10112012132148Foodborne Pathog DisFoodborne Pathog. Dis.Foodborne pathogens and disease1535-31411556-712524484290462065710.1089/fpd.2013.1670HHSPA731951ArticleCharacterization of blaCMY Plasmids and Their Possible Role in Source Attribution of Salmonella enterica Serotype Typhimurium InfectionsFolsterJ.P.1TolarB.1PecicG.12SheehanD.1RickertR.1HiseK.1ZhaoS.3Fedorka-CrayP. J.4McDermottP.3WhichardJ.M.1Division of Foodborne, Waterborne, and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, GeorgiaInternational Health Resources Consulting, Atlanta, GA, USADivision of Animal and Food Microbiology, Office of Research, Center for Veterinary Medicine, U.S. Food and Drug Administration, Laurel, MarylandBacterial Epidemiology and Antimicrobial Resistance Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Athens, Georgia2210201531120144201426102015114301306

Salmonella is an important cause of foodborne illness; however, identifying the source of these infections can be difficult. This is especially true for Salmonella serotype Typhimurium which is found in diverse agricultural niches. Extended spectrum cephalosporins (ESC) are one of the primary treatment choices for complicated Salmonella infections. In Salmonella, ESC resistance in the U.S. is mainly mediated by blaCMY genes carried on various plasmids. In this study, we examined whether the characterization of blaCMY plasmids, along with additional information, can help us identify potential sources of infection by Salmonella, and use serotype Typhimurium as a model. In the U.S., monitoring of retail meat, food animals, and ill persons for antimicrobial resistant Salmonella is conducted by the National Antimicrobial Resistance Monitoring System (NARMS). In 2008, 70 isolates (70/581;12.0 %) (34 isolates from retail meat, 23 food animal, and 13 human) were resistant to ceftriaxone and amoxicillin/clavulanic acid. All were PCR-positive for blaCMY and 59/70 (84.3%) of these genes were plasmid-encoded. PCR-based replicon typing (PBRT) identified 42/59 (71.2%) IncI1-blaCMY plasmids and 17/59 (28.8%) IncA/C-blaCMY plasmids. Isolates from chickens or chicken products with blaCMY plasmids primarily had IncI1-blaCMY plasmids (37/40; 92.5%), while all isolates from cattle had IncA/C-blaCMY plasmids. Isolates from humans had either IncA/C- blaCMY (n = 8/12; [66.7%]) or IncI1- blaCMY (n = 4/12 [33.3%]) plasmids. All of the IncI1-blaCMY plasmids were ST12 or were closely related to ST12. Antimicrobial susceptibility patterns (AST) and pulsed-field gel electrophoresis (PFGE) patterns of the isolates were also compared and differences were identified between isolate sources. When the source of a Typhimurium outbreak or sporadic illness is unknown, characterizing outbreak isolate’s blaCMY plasmids, AST, and PFGE patterns may help identify it.

Introduction

Salmonella is an important cause of foodborne illness in the United States resulting in approximately 1.2 million cases of salmonellosis a year (Scallan E 2011). Although salmonellosis is usually self-limiting, severe infections typically require antimicrobial treatment (RedBook 2012). Infections are commonly associated with consuming contaminated food or water. However, identifying the source of these infections can be difficult. This is especially true for Salmonella enterica serotype Typhimurium, which is found in diverse agricultural niches and is typically among the top serotypes associated with salmonellosis each year (Centers for Disease and Prevention 2010).

The National Antimicrobial Resistance Monitoring System (NARMS) determines antimicrobial susceptibility of Salmonella from humans, retail meats, and food animals. In 2008, Salmonella ser. Typhimurium was the most common serotype isolated from retail chicken breasts, the fourth most common from chickens, the fifth most common from cattle, and the second most common serotype from humans (United States Department of Agriculture 2009, United States Food and Drug Administration (A) 2009, Centers for Disease Control and Prevention 2010). Antimicrobial resistance in serotype Typhimurium is associated with bloodstream infection which is of concern because these patients are more likely to require antimicrobial treatment (Crump, Medalla et al. 2011). Extended spectrum cephalosporins (ESC), such as ceftriaxone, are one of the primary treatment choices for invasive salmonellosis (RedBook 2012). However, ESC resistance among Salmonella is on the rise in the U.S. and threatens to complicate treatment options (United States Food and Drug Administration (B) 2009). Among ceftriaxone resistant Salmonella collected in the United States in 2008, Typhimurium was the second most common serotype found in humans, the first from chicken retail meat, the second from chickens, and the fourth from cattle.

Considerable research has been performed on identifying the mechanisms of cephalosporin resistance in Salmonella. In the U.S., cephalosporin resistance is primarily mediated by AmpC β-lactamases, encoded by blaCMY genes (Philippon, Arlet et al. 2002, Folster, Pecic et al. 2010, Folster, Pecic et al. 2011). These genes are commonly carried on various types of plasmids, which can be distinguished by their incompatibility/replicon type (Carattoli, Bertini et al. 2005). Previous studies identified two major types of blaCMY plasmids among Salmonella in the U.S.; IncA/C and IncI1 (Folster, Pecic et al. 2010, Folster, Pecic et al. 2011). In this study, we examined blaCMY-positive Typhimurium isolates from retail meat, food animals, and humans to determine whether the phenotypic and genotypic characteristics of blaCMY plasmids can help us identify possible sources of infection by Typhimurium. We chose to focus on serotype Typhimurium isolates due to their commonality and widespread sources, however it is our hope that this study will serve as a model of all blaCMY-positive Salmonella serotypes.

MethodsIsolate collection and testing

Salmonella isolates from ill persons were obtained from specimens submitted to clinical laboratories in the United States and forwarded to state public health laboratories. Participating state public health laboratories serotyped and submitted every twentieth non-typhoidal Salmonella (NTS) to the CDC NARMS laboratory for susceptibility testing. NARMS retail meat monitoring was conducted by the United States FDA-Center for Veterinary Medicine as previously described (Zhao, White et al. 2008). NARMS monitoring of food animals at slaughter was conducted by the USDA Bacterial Epidemiology and Antimicrobial Resistance Research Unit (BEAR) of the Agricultural Research Service (ARS) as previously described (Frye, Fedorka-Cray et al. 2008). Broth microdilution (Sensititre®, Trek Diagnostics Systems, Thermo Fisher Scientific Inc., Cleveland, OH) was used to determine the minimum inhibitory concentrations (MIC) for 15 antimicrobial agents. Resistance was defined by the Clinical and Laboratory Standards Institute (CLSI) interpretive standards, when available (CLSI 2013). For streptomycin, where no CLSI interpretive criteria for human isolates exist, the resistance breakpoint is 64 µg/ml (United States Food and Drug Administration (B) 2009). Testing was performed according to the manufacturer’s instructions; the following quality control strains were used: E. coli ATCC 25922, Staphylococcus aureus ATCC 29213, E. coli ATCC 35218, and Pseudomonas aeruginosa ATCC 27853.

PCR amplification of <italic>bla</italic><sub>CMY</sub> genes

DNA templates for PCR was prepared by lysing the bacteria at 95°C and collecting the supernatant following centrifugation for 10 min at 20,000 g (Sorvall RC5B Plus, SS-34 rotor, Thermo Fischer Scientific Inc., Waltham, MA). PCR reactions contained 2x HotStar PCR Master Mix (Qiagen Inc., Valencia, CA), 0.4µM of each primer, 5µl template DNA and sterile PCR water to a final volume of 50µl. Thermal cycling was performed using the following conditions: 15 min at 95°C, followed by 30 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 90 s. To determine the presence of blaCMY genes, primers ampC1 (5′-ATGATGAAAAAATCGTTATGC-3′) and ampC2 (5′-TTGCAGCTTTTCAAGAATGCGC-3′) were used (Winokur, Vonstein et al. 2001).

Plasmid purification and characterization

Plasmids were purified using the QiaFilter Midi kit (Qiagen Inc.), following a modified manufacturer’s protocol (Folster, Pecic et al. 2010). Electroporation of each plasmid into E. coli DH10B Electromax competent cells (Invitrogen, Carlsbad, CA) was performed as previously described (Folster, Pecic et al. 2010). Cells were plated on LB agar plates containing 100 mg/L of ampicillin or 4 mg/L ceftriaxone (Sigma-Aldrich, St. Louis, MO). All transformants were confirmed as blaCMY positive by PCR analysis using primers ampC1 and ampC2. DNA templates for PCR from transformants were prepared as described above. Plasmid PCR-based replicon typing (PBRT) was performed as previously described (Carattoli, Bertini et al. 2005) on the transformants. Plasmid multi-locus sequence typing was performed on IncI1 plasmids as previously described (Garcia-Fernandez, Chiaretto et al. 2008). Sequencing was performed using Big Dye version 3.1 (Applied Biosystems, Foster City, CA) and sequence reactions were cleaned with Centri-sep plates (Princeton Separations, Adelphia, NJ). The reactions were electrophoresed through POP-7 polymer (Applied Biosystems) on a 3730 DNA Analyzer (Applied Biosystems) equipped with a 48-capillary, 50 cm array. Sequence analysis was performed using Lasergene 8 software (DNASTAR Inc, Madison, WI). Sequences were submitted to the plasmid multi locus sequence type (pMLST) web page (http://pubmlst.org/plasmid/) and the sequence type was determined.

Pulsed-Field Gel Electrophoresis (PFGE)

Two enzyme (XbaI and BlnI) PFGE was performed according to the CDC PulseNet protocol(Ribot, Fair et al. 2006, Jackson, Fedorka-Cray et al. 2007) Isolates were grown overnight on Trypticase Soy Agar with 5% defibrinated sheep blood (TSA-SB) (Becton Dickinson Biosciences). Bacterial cell concentration was adjusted by diluting with sterile cell suspension buffer (100 mM Tris, 100 mM EDTA, pH 8.0) to a turbidity measurement of 0.48–0.52 (Dade Microscan Tubidity Meter). Agarose-embedded cells were lysed by proteinase K treatment and extensively washed. Agarose plugs containing genomic DNA were digested with 50U of XbaI and BlnI restriction enzymes (New England Biolabs, Ipswich, MA) and incubated at 37°C for 2 hours. The fragments were then separated by PFGE using a CHEF Mapper (Bio-Rad Laboratories) with the following conditions and reagents: 1% SeaKem Gold agarose in 0.5% TBE buffer, voltage at 6 V/cm, run time at 18 hours with switch times ranging from 2.16 to 63.8 seconds, temperature at 14°C. Salmonella enterica ser. Braenderup H9812 was used as a molecular reference marker. PFGE profiles generated were submitted to the PulseNet national database administered by CDC (NARMS-FDA and NARMS-CDC) or USDA VetNet (NARMS-USDA). Gel images were captured using the GelDoc XR system (Bio-Rad Laboratories) and Quantity One 1-D analysis software (Bio-Rad Laboratories). Pattern analysis and UPGMA dendrogram generation were performed using BioNumerics software (Applied Maths, Saint-Martens-Latem, Belgium) with the Dice coefficient and tolerance of 1.5%.

ResultsIdentification of <italic>bla</italic><sub>CMY</sub>-positive <italic>Salmonella</italic> ser. Typhimurium isolates

NARMS received and performed antimicrobial susceptibility testing on 581 isolates of Salmonella Typhimurium from food animals, retail meat, and humans in 2008. Of these, 70 (12.0%) displayed resistance to ceftriaxone and amoxicillin-clavulanic acid, suggesting the presence of a blaCMY gene. Of the 70 isolates, 34 (48.6%) were from retail meat, specifically, chicken breasts. Twenty-three isolates (23/70 [32.9%]) were from food animal samples; 17/23 [73.9%] from chickens and 6/23 [26.1%] from cattle. Thirteen isolates were recovered from clinically-ill humans; 2/13 [15.4%] from male patients and 11/13 [84.6%] from females. The median age was 29.9 years with a range of less than 1 year to 77 years. PCR-analysis confirmed that all 70 resistant isolates carried a blaCMY gene. Besides resistance to ceftriaxone and amoxicillin/clavulanic acid, all 70 isolates were resistant to the additional β-lactams tested (ampicillin, cefoxitin, and ceftiofur) (Figure 1). The most common additional resistance observed among the isolates was to sulfisoxazole (n=68), tetracycline (n=64), streptomycin (n=24), and chloramphenicol (n=14). Resistance to kanamycin (n=9) and gentamicin (n=3) was less common and resistance to amikacin, ciprofloxacin, nalidixic acid, and sulphamethoxazole/trimethoprim was not observed.

Characterization of the <italic>bla</italic><sub>CMY</sub> plasmids

To determine if the blaCMY genes were located on a plasmid or the chromosome, plasmid DNA preparations were used to transform competent E. coli by electroporation. PCR-analysis showed successful transfer of blaCMY genes to E. coli for 59/70 (84.3%) from the ESC resistant Typhimurium isolates, suggesting that 11 isolates likely carried blaCMY chromosomally. PCR-based replicon typing (PBRT) performed on the transformants revealed that all 59 blaCMY-plasmid positive Typhimurium isolates carried the blaCMY gene on one of two plasmid types; 42/59 (71.2%) had IncI1- and 17/59 (28.8%) had IncA/C-blaCMY plasmids (Figure 1). Among the 51 chicken/chicken breast isolates, 38 had IncI1-blaCMY plasmids, 3 had IncA/C-blaCMY plasmids, and 10 isolates had no blaCMY plasmids. Among the six cattle isolates, all had IncA/C-blaCMY plasmids. Among the 13 isolates from humans, 4 isolates had IncI1-blaCMY plasmids, 8 isolates had IncA/C-blaCMY plasmids, and one had no blaCMY plasmid. IncI1 plasmids were compared using plasmid multi-sequence typing (pMLST) (Garcia-Fernandez, Chiaretto et al. 2008). All 42 IncI1-blaCMY plasmids were sequence type 12 or very closely related. Three isolates had plasmids with an identical point mutation in the pilL allele (G56 to A56) while 2 other isolates had plasmids two identical point mutations in the sogS allele (A175 to G175 and T177 to A177). All 5 five of these isolates were isolated from chicken breasts.

Determining similarity by PFGE of the <italic>bla</italic><sub>CMY</sub>-positive isolates

Two-enzyme PFGE was used to evaluate the genetic relatedness of the blaCMY-positive Typhimurium isolates from different sources (Figure 1). Of the 70 isolates, 56 (80%) had unique 2-enzyme patterns suggesting very little clonal spread. There were 9 groups of isolates with indistinguishable patterns and the largest group contained 4 isolates. The isolates grouped into 3 main clusters (labeled A, B, and C) and one outlier (bottom of dendrogram). The largest group, cluster C, contained closely related (> 75% with 2-enzymes) isolates from poultry sources (chicken breast [n=31] and chickens [n=16]) and humans [n=3]. None of these isolates displayed resistance to chloramphenicol. Isolates in cluster C primarily contained IncI1-blaCMY plasmids (n=39/50; 78%) or no blaCMY plasmids (8/50; 16%). Only three isolates contained IncA/C-blaCMY plasmids. Cluster A was the next largest cluster with 15 isolates. Although not as related (< 75%) as cluster C, cluster A contained almost all of the isolates with chloramphenicol resistance (12/14; 85.7%) and most of the isolates with streptomycin resistance (14/24; 58.3%), meeting the definition of MDR-AmpC (Harbottle, White et al. 2006). Isolates in cluster A primarily contained isolates of either human (n=9) or cattle (n=5) sources and only a single poultry isolate. Cluster A also consisted primarily of isolates with IncA/C-blaCMY plasmids (12/15; 80%) with only two IncI1-blaCMY containing isolates and a single isolate with no blaCMY plasmid. Cluster B contains only 4 isolates and is mixed with respect to antimicrobial resistance patterns (two isolates displayed chloramphenicol resistance), isolate source (two isolates from chicken breast, one cattle, and one human source) and blaCMY plasmid type (two IncA/C-blaCMY, one IncI1-blaCMY, and one without a blaCMY plasmid).

Discussion

Laboratory-based disease surveillance and foodborne outbreak detection and investigation requires high quality epidemiological data and detailed agent information. With the evolution of new strain typing techniques, investigators have sought to combine various tools to provide more specific agent information in an effort to improve the surveillance and investigative processes. We examined the value of combining phenotypic data on antimicrobial resistance and serotype with genetic data represented by PFGE patterns and differences in plasmid content.

IncI1 plasmids are a narrow-host-range plasmid type and are limited to enteric bacteria (Johnson, Shepard et al. 2011). They are commonly associated with Salmonella and E. coli from avian and porcine sources, and are more frequent among pathogenic than commensal E. coli strains found among avian and human sources in the U.S. (Johnson, Wannemuehler et al. 2007). IncI1 plasmids commonly carry genes conferring ESC resistance, including blaCMY and blaCTX-M. Previous studies have identified IncI1 plasmids as the primary plasmid type carrying blaCMY among humans with Salmonella serotypes commonly associated with poultry and IncI1 is the most prominent blaCMY plasmid type among serotype Heidelberg isolates from humans and poultry sources (Folster, Pecic et al. 2010, Folster, Pecic et al. 2011).

IncA/C plasmids have a broad host range and have been isolated from diverse groups of Proteobacteria found in the environment, animals, and humans (Lang, Danzeisen et al. 2012). IncA/C plasmids are commonly large, multidrug resistant, and have been identified among isolates from food animals including beef, chicken, turkey and pork, suggesting that they may be responsible for the spread of MDR from food animals to humans (Mulvey, Susky et al. 2009). IncA/C plasmids are one of the most frequent plasmid types carrying blaCMY in the United States (Giles, Benson et al. 2004). IncA/C plasmids are the most common plasmid type carrying blaCMY among humans with Salmonella serotypes usually associated with cattle and beef sources, including Newport and Dublin (Folster, Pecic et al. 2010). A study of blaCMY plasmids from E. coli and Salmonella in Canada found that bacteria from cattle and beef all had IncA/C plasmids (Martin, Weir et al. 2012).

In this study, we identified and characterized 70 ceftriaxone and amoxicillin/clavulanic acid resistant Salmonella ser. Typhimurium isolates. All of the isolates contained a blaCMY gene and over 80% were plasmid encoded. We identified two blaCMY plasmid types, IncI1 and IncA/C, with IncI1 comprising greater than 70% of blaCMY plasmids identified. When we compared plasmid type to the source of each non-clinical isolate we found that nearly all of the isolates with blaCMY plasmids from chicken or chicken products had IncI1-blaCMY plasmids (92.5%), while all of the cattle isolates had IncA/C-blaCMY plasmids. This shows a correlation between the animal source of Typhimurium isolates and the replicon type of blaCMY plasmid they carry. However, a larger study set of isolates over several years is needed to confirm this observation. Clinical isolates from humans with blaCMY plasmids had both IncA/C-blaCMY (n=8) and IncI1-blaCMY (n=4) plasmids; however, no information was available regarding the source of infection of these routine surveillance isolates.

When we compared plasmid type, source, and antimicrobial resistance patterns we found that all of the IncA/C-blaCMY isolates from cattle and humans were resistant to chloramphenicol. Chloramphenicol resistance is commonly conferred by IncA/C plasmids (Lindsey, Frye et al. 2011). However, the three IncA/C-blaCMY isolates from chicken breasts were all chloramphenicol susceptible and none of the IncI1-blaCMY positive isolates from chicken or chicken breasts were chloramphenicol resistant. This suggests that chloramphenicol resistance, when observed along with the blaCMY mediated resistance phenotype, may point to a possible cattle/beef source.

PFGE analysis showed a significant amount of genetic variation among all 70 isolates but the isolates grouped into 3 main clusters (A, B, and C). When we compared this to isolate source, blaCMY plasmid type, and chloramphenicol resistance, we again found a strong correlation. Cluster C, the largest cluster with 50 isolates, contained 92.2% of the poultry isolates and no cattle isolates, 91.6% of the IncI1-blaCMY plasmids, and no chloramphenicol resistant isolates. In contrast, cluster A, containing 15 isolates, had five out of six isolates from cattle, 70.6% (12/17) of the IncA/C-blaCMY plasmids, and 85.7% (12/14) of the chloramphenicol resistant isolates. If we further divide cluster A, the bottom group (containing seven isolates) contains isolates with an identical resistance profile and plasmid type (IncA/C) but isolates from both cattle (n=4) and humans (n=3), suggesting that the human isolates were likely acquired from a beef source.

Interestingly, even though Salmonella is rarely isolated from ground beef and none of the isolates in this study were isolated from ground beef, the majority of isolates from humans had IncA/C-blaCMY plasmids, which we interpret to indicate that humans were more likely to acquire blaCMY isolates with plasmids that resemble those from cattle sources than poultry sources. This suggests that human infections with blaCMY positive Typhimurium isolates may be from another beef source which is not being sampled or that current sampling and/or isolation methods are not detecting Salmonella in ground beef. The latter may be due to processing events specific for ground beef which could result in a temporary decrease in Salmonella numbers at the time of sampling (Harris, Brashears et al. 2012). It is also possible that humans are acquiring blaCMY positive Salmonella ser. Typhimurium from unsampled non-meat sources, including vegetables.

Conclusions

Although the numbers of isolates in the study were small, especially from cattle, we did find associations between blaCMY plasmid type and food animal source (IncA/C and cattle, IncI1 and poultry). This work may be useful in generating hypotheses about the sources for foodborne outbreaks and sporadic illness caused by Salmonella. However, resistance plasmid type needs to be examined along with careful consideration of all of the available information. This is particularly important for some serotypes like Typhimurium which has great genetic diversity and wide range of hosts. Further work is necessary to determine if these correlations occur with other Salmonella serotypes, other enteric bacteria, and additional antimicrobial resistance plasmids. Analysis of a larger set of isolates over a longer period of time is needed to determine how best to combine plasmid, PFGE, serotype, and susceptibility data to effectively guide investigations of foodborne disease.

Acknowledgements

We thank the NARMS participating public health laboratories for submitting the isolates, Anne Whitney for DNA sequencing, Alessandra Carattoli for the plasmid incompatibility typing control strains. This work was partially supported by an interagency agreement between CDC, USDA, and the FDA Center for Veterinary Medicine.

The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the CDC, FDA or USDA.

ReferencesCarattoliABertiniAVillaLFalboVHopkinsKLThrelfallEJIdentification of plasmids by PCR-based replicon typingJ Microbiol Methods200563321922815935499Centers for Disease, C. and PreventionPreliminary FoodNet data on the incidence of infection with pathogens transmitted commonly through food - 10 states, 2009MMWR Morb Mortal Wkly Rep2010591441842220395935Centers for Disease Control and PreventionNational Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS): Enteric Bacteria Annual Report2010Atlanta, GACDC2010b.CLSIPerformance Standards for Antimicrobial Susceptibility Testing; Twenty-first Informational Supplement. CLSI Document M100-S21Clinical and Laboratory Standards Institute2013CrumpJAMedallaFMJoyceKWKruegerALHoekstraRMWhichardJMBarzilayEJAntimicrobial resistance among invasive nontyphoidal Salmonella enterica isolates in the United States: National Antimicrobial Resistance Monitoring System, 1996 to 2007Antimicrob Agents Chemother20115531148115421199924FolsterJPPecicGBolcenSTheobaldLHiseKCarattoliAZhaoSMcDermottPFWhichardJMCharacterization of extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg isolated from humans in the United StatesFoodborne Pathog Dis20107218118719785533FolsterJPPecicGMcCulloughARickertRWhichardJMCharacterization of bla(CMY)-Encoding Plasmids Among Salmonella Isolated in the United States in 2007Foodborne Pathog Dis2011FryeJGFedorka-CrayPJJacksonCRRoseMAnalysis of Salmonella enterica with reduced susceptibility to the third-generation cephalosporin ceftriaxone isolated from U.S. cattle during 2000–2004Microb Drug Resist200814425125819025468Garcia-FernandezAChiarettoGBertiniAVillaLFortiniDRicciACarattoliAMultilocus sequence typing of IncI1 plasmids carrying extended-spectrum beta-lactamases in Escherichia coli and Salmonella of human and animal originJ Antimicrob Chemother20086161229123318367460GilesWPBensonAKOlsonMEHutkinsRWWhichardJMWinokurPLFeyPDDNA sequence analysis of regions surrounding blaCMY-2 from multiple Salmonella plasmid backbonesAntimicrob Agents Chemother20044882845285215273090HarbottleHWhiteDGMcDermottPFWalkerRDZhaoSComparison of multilocus sequence typing, pulsed-field gel electrophoresis, and antimicrobial susceptibility typing for characterization of Salmonella enterica serotype Newport isolatesJ Clin Microbiol20064472449245716825363HarrisDBrashearsMMGarmynAJBrooksJCMillerMFMicrobiological and organoleptic characteristics of beef trim and ground beef treated with acetic acid, lactic acid, acidified sodium chlorite, or sterile water in a simulated commercial processing environment to reduce Escherichia coli O157:H7 and SalmonellaMeat Sci201290378378822122990JacksonCRFedorka-CrayPJWinelandNTanksonJDBarrettJBDourisAGreshamCPJackson-HallCMcGlincheyBMPriceMVIntroduction to United States Department of Agriculture VetNet: status of Salmonella and Campylobacter databases from 2004 through 2005Foodborne Pathog Dis20074224124817600492JohnsonTJShepardSMRivetBDanzeisenJLCarattoliAComparative genomics and phylogeny of the IncI1 plasmids: a common plasmid type among porcine enterotoxigenic Escherichia coliPlasmid201166314415121843549JohnsonTJWannemuehlerYMJohnsonSJLogueCMWhiteDGDoetkottCNolanLKPlasmid replicon typing of commensal and pathogenic Escherichia coli isolatesAppl Environ Microbiol20077361976198317277222LangKSDanzeisenJLXuWJohnsonTJTranscriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C plasmidAppl Environ Microbiol20127893379338622344651LindseyRLFryeJGFedorka-CrayPJMeinersmannRJMicroarray-based analysis of IncA/C plasmid-associated genes from multidrug-resistant Salmonella entericaAppl Environ Microbiol201177196991699921841024MartinLCWeirEKPoppeCReid-SmithRJBoerlinPCharacterization of blaCMY-2 plasmids in Salmonella and Escherichia coli isolates from food animals in CanadaAppl Environ Microbiol20127841285128722156427MulveyMRSuskyEMcCrackenMMorckDWReadRRSimilar cefoxitin-resistance plasmids circulating in Escherichia coli from human and animal sourcesVet Microbiol20091343–427928718824313PhilipponAArletGJacobyGAPlasmid-determined AmpC-type beta-lactamasesAntimicrob Agents Chemother200246111111751104RedBookLarryKPSalmonella InfectionsRed Book2012Village, ILAmerican Academy of Pediatrics635640RibotEMFairMAGautomRCameronDNHunterSBSwaminathanBBarrettTJStandardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNetFoodborne Pathog Dis200631596716602980ScallanEHRAnguloFJTauxeRVWiddowsonM-ARoySLFoodborne illness acquired in the United States—major pathogensEmerging Infectious Disease2011[serial on the internet.United States Department of AgricultureNational Antimicrobial Resistance Monitoring System for Enteric Bacteria (NARMS): Animal Arm Annual Report2009Washington DCUnited States Food and Drug Administration (A)NARMS Retail Meat Annual Report2009Rockville, MDUnited States Food and Drug Administration (B)NARMS excutive report2009Rockville, MDWinokurPLVonsteinDLHoffmanLJUhlenhoppEKDoernGVEvidence for transfer of CMY-2 AmpC beta-lactamase plasmids between Escherichia coli and Salmonella isolates from food animals and humansAntimicrob Agents Chemother200145102716272211557460ZhaoSWhiteDGFriedmanSLGlennABlickenstaffKAyersSLAbbottJWHall-RobinsonEMcDermottPFAntimicrobial resistance in Salmonella enterica serovar Heidelberg isolates from retail meats, including poultry, from 2002 to 2006Appl Environ Microbiol200874216656666218757574

PFGE patterns of blaCMY-positive Salmonella enterica ser. Typhimurium isolated from food animals, retail meat, and humans in the United States in 2008. Dendrogram of percent genetic similarity by PFGE was generated using BioNumerics based on XbaI and BlnI restriction digestion. Pattern analysis and UPGMA dendrogram generation were performed using BioNumerics software (Applied Maths, Saint-Martens-Latem, Belgium) with the Dice coefficient and tolerance of 1.5%. Percent similarity is located above dendrogram. Antibiogram displays the antimicrobial resistance profile of the isolates; a black box indicates resistance to that antimicrobial. AMK, amikacin; AUG, amoxicillin/clavulanic acid; AMP, ampicillin; FOX, cefoxitin; AXO, ceftriaxone; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; STR, streptomycin; FIS, sulfisoxazole; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole; TIO, ceftiofur. Isolate number, source, and plasmid incompatibility type are listed to the right of the antibiogram. The “-“symbol represents that no blaCMY plasmid was found. The isolates are groups into three clusters, labeled A, B, and C.