MECHANISMS OF RESISTANCE
crossm Identification of Plasmid-Mediated Quinolone Resistance in Salmonella Isolated from Swine Ceca and Retail Pork Chops in the United States Gregory H. Tyson,a Heather P. Tate,a Shaohua Zhao,a Cong Li,a Uday Dessai,b Mustafa Simmons,c Patrick F. McDermotta U.S. Food and Drug Administration, Center for Veterinary Medicine, Office of Research, Laurel, Maryland, USAa; U.S. Department of Agriculture, Food Safety and Inspection Service, Office of Public Health Science, Washington, DC, USAb; U.S. Department of Agriculture, Food Safety and Inspection Service, Office of Public Health Science, Athens, Georgia, USAc
ABSTRACT Fluoroquinolones are important antimicrobial drugs used to treat human Salmonella infections, and resistance is rare in the United States for isolates from human and animal sources. Recently, a number of Salmonella isolates from swine cecal contents and retail pork products from National Antimicrobial Resistance Monitoring System (NARMS) surveillance exhibited decreased susceptibility to ciprofloxacin. We identified two qnrB19 quinolone resistance plasmids that are predominantly responsible for this phenomenon and found them distributed among several Salmonella serotypes isolated throughout the United States. KEYWORDS antimicrobial resistance, fluoroquinolones, PMQR, Salmonella
A
ntimicrobial resistance in foodborne pathogens is a significant threat to public health. This is particularly true with nontyphoidal Salmonella, the most common bacterial foodborne pathogen in the United States (1). The Centers for Disease Control and Prevention (CDC) deemed Salmonella resistance to fluoroquinolones to be a serious threat to public health (2), and the Food and Drug Administration (FDA) Guidance for Industry 152 identified fluoroquinolones as critically important drugs for human health (3). Therefore, any findings of decreased susceptibility to fluoroquinolones or identification of new genetic determinants conferring fluoroquinolone resistance in Salmonella may be a public health concern. Gram-negative infections with decreased susceptibility to fluoroquinolones are also associated with more persistent infections and longer hospital stays (4). Historically, mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC have been the most common mediators of quinolone resistance in Gram-negative bacteria (5). Although these mutations can impede the ability to treat infections, they are not typically horizontally transmissible, thus limiting the rate and range of resistance spread in bacterial populations. In recent years, however, plasmidmediated quinolone resistance (PMQR) has become a threat to the effective therapeutic use of quinolones (6). The quinolone resistance qnr genes identified from plasmids include qnrA, qnrB, qnrS, qnrD, and qnrS (7), and the presence of these resistance determinants among Salmonella isolates is thought to be rare in the United States (8). Using broth microdilution in vitro susceptibility methods, the National Antimicrobial Resistance Monitoring System (NARMS) screens Salmonella isolated from food-producing animals, retail meats, and human patients for resistance to 14 antimicrobials (9). Recently, NARMS observed an increase in Salmonella nonsusceptibility to ciprofloxacin (MIC, ⱖ0.12 g/ml, according to CLSI breakpoints) among isolates collected from swine October 2017 Volume 61 Issue 10 e01318-17
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Received 28 June 2017 Returned for modification 25 July 2017 Accepted 31 July 2017 Accepted manuscript posted online 7 August 2017 Citation Tyson GH, Tate HP, Zhao S, Li C, Dessai U, Simmons M, McDermott PF. 2017. Identification of plasmid-mediated quinolone resistance in Salmonella isolated from swine ceca and retail pork chops in the United States. Antimicrob Agents Chemother 61:e01318-17. https://doi.org/10.1128/AAC.01318-17. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply. Address correspondence to Gregory H. Tyson, [email protected].
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TABLE 1 List of ciprofloxacin-nonsusceptible isolates from porcine sources Strain N54262 N56916F N56949F N56967F N57007F N57010F N57229F N57607F N57811F N57952F N58036 N58621F N45337F N44358F N44364F N47616F N48215F N48456F N49300F N49382F N49614F N57076F N57698F N57957F N44134F N50969F N57645F N48323F N47630F N52019
Accession no. SRR2407688 SRR3295634 SRR3295644 SRR3295650 SRR3295659 SRR3295661 SRR3295726 SRR3295834 SRR3295907 SRR3295950 SRR3295618 SRR3295964 SRR5235473 SRR5235471 SRR5235472 SRR5235474 SRR5235476 SRR5235478 SRR5235479 SRR5235480 SRR5235481 SRR3295681 SRR3295870 SRR3295953 SRR5235470 SRR5235482 SRR3295850 SRR5235477 SRR5235475 SRR2407540
Serotype Derby Derby Anatum Anatum Anatum Anatum London Adelaide Derby Anatum Derby I 4,[5],12:i:Typhimurium Muenchen Rissen Muenchen Muenchen Muenchen Muenster Muenchen Muenchen Muenchen Muenchen Brandenburg Heidelberg Give Derby Senftenberg Rough/nonmotile Derby
Yr 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2015 2014 2013 2013 2013 2013 2013 2013 2013 2013 2013 2014 2014 2014 2013 2013 2014 2013 2013 2014
Source Pork chops Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Pork chops Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Pork chops
State NM WI IL CA PA CA NE SD KS PA WA SC NE NC NC IL KY TX OH VA TN MS IA KS CA TX TN MO CO
Quinolone genotype qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b; GyrA (D87N); ParC(S80I) qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c GyrA (D87N) GyrA (D87N) GyrA (S83F) qnrB6; aac(6=)Ib-cr qnrS2 qnrS2
CIP MICa (g/ml) 0.5 0.5 0.5 0.5 1 0.5 0.5 1 0.5 0.5 0.5 0.5 ⬎4 0.25 0.5 0.5 0.5 0.25 0.5 0.5 0.5 0.5 0.5 1 0.5 0.25 0.25 4 2 2
aCIP,
ciprofloxacin. (3,071 bp). cPlasmid (2,699 bp). bPlasmid
sources. In 2013, the first year of swine cecal sampling at slaughter by the Food Safety and Inspection Service (FSIS), 2.7% (15/552) of Salmonella isolates were nonsusceptible to ciprofloxacin (9); in 2014, ciprofloxacin nonsusceptibility was 3.8% (23/603). Among the NARMS retail pork chop samples, 3 (7.7%) of 39 Salmonella isolates from 2014 to 2015 were nonsusceptible to ciprofloxacin, whereas all of the 176 Salmonella isolates from 2002 to 2013 were susceptible. A direct programmatic and temporal comparison cannot be made between cecal and retail samples. However, these observations may collectively point toward a potential increase in ciprofloxacin nonsusceptibility. To further understand the ciprofloxacin nonsusceptibility phenomenon, 30 of the ciprofloxacin-nonsusceptible isolates from swine cecal and retail pork chop samples collected in 2013 to 2015 were sequenced on the Illumina MiSeq system using v3 reagent kits. Sequences were assembled de novo using CLC Genomics Workbench version 9.0.1 and were analyzed for mutations in the QRDRs of gyrA, gyrB, parC, and parE and for the presence of PMQR genes in the ResFinder database, including qnr genes, aac(6=)-Ib-cr, qepA, and oqxAB (10). Of the 30 sequenced isolates, 27 had PMQR genes and 24 had qnrB19 genes, with one of the latter isolates having additional gyrA and parC mutations (Table 1). Two isolates had qnrS2 genes—the first known report of this gene in Salmonella in the United States. A Salmonella Senftenberg isolate from cecal contents had both aac(6=)Ib-cr and qnrB6, a combination that is known to substantially increase the ciprofloxacin MIC (Table 1) (11). Presence of the PMQR gene aac(6=)-Ib-cr in a Salmonella isolate from any animal source in the United States is also a novel finding. qepA and oqxAB were not identified in any isolates. The MICs and the National Center for Biotechnology Information (NCBI) sequence database (GenBank) accession numbers for all strains used in this study are shown in Table 1. Note that of all the retail pork isolates sequenced in 2002 to 2013 (176 total), none had fluoroquinOctober 2017 Volume 61 Issue 10 e01318-17
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FIG 1 Geographic distribution of two qnrB19-containing plasmids in the United States. Isolates in New Mexico and Washington are from pork chop samples collected in local grocery stores, and the remaining isolates are from swine ceca sampled at slaughter plants. These geographic distributions do not necessarily reflect where animals were raised.
olone resistance mechanisms. As the annual rate of retail pork sampling has not changed since 2013, the presence of PMQR genes in Salmonella isolates from U.S. swine sources may be an emerging phenomenon of recent origin. The contigs containing the qnrB19 genes were of very high coverage, and by mapping reads back to the contigs, we were able to close each of the plasmid sequences, confirming that all 24 of the qnrB19-containing sequences were on ColEtype plasmids. Thirteen of these were identical 3,071-bp plasmids, and the remaining 11 were identical 2,699-bp plasmids. Using nucleotide BLAST analysis, we found that the smaller of the two plasmids was identical to ColE-type plasmids that were identified in Escherichia coli isolates from Peru (12) and Salmonella isolates from Argentina and Germany (13, 14). The larger plasmid contained only two single nucleotide polymorphisms (SNPs) relative to an E. coli plasmid from Bolivia (12), the only BLAST hit in GenBank within 10 SNPs. Although several larger qnrB19-containing conjugative plasmids have been described among Enterobacteriaceae (15, 16), these ColE-type plasmids do not share extensive homology with them, demonstrating a potentially distinct path of PMQR dissemination. The 3,071-bp plasmid and the 2,699-bp plasmid share ⬃2,300 bp of alignment and, aside from the qnrB19 and plasmid maintenance genes, contain relatively few additional genes. As mentioned earlier, these two plasmids were identified in South American E. coli isolates and have been compared and described extensively (12). Isolates containing the plasmids were diverse, with six different Salmonella serotypes possessing the 3,071-bp plasmid and four different serotypes containing the 2,699-bp plasmid, the most common serotypes being Muenchen, Anatum, and Derby (Table 1). Although geographic inferences cannot be made directly because of the complexity associated with animal production to slaughter practices and varied product distribution, note that each of the two plasmids was isolated from swine cecal contents or retail pork samples in at least nine states (Fig. 1). Despite the diversity of isolates with these plasmids, all copies of the two plasmids were clonal, as determined by BLAST analysis, potentially pointing to a common source of dissemination. The reason underlying the evident increase in PMQRs in swine is not clear, as there is no known factor selecting for increased resistance. The fluoroquinolone drug enrofloxacin October 2017 Volume 61 Issue 10 e01318-17
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is approved for use in swine to treat and control colibacillosis and swine respiratory disease (https://animaldrugsatfda.fda.gov), but extralabel use is prohibited. In summary, we identified the first PMQR Salmonella from swine sources in the United States, primarily in two clonal qnrB19-containing plasmids. On average, ⬃47% of swine cecal samples in the United States are positive for Salmonella (9), leading to the potential for increased fluoroquinolone nonsusceptibility in Salmonella in food and the environment through horizontal transmission of qnrB19 plasmids. Although Salmonella contamination of retail pork remains below 5% (9), the presence of strains with these plasmids in retail meat products is of particular concern due to the potential for spread in bacterial populations in ecogeographic niches, resulting in elevated consumer exposure through contaminated food products. Additional work is needed to determine whether Salmonella isolates with these qnrB19-containing plasmids have been transmitted to humans through the U.S. food supply to result in human infections; the NARMS surveillance program will continue to monitor qnr gene dissemination through phenotypic and genotypic approaches. The recent findings of IncR-type qnrB19 plasmids in dairy cattle from Texas along with our data from swine sources highlight the importance of gene-level resistance surveillance (17), and these results demonstrate the need to monitor the movement of resistant strains and genes through the farm-to-fork continuum to further strengthen food safety and public health. Accession number(s). We submitted the 3,071-bp and 2,699-bp plasmid sequences to GenBank, with accession numbers KY991369 and KY991368, respectively. ACKNOWLEDGMENTS We acknowledge the work of FSIS OPHS Eastern Laboratory Microbiology Screening Branch and Microbiology Characterization Branch in the isolation and characterization of NARMS cecal isolates and the FSIS OPHS Applied Epidemiology staff for their support. We also acknowledge Claudia Lam of FDA CVM for submitting the sequence data to GenBank. The views expressed in this article are those of the authors and do not necessarily reflect the official policy of the Department of Health and Human Services, the U.S. Food and Drug Administration, or the U.S. Government. Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
REFERENCES 1. Huang JY, Henao OL, Griffin PM, Vugia DJ, Cronquist AB, Hurd S, TobinD’Angelo M, Ryan P, Smith K, Lathrop S, Zansky S, Cieslak PR, Dunn J, Holt KG, Wolpert BJ, Patrick ME. 2016. Infection with pathogens transmitted commonly through food and the effect of increasing use of culture-independent diagnostic tests on surveillance–foodborne diseases active surveillance network, 10 U.S. sites, 2012-2015. MMWR Morb Mortal Wkly Rep 65:368 –371. https://doi.org/10.15585/mmwr .mm6514a2. 2. Centers for Disease Control and Prevention. 2013. Antibiotic resistance threats in the United States, 2013. https://www.cdc.gov/drugresistance/ threat-report-2013/index.html. 3. U.S. Food and Drug Administration. 2003. Guidance for industry 152: evaluating the safety of antimicrobial new animal drugs with regard to their microbiological effects on bacteria of human health concern. https://www .fda.gov/downloads/AnimalVeterinary/GuidanceComplianceEnforcement/ GuidanceforIndustry/UCM052519.pdf. 4. Defife R, Scheetz MH, Feinglass JM, Postelnick MJ, Scarsi KK. 2009. Effect of differences in MIC values on clinical outcomes in patients with bloodstream infections caused by Gram-negative organisms treated with levofloxacin. Antimicrob Agents Chemother 53:1074–1079. https://doi.org/10.1128/AAC .00580-08. 5. Reyna F, Huesca M, Gonzalez V, Fuchs LY. 1995. Salmonella Typhimurium gyrA mutations associated with fluoroquinolone resistance. Antimicrob Agents Chemother 39:1621–1623. https://doi.org/10.1128/AAC.39.7 .1621. 6. Rodriguez-Martinez JM, Machuca J, Cano ME, Calvo J, Martinez-Martinez October 2017 Volume 61 Issue 10 e01318-17
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L, Pascual A. 2016. Plasmid-mediated quinolone resistance: two decades on. Drug Resist Updat 29:13–29. https://doi.org/10.1016/j.drup.2016.09 .001. Strahilevitz J, Jacoby GA, Hooper DC, Robicsek A. 2009. Plasmidmediated quinolone resistance: a multifaceted threat. Clin Microbiol Rev 22:664 – 689. https://doi.org/10.1128/CMR.00016-09. Sjolund-Karlsson M, Howie R, Rickert R, Krueger A, Tran TT, Zhao S, Ball T, Haro J, Pecic G, Joyce K, Fedorka-Cray PJ, Whichard JM, McDermott PF. 2010. Plasmid-mediated quinolone resistance among non-Typhi Salmonella enterica isolates, USA. Emerg Infect Dis 16:1789 –1791. https://doi .org/10.3201/eid1611.100464. U.S. Food and Drug Administration. 2016. 2014 NARMS integrated report. The National Antimicrobial Resistance Monitoring System for enteric bacteria (NARMS). U.S. Food and Drug Administration, Rockville, MD. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, Aarestrup FM, Larsen MV. 2012. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 67:2640 –2644. https://doi .org/10.1093/jac/dks261. Robicsek A, Strahilevitz J, Jacoby GA, Macielag M, Abbanat D, Park CH, Bush K, Hooper DC. 2006. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 12:83– 88. https://doi.org/10.1038/nm1347. Pallecchi L, Riccobono E, Sennati S, Mantella A, Bartalesi F, Trigoso C, Gotuzzo E, Bartoloni A, Rossolini GM. 2010. Characterization of small ColE-like plasmids mediating widespread dissemination of the qnrB19 aac.asm.org 4
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gene in commensal enterobacteria. Antimicrob Agents Chemother 54: 678 – 682. https://doi.org/10.1128/AAC.01160-09. 13. Hammerl JA, Beutlich J, Hertwig S, Mevius D, Threlfall EJ, Helmuth R, Guerra B. 2010. pSGI15, a small ColE-like qnrB19 plasmid of a Salmonella enterica serovar Typhimurium strain carrying Salmonella genomic island 1 (SGI1). J Antimicrob Chemother 65:173–175. https://doi.org/10.1093/ jac/dkp383. 14. Tran T, Andres P, Petroni A, Soler-Bistue A, Albornoz E, Zorreguieta A, Reyes-Lamothe R, Sherratt DJ, Corso A, Tolmasky ME. 2012. Small plasmids harboring qnrB19: a model for plasmid evolution mediated by site-specific recombination at oriT and Xer sites. Antimicrob Agents Chemother 56:1821–1827. https://doi.org/10.1128/AAC.06036-11. 15. Schink AK, Kadlec K, Schwarz S. 2012. Detection of qnr genes among
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Escherichia coli isolates of animal origin and complete sequence of the conjugative qnrB19-carrying plasmid pQNR2078. J Antimicrob Chemother 67:1099 –1102. https://doi.org/10.1093/jac/dks024. 16. Hordijk J, Bosman AB, van Essen-Zandbergen A, Veldman K, Dierikx C, Wagenaar JA, Mevius D. 2011. qnrB19 gene bracketed by IS26 on a 40-kilobase IncR plasmid from an Escherichia coli isolate from a veal calf. Antimicrob Agents Chemother 55:453– 454. https://doi.org/10.1128/AAC .00866-10. 17. Cummings KJ, Rodriguez-Rivera LD, Norman KN, Ohta N, Scott HM. 2016. Identification of a plasmid-mediated quinolone resistance gene in Salmonella isolates from Texas dairy farm environmental samples. Zoonoses Public Health 64:305–307. https://doi.org/10.1111/zph .12318.
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crossm Identification of Plasmid-Mediated Quinolone Resistance in Salmonella Isolated from Swine Ceca and Retail Pork Chops in the United States Gregory H. Tyson,a Heather P. Tate,a Shaohua Zhao,a Cong Li,a Uday Dessai,b Mustafa Simmons,c Patrick F. McDermotta U.S. Food and Drug Administration, Center for Veterinary Medicine, Office of Research, Laurel, Maryland, USAa; U.S. Department of Agriculture, Food Safety and Inspection Service, Office of Public Health Science, Washington, DC, USAb; U.S. Department of Agriculture, Food Safety and Inspection Service, Office of Public Health Science, Athens, Georgia, USAc
ABSTRACT Fluoroquinolones are important antimicrobial drugs used to treat human Salmonella infections, and resistance is rare in the United States for isolates from human and animal sources. Recently, a number of Salmonella isolates from swine cecal contents and retail pork products from National Antimicrobial Resistance Monitoring System (NARMS) surveillance exhibited decreased susceptibility to ciprofloxacin. We identified two qnrB19 quinolone resistance plasmids that are predominantly responsible for this phenomenon and found them distributed among several Salmonella serotypes isolated throughout the United States. KEYWORDS antimicrobial resistance, fluoroquinolones, PMQR, Salmonella
A
ntimicrobial resistance in foodborne pathogens is a significant threat to public health. This is particularly true with nontyphoidal Salmonella, the most common bacterial foodborne pathogen in the United States (1). The Centers for Disease Control and Prevention (CDC) deemed Salmonella resistance to fluoroquinolones to be a serious threat to public health (2), and the Food and Drug Administration (FDA) Guidance for Industry 152 identified fluoroquinolones as critically important drugs for human health (3). Therefore, any findings of decreased susceptibility to fluoroquinolones or identification of new genetic determinants conferring fluoroquinolone resistance in Salmonella may be a public health concern. Gram-negative infections with decreased susceptibility to fluoroquinolones are also associated with more persistent infections and longer hospital stays (4). Historically, mutations in the quinolone resistance-determining regions (QRDRs) of gyrA and parC have been the most common mediators of quinolone resistance in Gram-negative bacteria (5). Although these mutations can impede the ability to treat infections, they are not typically horizontally transmissible, thus limiting the rate and range of resistance spread in bacterial populations. In recent years, however, plasmidmediated quinolone resistance (PMQR) has become a threat to the effective therapeutic use of quinolones (6). The quinolone resistance qnr genes identified from plasmids include qnrA, qnrB, qnrS, qnrD, and qnrS (7), and the presence of these resistance determinants among Salmonella isolates is thought to be rare in the United States (8). Using broth microdilution in vitro susceptibility methods, the National Antimicrobial Resistance Monitoring System (NARMS) screens Salmonella isolated from food-producing animals, retail meats, and human patients for resistance to 14 antimicrobials (9). Recently, NARMS observed an increase in Salmonella nonsusceptibility to ciprofloxacin (MIC, ⱖ0.12 g/ml, according to CLSI breakpoints) among isolates collected from swine October 2017 Volume 61 Issue 10 e01318-17
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Received 28 June 2017 Returned for modification 25 July 2017 Accepted 31 July 2017 Accepted manuscript posted online 7 August 2017 Citation Tyson GH, Tate HP, Zhao S, Li C, Dessai U, Simmons M, McDermott PF. 2017. Identification of plasmid-mediated quinolone resistance in Salmonella isolated from swine ceca and retail pork chops in the United States. Antimicrob Agents Chemother 61:e01318-17. https://doi.org/10.1128/AAC.01318-17. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply. Address correspondence to Gregory H. Tyson, [email protected].
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TABLE 1 List of ciprofloxacin-nonsusceptible isolates from porcine sources Strain N54262 N56916F N56949F N56967F N57007F N57010F N57229F N57607F N57811F N57952F N58036 N58621F N45337F N44358F N44364F N47616F N48215F N48456F N49300F N49382F N49614F N57076F N57698F N57957F N44134F N50969F N57645F N48323F N47630F N52019
Accession no. SRR2407688 SRR3295634 SRR3295644 SRR3295650 SRR3295659 SRR3295661 SRR3295726 SRR3295834 SRR3295907 SRR3295950 SRR3295618 SRR3295964 SRR5235473 SRR5235471 SRR5235472 SRR5235474 SRR5235476 SRR5235478 SRR5235479 SRR5235480 SRR5235481 SRR3295681 SRR3295870 SRR3295953 SRR5235470 SRR5235482 SRR3295850 SRR5235477 SRR5235475 SRR2407540
Serotype Derby Derby Anatum Anatum Anatum Anatum London Adelaide Derby Anatum Derby I 4,[5],12:i:Typhimurium Muenchen Rissen Muenchen Muenchen Muenchen Muenster Muenchen Muenchen Muenchen Muenchen Brandenburg Heidelberg Give Derby Senftenberg Rough/nonmotile Derby
Yr 2014 2014 2014 2014 2014 2014 2014 2014 2014 2014 2015 2014 2013 2013 2013 2013 2013 2013 2013 2013 2013 2014 2014 2014 2013 2013 2014 2013 2013 2014
Source Pork chops Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Pork chops Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Cecal swine Pork chops
State NM WI IL CA PA CA NE SD KS PA WA SC NE NC NC IL KY TX OH VA TN MS IA KS CA TX TN MO CO
Quinolone genotype qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b qnrB19b; GyrA (D87N); ParC(S80I) qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c qnrB19c GyrA (D87N) GyrA (D87N) GyrA (S83F) qnrB6; aac(6=)Ib-cr qnrS2 qnrS2
CIP MICa (g/ml) 0.5 0.5 0.5 0.5 1 0.5 0.5 1 0.5 0.5 0.5 0.5 ⬎4 0.25 0.5 0.5 0.5 0.25 0.5 0.5 0.5 0.5 0.5 1 0.5 0.25 0.25 4 2 2
aCIP,
ciprofloxacin. (3,071 bp). cPlasmid (2,699 bp). bPlasmid
sources. In 2013, the first year of swine cecal sampling at slaughter by the Food Safety and Inspection Service (FSIS), 2.7% (15/552) of Salmonella isolates were nonsusceptible to ciprofloxacin (9); in 2014, ciprofloxacin nonsusceptibility was 3.8% (23/603). Among the NARMS retail pork chop samples, 3 (7.7%) of 39 Salmonella isolates from 2014 to 2015 were nonsusceptible to ciprofloxacin, whereas all of the 176 Salmonella isolates from 2002 to 2013 were susceptible. A direct programmatic and temporal comparison cannot be made between cecal and retail samples. However, these observations may collectively point toward a potential increase in ciprofloxacin nonsusceptibility. To further understand the ciprofloxacin nonsusceptibility phenomenon, 30 of the ciprofloxacin-nonsusceptible isolates from swine cecal and retail pork chop samples collected in 2013 to 2015 were sequenced on the Illumina MiSeq system using v3 reagent kits. Sequences were assembled de novo using CLC Genomics Workbench version 9.0.1 and were analyzed for mutations in the QRDRs of gyrA, gyrB, parC, and parE and for the presence of PMQR genes in the ResFinder database, including qnr genes, aac(6=)-Ib-cr, qepA, and oqxAB (10). Of the 30 sequenced isolates, 27 had PMQR genes and 24 had qnrB19 genes, with one of the latter isolates having additional gyrA and parC mutations (Table 1). Two isolates had qnrS2 genes—the first known report of this gene in Salmonella in the United States. A Salmonella Senftenberg isolate from cecal contents had both aac(6=)Ib-cr and qnrB6, a combination that is known to substantially increase the ciprofloxacin MIC (Table 1) (11). Presence of the PMQR gene aac(6=)-Ib-cr in a Salmonella isolate from any animal source in the United States is also a novel finding. qepA and oqxAB were not identified in any isolates. The MICs and the National Center for Biotechnology Information (NCBI) sequence database (GenBank) accession numbers for all strains used in this study are shown in Table 1. Note that of all the retail pork isolates sequenced in 2002 to 2013 (176 total), none had fluoroquinOctober 2017 Volume 61 Issue 10 e01318-17
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FIG 1 Geographic distribution of two qnrB19-containing plasmids in the United States. Isolates in New Mexico and Washington are from pork chop samples collected in local grocery stores, and the remaining isolates are from swine ceca sampled at slaughter plants. These geographic distributions do not necessarily reflect where animals were raised.
olone resistance mechanisms. As the annual rate of retail pork sampling has not changed since 2013, the presence of PMQR genes in Salmonella isolates from U.S. swine sources may be an emerging phenomenon of recent origin. The contigs containing the qnrB19 genes were of very high coverage, and by mapping reads back to the contigs, we were able to close each of the plasmid sequences, confirming that all 24 of the qnrB19-containing sequences were on ColEtype plasmids. Thirteen of these were identical 3,071-bp plasmids, and the remaining 11 were identical 2,699-bp plasmids. Using nucleotide BLAST analysis, we found that the smaller of the two plasmids was identical to ColE-type plasmids that were identified in Escherichia coli isolates from Peru (12) and Salmonella isolates from Argentina and Germany (13, 14). The larger plasmid contained only two single nucleotide polymorphisms (SNPs) relative to an E. coli plasmid from Bolivia (12), the only BLAST hit in GenBank within 10 SNPs. Although several larger qnrB19-containing conjugative plasmids have been described among Enterobacteriaceae (15, 16), these ColE-type plasmids do not share extensive homology with them, demonstrating a potentially distinct path of PMQR dissemination. The 3,071-bp plasmid and the 2,699-bp plasmid share ⬃2,300 bp of alignment and, aside from the qnrB19 and plasmid maintenance genes, contain relatively few additional genes. As mentioned earlier, these two plasmids were identified in South American E. coli isolates and have been compared and described extensively (12). Isolates containing the plasmids were diverse, with six different Salmonella serotypes possessing the 3,071-bp plasmid and four different serotypes containing the 2,699-bp plasmid, the most common serotypes being Muenchen, Anatum, and Derby (Table 1). Although geographic inferences cannot be made directly because of the complexity associated with animal production to slaughter practices and varied product distribution, note that each of the two plasmids was isolated from swine cecal contents or retail pork samples in at least nine states (Fig. 1). Despite the diversity of isolates with these plasmids, all copies of the two plasmids were clonal, as determined by BLAST analysis, potentially pointing to a common source of dissemination. The reason underlying the evident increase in PMQRs in swine is not clear, as there is no known factor selecting for increased resistance. The fluoroquinolone drug enrofloxacin October 2017 Volume 61 Issue 10 e01318-17
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is approved for use in swine to treat and control colibacillosis and swine respiratory disease (https://animaldrugsatfda.fda.gov), but extralabel use is prohibited. In summary, we identified the first PMQR Salmonella from swine sources in the United States, primarily in two clonal qnrB19-containing plasmids. On average, ⬃47% of swine cecal samples in the United States are positive for Salmonella (9), leading to the potential for increased fluoroquinolone nonsusceptibility in Salmonella in food and the environment through horizontal transmission of qnrB19 plasmids. Although Salmonella contamination of retail pork remains below 5% (9), the presence of strains with these plasmids in retail meat products is of particular concern due to the potential for spread in bacterial populations in ecogeographic niches, resulting in elevated consumer exposure through contaminated food products. Additional work is needed to determine whether Salmonella isolates with these qnrB19-containing plasmids have been transmitted to humans through the U.S. food supply to result in human infections; the NARMS surveillance program will continue to monitor qnr gene dissemination through phenotypic and genotypic approaches. The recent findings of IncR-type qnrB19 plasmids in dairy cattle from Texas along with our data from swine sources highlight the importance of gene-level resistance surveillance (17), and these results demonstrate the need to monitor the movement of resistant strains and genes through the farm-to-fork continuum to further strengthen food safety and public health. Accession number(s). We submitted the 3,071-bp and 2,699-bp plasmid sequences to GenBank, with accession numbers KY991369 and KY991368, respectively. ACKNOWLEDGMENTS We acknowledge the work of FSIS OPHS Eastern Laboratory Microbiology Screening Branch and Microbiology Characterization Branch in the isolation and characterization of NARMS cecal isolates and the FSIS OPHS Applied Epidemiology staff for their support. We also acknowledge Claudia Lam of FDA CVM for submitting the sequence data to GenBank. The views expressed in this article are those of the authors and do not necessarily reflect the official policy of the Department of Health and Human Services, the U.S. Food and Drug Administration, or the U.S. Government. Reference to any commercial materials, equipment, or process does not in any way constitute approval, endorsement, or recommendation by the Food and Drug Administration.
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