IntroductionSalmonella 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 testingSalmonella 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> genesDNA 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 characterizationPlasmids 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 isolatesNARMS 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> plasmidsTo 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 isolatesTwo-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).
DiscussionLaboratory-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.