuna was processed in Indonesia. Therefore, enhanced food safety programs for preventive controls in the food processing environment are consistently required. In this study, resistance to trimethoprim-sulfamethoxazole (16.4%, 18 of 110 NTS isolates), tetracycline (13.6%), nalidixic acid (9.1%), ampicillin (8.2%), and ciprofloxacin (0.9%) was relatively high compared with the NARMS data (11), in which the respective resistances were 1.4, 12.6, 2.8, 10.4, and 0.5%. The differences between these data sets may reflect a difference in the prevalent serotype among the NTS strains. Although the most prevalent Salmonella serovars in the present study were Weltevreden (19.1%, 21 of 110 isolates), Newport (8.2%), Senftenberg (8.2%), Virchow (6.4%), Enteritidis (5.5%), Bareilly (4.5%), and Typhimurium (4.5%), the most common serovars in the NARMS data set (9) were Enteritidis (16.5%), Typhimurium (13.4%), Newport (11.4%), and Javiana (6.4%). In that data set, 17.5% of Salmonella Enteritidis isolates and 14.9% of Salmonella Typhimurium isolates were resistant to antimicrobial agents (11), whereas in the present study only one Salmonella Weltevreden strain (0.9%) was resistant to antimicrobial agents (Table 1). In the present study, 12

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FIGURE 1. World map showing the countries exporting NTS-contaminated food to the United States from 2011 through 2013 (A) and a treemap representing the exporting countries (rectangles) sized by the number of NTS isolates and colored by the number of antimicrobial-resistant NTS isolates (B). Antimicrobial-resistant NTS strains were predominantly found in food products from Vietnam, Indonesia, Thailand, Philippines, Taiwan, and China.

strains (10.9%) were resistant to at least three different classes of antibiotics. The rate of NTS MAR strains (10.9%) is consistent with the NARMS data (10.0%) (11). Recently, we reported (7) that 2 of the same set of 110 NTS strains, PSS_913 and PSS_988 (1.8%), were highly resistant to b-lactams, including ampicillin and different generations of cephalosporins. In addition to b-lactam– resistant NTS strains, strains PSS_913, PSS_948, and PSS_955 were also resistant to quinolones or/and FQs (Table 1). Conjugation data revealed that b-lactam resistance in these strains was genotypically and phenotypically transferred to E. coli strain J53 (7), but quinolone resistance was not. The emergence of ESBL-producing NTS strains, harboring conjugative plasmid-borne ESBL genes, may threaten human and animal health through the global spread of ESBL-producing NTS strains and acquired resistance to b-lactams (30). All of the ampicillin resistant NTS strains in the present study were MAR strains. Similar antimicrobial resistance phenotype patterns were found in a previous study

(1). MAR was mediated by a plasmid harboring multiple antimicrobial resistance genes (1, 24). The results of these studies and the present study suggest that the plasmid harboring the antimicrobial resistance gene has played a major role in the dissemination of MAR. Some plasmids were not typeable by the PCR method we used (Table 1). For these unclassified plasmids, further study of new plasmid types is required regarding MAR in NTS strains (16, 24). The widespread heavy use of FQs has resulted in the rapid emergence of FQ resistance worldwide. Two main recognized resistance mechanisms, (i) chromosomal mutations in the genes responsible for topological changes of cellular DNA and transportation and (ii) PMQR determinants, can work either alone or together for a high level of quinolone resistance (19, 28). In FQ-resistant strain PSS_913 with no contribution of PMQR, the mutational combination of S83F and D87N in GyrA and S80R and T57S in ParC might be directly associated with the high


ANTIMICROBIAL-RESISTANT S. ENTERICA FROM IMPORTED FOODS

MIC of FQ. Amino acid substitutions of Ser-83 and Asp-87 in the QRDR of GyrA have been reported to play a vital role in increased resistance to quinolones (20, 28, 36). In ParC, a substitution to Arg at Ser-80 in the QRDR is also frequently observed with a high level of quinolone resistance (14, 25, 28). Introduction of hydrophobic bulky side chains on the ahelices in the DNA-FQ binding sites of GyrA and ParC seems to be essential to directly reduce the affinity of quinolone antibiotics for the enzyme-DNA complex. In this respect, when considering the structural location and no nucleophilic functional group change upon mutation, the mutation T57S in ParC in strain PSS_913, which has another mutation S80R in ParC, could have less of an epistatic impact on the binding of DNA and FQs and on the structural dimerization of ParC. Although the mutation T57S in ParC has been frequently observed, the precise structural and functional nature of this mutation has not been determined (14, 19, 25). Despite having qnrS and a mutation of T57S in ParC, strain PSS_948 was sensitive to ciprofloxacin. This observation further supports the idea that PMQR leads to a relatively low level of FQ resistance and that the mutation T57S in ParC leads to insignificant structural and functional changes (14, 19, 25, 28). In summary, 110 diverse NTS serovars were recovered from 3,840 imported food samples; 23 of the 110 isolates were resistant to antimicrobial agents, and the antimicrobial resistance determinants were identified. Twelve of the 23 antimicrobial-resistant strains were resistant to multiple antimicrobial agents. This study may provide a better understanding of the mechanism of FQ resistance in foodborne NTS isolates. Our monitoring data combined with molecular characterization of antimicrobial resistance determinants in Salmonella isolates from imported food products may contribute significantly to improvements in food safety and public health via surveillance and antimicrobial resistance monitoring systems of foodborne pathogens in a variety of animal, plant, and fish or fishery food products.

ACKNOWLEDGMENTS We thank Drs. Carl E. Cerniglia, John B. Sutherland, and M. S. Nawaz for critical review of the manuscript, Christine Summage-West for technical help, and Gwendolyn Anderson and Stephanie Horton for the Salmonella isolates. This research was supported in part by an appointment to the Research Participation Program at the National Center for Toxicological Research administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and the U.S. Food and Drug Administration. The views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration.

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