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Appl Environ Microbiol. 2009 December; 75(24): 7624–7630.
Published online 2009 October 23. doi:  10.1128/AEM.01158-09
PMCID: PMC2794113

β-Lactam Resistance in Salmonella Strains Isolated from Retail Meats in the United States by the National Antimicrobial Resistance Monitoring System between 2002 and 2006[down-pointing small open triangle]

Abstract

Ampicillin-resistant (Ampr) Salmonella enterica isolates (n = 344) representing 32 serotypes isolated from retail meats from 2002 to 2006 were tested for susceptibility to 21 other antimicrobial agents and screened for the presence of five beta-lactamase gene families (blaCMY, blaTEM, blaSHV, blaOXA, and blaCTX-M) and class 1 integrons. Among the Ampr isolates, 66.9% were resistant to five or more antimicrobials and 4.9% were resistant to 10 or more antimicrobials. Coresistance to other β-lactams was noted for amoxicillin-clavulanic acid (55.5%), ceftiofur (50%), cefoxitin (50%), and ceftazidime (24.7%), whereas less than 5% of isolates were resistant to piperacillin-tazobactam (4.9%), cefotaxime (3.5%), ceftriaxone (2%), and aztreonam (1.2%). All isolates were susceptible to cefepime, imipenem, and cefquinome. No Salmonella producing extended-spectrum beta-lactamases was found in this study. Approximately 7% of the isolates displayed a typical multidrug-resistant (MDR)-AmpC phenotype, with resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide, tetracycline, plus resistance to amoxicillin-clavulanic acid, cefoxitin, and ceftiofur and with decreased susceptibility to ceftriaxone (MIC ≥ 4 μg/ml). Pulsed-field gel electrophoresis results showed that several MDR clones were geographically dispersed in different types of meats throughout the five sampling years. Additionally, 50% of the isolates contained blaCMY, 47% carried blaTEM-1, and 2.6% carried both genes. Only 15% of the isolates harbored class I integrons carrying various combinations of aadA, aadB, and dfrA gene cassettes. The blaCMY, blaTEM, and class 1 integrons were transferable through conjugation and/or transformation. Our findings indicate that a varied spectrum of coresistance traits is present in Ampr Salmonella strains in the meat supply of the United States, with a continued predominance of blaCMY and blaTEM genes in β-lactam-resistant isolates.

Nontyphoid Salmonella enterica is one of the most important food-borne pathogens and represents a significant public health hazard worldwide. It is estimated that 1.4 million people in the United States are infected with non-Typhi Salmonella annually, resulting in 15,000 hospitalizations and more than 400 deaths (28). Salmonella infections in humans often result from the ingestion of contaminated foods, such as poultry, beef, pork, eggs, milk, seafood, and produce (10). Salmonellosis following direct contact with animals and dog treats has also been reported (3, 6, 7). Human salmonellosis usually results in a self-limiting diarrhea that does not require antimicrobial therapy. However, in severe cases of enteritis and systemic infections, fluoroquinolones and extended-spectrum cephalosporins such as ceftriaxone (AXO) are used as first-line therapeutics (12, 27).

Multidrug-resistant (MDR) Salmonella strains have been detected in many serotypes, such as S. enterica serotype Typhimurium (9, 26), S. enterica serotype Agona, S. enterica serotype Anatum, S. enterica serotype Choleraesuis, S. enterica serotype Dublin, S. enterica serotype Heidelberg, S. enterica serotype Kentucky, S. enterica serotype Newport, S. enterica serotype Schwarzengrund, S. enterica serotype Senftenberg, and S. enterica serotype Uganda, among others (14, 33, 35) (http://internet-dev/cvm/2005NARMSExeRpt.htm). The most common MDR pattern, which first emerged in S. Typhimurium, has been a pattern of resistance to ampicillin (AMP), chloramphenicol (CHL), streptomycin (STR), sulfonamides, and tetracycline (TET) (ACSSuT). More recently, strains exhibiting the ACSSuT pattern also have acquired MDR plasmids carrying the blaCMY gene and others (30) that can spread readily to different members of the Enterobacteriaceae. The strains demonstrate extensive resistances, which, in addition to the ACSSuT phenotype, may include resistance to amoxicillin-clavulanic acid (AUG), cefoxitin (FOX), and ceftiofur (TIO) and decreased susceptibility to AXO (MIC ≥ 4 μg/ml). TIO is a third-generation cephalosporin that was approved for use in animals in 1998. Tior Salmonella isolates often show resistance or decreased susceptibility to AXO (also a third-generation cephalosporin used to treat human infections). Some strains may also display resistance to gentamicin (GEN), kanamycin (KAN), and trimethoprim-sulfamethoxazole ([SMX] COT) as well as resistance to disinfectants and heavy metals. Resistance to third-generation cephalosporins in Salmonella strains is of interest because these are the drugs of choice for treating salmonellosis in children, where fluoroquinolones are contraindicated (13).

To date, more than 340 beta-lactamases have been described (11). The most common genes, such as blaTEM, blaSHV, blaCTX-M, blaOXA, blaPER, blaPSE, and blaCMY, have been detected in Salmonella, with the prevalence of these genes varying by region (32). Extended-spectrum beta-lactamases (ESBLs) are less prevalent in Salmonella strains than in other gram-negative bacteria such as Klebsiella, Escherichia coli, and Proteus. The ESBLs are β-lactamases capable of conferring bacterial resistance to the penicillins; to first-, second-, and third-generation cephalosporins; and to aztreonam (ATM) (but not to the cephamycins or carbapenems) by hydrolysis of these antibiotics, which are inhibited by β-lactamase inhibitors such as clavulanic acid (21). Most ESBL-carrying Salmonella strains have been reported in Latin America, the Western Pacific, and Europe (32), with only a few reports from North America. In the United States the first case was reported in 1994, when blaCTX-5 was detected in an S. Typhimurium var. Copenhagen strain from an infant adopted from Russia (25). Additional ESBL Salmonella strains have been reported recently, one from a horse (blaSHV-12) and another from a 3-month-old child (blaCTX-M-5) (23, 25). Carbapenem resistance in Salmonella is also rare in the United States but has been detected in S. enterica serotype Cubana associated with a plasmid-mediated blaKPC-2 gene (18). In contrast to the low prevalence of ESBL-carrying Salmonella strains in the United States, AmpC resistance mediated by blaCMY has been emerging in both humans and food animals. The blaCMY encodes a cephalomycinase that exhibits extended resistance to many beta-lactams, including first-, second-, and third-generation cephalosporins (36).

The objectives of this study were to determine the genetic basis of beta-lactam resistance and to examine the extent of coresistance to other antimicrobials among 344 Ampr Salmonella isolates obtained from retail meats. We screened for the presence of five beta-lactam resistance gene families (blaCMY, blaTEM, blaSHV, blaOXA, and blaCTX-M) and the presence of class 1 integrons. The range of resistance phenotypes borne on plasmids was examined by filter mating and electroporation, and all isolates were characterized for genetic relatedness using pulsed-field gel electrophoresis (PFGE).

MATERIALS AND METHODS

Bacterial isolates.

All Ampr Salmonella isolates (n = 344), representing 32 serotypes, recovered from retail meats from 2002 to 2006 by the National Antimicrobial Resistance Monitoring System (NARMS) were used in this study. Among the 344 Ampr isolates, 28 (8.1%) were isolated in 2002, 66 (19.2%) were from 2003, 81 (23.5%) were from 2004, 94 (27.3%) were from 2005, and 75 (21%) were from 2006. Nearly all of the isolates were recovered from poultry meats, with 162 (47%) from ground turkey and 158 (45.9%) from chicken breast; 13 (3.8%) were from ground beef, and 11 (3.2%) were from pork chops. Among the 32 serotypes, the most five common serotypes were S. Typhimurium (including S. Typhimurium var. 5-; n = 84; 24.4%), S. Heidelberg (n = 56; 16.3%), S. enterica serotype Saintpaul (n = 53; 15.4%), S. Kentucky (n = 45; 13.1%), and S. Senftenberg (n = 16; 4.7%). Detailed information on sampling, isolation, identification, and serotyping can be found at http://www.fda.gov/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/default.htm. Bacteria were stored in Trypticase soy broth containing 15% glycerol at −80°C until use.

Antimicrobial susceptibility testing.

Antimicrobial MICs were determined using a Sensititre automated antimicrobial susceptibility system in accordance with the manufacturer's instructions (Trek Diagnostic Systems, Cleveland, OH). Initially, all strains were tested using a panel designed for NARMS, which included AXO, TIO, AUG, AMP, FOX, ciprofloxacin (CIP), nalidixic acid (NAL), amikacin (AMI), GEN, STR, KAN, SMX, COT, TET, and CHL. All Ampr strains were tested with a secondary panel of β-lactam antimicrobials that included ATM, cefquinome (CQN), imipenem (IMI), cefepime (FEP), piperacillin-tazobactam (P/T), ceftazidime (TAZ), ceftazidime-clavulanic acid (T/C), cefotaxime (FOT), and cefotaxime-clavulanic acid (F/C). Results were interpreted in accordance with CLSI criteria with the exception of STR (resistance breakpoint, ≥64 μg/ml) and CQN (resistance breakpoint, ≥32 μg/ml) (5). E. coli ATCC 25922, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853 were used as quality control organisms in the antimicrobial MIC determinations.

DNA isolation, PCR, and DNA sequencing.

The presence of blaCMY, blaTEM, blaSHV, blaOXA, blaCTX-M, and class I integrons was detected by PCR using previously published methods (1, 24, 34). DNA templates were prepared using MoBio Ultraclean DNA isolation kits (MoBio Laboratories, Inc., Carlsbad, CA) following the manufacturer's instructions. Amplifications were carried out using 200 ng of DNA template, 250 μM of each deoxynucleoside triphosphate, 1.5 mM MgCl2, 50 pmol of each primer, and 1 U of Amplitaq Gold Taq polymerase (Applied Biosystems, Foster City, CA). The amplified products were separated by gel electrophoresis on 1.0% agarose gels and stained with ethidium bromide. The gels were visualized under UV light. Each positive PCR product was sequenced using an ABI 3730 automated sequencer. Sequences were analyzed using the National Center for Biotechnology Information's BLAST network service (http://www.ncbi.nlm.nih.gov/BLAST/). Each gene was identified by comparison to sequences in the GenBank database of accession numbers AB126601, DQ238100, DQ388125, DQ069244, AJ809407, AB282997, and EU113221.

Conjugation and transformation.

To assess the transfer mechanism and the extent of resistance phenotypes carried on plasmids, conjugations and transformations were carried out by filter mating and electroporation, respectively. Nineteen blaCMY-positive Salmonella isolates representing 14 serotypes from different sources were selected as donors, and ElectroMax DH5α E. coli cells (Invitrogen, Carlsbad, CA) were used as recipients (Table (Table1).1). TIO and NAL were used as the selective agents for the donor and recipient strains, respectively. Filter mating was carried out as previously described (4). For electroporation, plasmids were prepared using Qiagen HiSpeed plasmid purification kits (Qiagen, Valencia, CA). In a microcentrifuge tube, 20 μl of thawed DH5α cells was added to 200 ng (2 μl) of plasmid DNA. The mixture was pipetted into a chilled cuvette and electroporated at 2.0 kV, 200 Ω, and 25 μF. After electroporation, 1 ml of SOC medium (Sigma-Aldrich, St. Louis, MO) was added, and the suspension was incubated at 37°C with shaking for 1 h. The mixture was streaked on selective plates containing 4 μg/ml of TIO and examined for bacterial growth after 24 h of incubation at 37°C.

TABLE 1.
Salmonella isolates selected as donors in conjugation and electroporation experimentsa

PFGE.

PFGE was performed according to the protocol developed by the Centers for Disease Control and Prevention (http://www.cdc.gov/pulsenet/protocols.htm), using S. enterica serovar Braenderup H9812 as the control strain. Agarose-embedded DNA was digested with 50 U of XbaI or Bln1 (Boehringer Mannhein, Indianapolis, IN) for least 4 h in a water bath at 37°C. The restriction fragments were separated by electrophoresis in 0.5× Tris-borate-EDTA buffer (Invitrogen, Carlsbad, CA) at 14°C for 18 h using a Chef Mapper electrophoresis system (Bio-Rad, Hercules, CA) with pulse times of 2.16 to 63.8 s. Isolates showing DNA smears were retested using plugs digested with XbaI or BlnI and electrophoresis buffer containing 50 μM thiourea in 0.5× Tris-borate-EDTA buffer. The gels were stained with ethidium bromide, and DNA bands were visualized with UV transillumination (Bio-Rad). PFGE results were analyzed using BioNumerics software (Applied Maths, Kortrijk, Belgium), and banding pattern similarity was compared using an average of the results of two enzymes with a 1.5% band position tolerance. All PFGE profiles generated from this study were submitted to the national CDC PulseNet database for comparison with isolates from clinical human salmonellosis cases.

RESULTS

Antimicrobial susceptibility profiles.

A wide range of susceptibility patterns were noted among the Ampr isolates, with 66.9% of isolates resistant to at least 5 antimicrobials and 4.9% resistant to at least 10 antimicrobials. In addition to resistance to AMP, cross-resistance to other beta-lactams was noted for AUG (55.5%), TIO (50%), FOX (50%), and TAZ (24.7%), whereas less than 5% of isolates were resistant to P/T (4.9%), FOT (3.5%), AXO (2%), and ATM (1.2%). All isolates were susceptible to FEP, IMI, and CQN. For aminoglycosides, the most common coresistance was to STR (55.5%), followed by KAN (30.8%) and GEN (24.4%). All isolates were susceptible to AMI. For quinolones and fluoroquinolones, 3.3% were resistant to NAL, and none were resistant to CIP. Additionally, 57.8% of isolates were resistant to TET, 55.5% to sulfonamides, 10.8% to CHL, and 1.7% to COT (Table (Table2).2). Based on susceptibility to clavulanic acid with TAZ and FOT, no ESBL-producing Salmonella strains were detected in this study.

TABLE 2.
Antimicrobial resistance phenotypes of Ampr Salmonella isolates from NARMS retail meats from 2002 to 2006

MDR patterns varied within and between serotypes. For example, the most prevalent MDR pattern in S. Typhimurium was AMP/AUG/FOX/TIO/SMX/TET (n = 25; 30%), followed by AMP/AUG/FOX/TIO (n = 19; 23%), AMP/AUG/FOX/TIO/KAN/SMX/TET (n = 17; 20%), and ACSSuT (AMP/CHL/STR/SMX/TET) (n = 8; 10%). By contrast, the dominant MDR pattern in S. Kentucky was AMP/AUG/FOX/TIO/STR/TET (n = 35; 78%), followed by AMP/AUG/FOX/TIO/CHL/GEN/KAN/STR/SMX/TET (n = 4; 9%); all nine strains of S. Newport displayed the typical MDR-AmpC phenotype with resistance to ACSSuT, plus resistance to AUG/FOX/TIO and decreased susceptibility to AXO (MIC ≥ 4 μg/ml). Four S. Newport isolates showed additional resistance to COT. The top MDR profiles among Tior isolates are listed in Table Table3.3. Some serotypes such as S. Heidelberg showed very diverse resistance patterns such that 20 resistance patterns were observed among 56 Ampr isolates. Overall, 7% of the isolates displayed the typical MDR-AmpC phenotype, and several of these showed resistance also to P/T, ATM, TAZ, and FOT, as well as other drug classes represented by GEN, KAN, and COT. The typical MDR-AmpC phenotype was present in S. Dublin, S. enterica serotype Enteritidis, S. Heidelberg, S. enterica serotype I 4,12:r:−, S. Kentucky, S. Newport, S. Saintpaul, and S. Typhimurium var. Copenhagen. Additional information regarding antimicrobial resistance and serotypes of Salmonella from retail meats is available at the NARMS website (http://www.fda.gov/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/NationalAntimicrobialResistanceMonitoringSystem/default.htm).

TABLE 3.
Most common TIO resistance profiles of Salmonella isolated from retail meatsa

Beta-lactam resistance genes and class 1 integrons.

For the beta-lactam resistance genes, 50% (n = 163) of the Ampr isolates carried blaCMY, 47% (n = 151) carried blaTEM-1, and 2.6% (n = 9) carried both genes. No strains were found to carry blaSHV, blaOXA, or blaCTX-M. All blaCMY-positive isolates were Tior with decreased susceptibility or resistance to AXO, whereas all isolates carrying blaTEM-1 and not blaCMY were susceptible to TIO. All isolates carrying both blaTEM-1 and blaCMY displayed MDR phenotypes with resistance to 8 to 11 antimicrobials of different classes, except for one S. Typhimurium var. Copenhagen isolate from chicken breast that showed resistance only to beta-lactams (AMP/AUG/FOX/TIO/TAZ). Twenty-one Ampr isolates did not contain any of the five beta-lactam resistance genes tested. Fifteen percent of isolates (n = 52) contained class I integrons carrying various combinations of aadA, aadB, and dfrA gene cassettes encoding resistance to STR, GEN/KAN, and COT, respectively.

Conjugation and transformation.

Nineteen blaCMY-positive Salmonella isolates representing 14 serotypes were selected as donor strains in conjugation and transformation based on the combination of resistance profile, serotype, and source and year of isolation (Table (Table1).1). The results showed that the blaCMY gene can be transferred by both filter mating and electroporation. However, filter mating showed more transfer than electroporation. Sixty-eight percent (13/19) of isolates successfully transferred blaCMY by filter mating and only 37% (7/19) by electroporation. Other resistance phenotypes such as resistance to CHL, TET, GEN, SMX, KAN, and STR were also cotransferred in some isolates by filter mating but in no cases by electroporation. Six isolates including serotypes S. Anatum, S. Agona, S. Derby, S. Kentucky, S. Typhimurium, and S. I 4,5,12: nonmotile successfully transferred blaCMY by both filter mating and electroporation, whereas four isolates comprised of S. Dublin, S. Enteritidis, S. Newport, and S. I 4,12:r:− did not transfer blaCMY by either technique. PCR confirmed that all 13 transconjugates carried blaCMY; one also carried a class 1 integron, and another carried both blaTEM-1 and a class 1 integron. Seven transformants showed the presence of only blaCMY.

PFGE profiles.

Genomic DNA digestion with XbaI and Bln1 generated a total of 249 PFGE profiles among the 344 Ampr isolates (data not shown). PFGE showed a good correlation with Salmonella serotypes among most isolates, where isolates of the same serotype clustered together. PFGE profiles showed that some serotypes were genetically more diverse than others (data not shown). For example, some isolates of S. Typhimurium and S. enterica serotype Bredeney formed multiple distinct clusters with 50 to 60% pattern similarity.

Among the 249 strain types identified by PFGE, some were recovered repeatedly from different states over the 5-year sampling time period (Fig. (Fig.1).1). Most clones were seen in only one type of meat product (Fig. (Fig.1,1, clusters A1, A2, B, and C), but several were seen from different meat products. In clone D, a beef isolate of S. Saintpaul shared the same PFGE profile with 12 turkey S. Saintpaul isolates (Fig. (Fig.1).1). Similarly, in clone C1 (Fig. (Fig.2),2), one turkey S. Newport isolate showed the same PFGE profile as two pork S. Newport isolates. This may be the result of cross-contamination at the retail outlet, as suggested by sampling time and place. Some PFGE clusters displayed good correlation with their antimicrobial resistance profiles (Fig. (Fig.2).2). For example, 21 S. Typhimurium isolates formed three clusters, each containing strains sharing 90 to 100% PFGE pattern similarity (Fig. (Fig.1,1, clusters A1, A2, and A3) and specific resistance profiles (AMP/AUG/FOX/TIO/KAN/SMX/TET, AMP/AUG/FOX/TIO/SMX/TET, and AMP/CHL/STR/SMX/TET). Such correlations were also seen in serotypes Kentucky, Newport, and Reading (Fig. (Fig.2,2, clusters B, C, and D).

FIG. 1.
XbaI/Bln1 PFGE profiles of selected of Ampr clones from Salmonella serotypes Heidelberg, Kentucky, Saintpaul, and Typhimurium.
FIG. 2.
PFGE and antimicrobial resistance profiles of selected Ampr Salmonella isolates. A black box in the antibiogram column indicates resistance to a particular antimicrobial. AMC, amoxicillin-clavulanic acid; SMX/FIS, SMX-sulfisoxazole.

DISCUSSION

Because of the importance of extended-spectrum cephalosporins for treating salmonellosis, resistance to this class of compounds in Salmonella is monitored. According to CDC NARMS data, resistance to extended-spectrum cephalosporins in human clinical isolates increased from 0.2% in 1996 to 4.9% in 2003 but declined to 2.9% in 2005 (http://www.cdc.gov/narmsAnnualReport2005.pdf). Our analysis of NARMS retail meat monitoring data showed that 50% of all Ampr Salmonella isolates were resistant to extended-spectrum cephalosporins, which accounted for 12.7% of all Salmonella recovered during 2002 to 2006. By source, Salmonella isolated from chicken breast showed the highest overall prevalence of Tior (10% to 25.3%) during the 5 years of sampling, followed by ground turkey (5% to 8.1%). In addition, certain serotypes were more likely to display Tior than others, such as S. Newport, S. Typhimurium, S. Kentucky, S. Heidelberg, S. Dublin, S. Agona, and S. Uganda (http://www.fda.gov/cvm/2006 NARMSAnnualRpt.htm) (33). Due to the limited number of isolates from ground beef and pork chops, it was difficult to assess the prevalence of Tior in these two commodities. However, there were no Tior Salmonella strains detected from 27 ground beef isolates and 17 pork chop isolates recovered in 2005 and 2006. Compared with USDA NARMS data on Salmonella from food animals during the same period (2002 to 2006), Tior was most prevalent in cattle isolates (13.3% to 21.6%), followed by chicken (9.8% to 12.8%), turkey (2.5% to 5.3%), and swine (2% to 4.3%) (http://www.ars.usda.gov/Main/docs.htm?docid=17320). The difference in the prevalence of Tior Salmonella between retail meats and food animals was most likely due to the sampling scheme. The NARMS animal program took samples from both slaughter house and veterinary clinics. As the Tior phenotype was often related to Salmonella serotypes and as specific serotypes such as S. Dublin, S. Agona, S. Reading, and S. Uganda, with high percentages of the Tior phenotype, were often isolated from sick cattle, the prevalence of Tior was likely greater in Salmonella isolates from clinical samples than from retail meats.

No ESBLs were found in Salmonella isolates from this study. However, all Tior isolates showed cross-resistance or decreased susceptibility to other beta-lactams. Some were coresistant to other classes of antimicrobials, including aminoglycosides, sulfonamides, COT, TET, and CHL, with 67% of isolates showing resistance to at least 5 antimicrobials and 5% of isolates showing resistance to at least 10 antimicrobials. Many of the conjugative plasmids in our study showed that they are MDR plasmids; in addition to carrying blaCMY, they also carried resistance to combinations of CHL, TET, GEN, SMX, KAN, and STR. These plasmids are often large, which makes them difficult to transfer by electroporation. A study by Welch et al. (30) showed that MDR-AmpC plasmid (pSN254) from S. Newport was larger than 170 kb and contained 12 resistance genes belonging to six different classes, including beta-lactams, aminoglycosides, SMX, TET, phenicols, and quaternary ammonium compounds. Sequence data showed that the plasmid contains two copies each of blaCMY, sul, and sugE. The sul and sugE genes encode resistance to SMX and to a subset of toxic quaternary ammonium compounds (including cetylpyridinium chloride), respectively. The plasmid backbone of pSN254 showed 99% DNA sequence homology with the IncA/C plasmid backbone commonly present in many members of the Enterobacteriaceae such as Yesinia, Aeromonas, and Vibrio (17, 20). This plasmid appears to be nearly universally present in Tior meat isolates of Salmonella (29) and is also present in MDR E. coli and Klebsiella from retail meats (30) The IncA/C backbone is also similarly detected in Tior Salmonella isolated from varieties of animal species (16).

Our study showed that the blaCMY and blaTEM-1 genes were the major contributors to beta-lactam resistance in food isolates of Salmonella. A similar finding was reported by Frye et al. using USDA NARMS cattle isolates from 2000 to 2004 (8), where the IncA/C plasmid also predominated. Screening human clinical non-Typhi Salmonella isolates with decreased susceptibility to quinolones and extended-spectrum cephalosporins recovered from 1996 to 2004, Whichard et al. reported the presence of blaSHV and blaOXA in addition to blaCMY and blaTEM-1 (31). In our study, 21 Ampr isolates from retail meats did not contain any of the five beta-lactam resistance genes tested. This may be due to the presence of other mechanisms such as efflux pumps (2), cell wall changes (22), or other undetected β-lactamases. It is not surprising that no ESBL-carrying Salmonella were identified since they are relatively rare in North America compared to other species in the Enterobacteriaceae family (19). Previous studies demonstrated that blaCMY-2 confers resistance to a spectrum of beta-lactam compounds and is responsible for resistance to first-, second-, and third-generation cephalosporins (36). It has been noticed in our study that blaCMY and blaTEM-1 are almost mutually exclusive, with only 2.6% isolates carrying both genes. We observed a 100% correlation between the presence of blaCMY and resistance to TIO, FOX, and AUG plus resistance/decreased susceptibility to AXO (4 to 64 μg/ml), TAZ (16 to 64 μg/ml), and FOT (8 to 64 μg/ml). The MIC differences for AXO, TAZ, and FOT beta-lactams among individual blaCMY-positive strains could be due to allelic differences. Among the 43 blaCMY alleles identified so far, relatively few have been characterized for their role is resistance to different beta-lactams (15). Alternatively, these differences may be due to synergistic or additive effects between blaCMY and other loci or undetected β-lactamases.

Our data suggest that Salmonella contamination of food can occur at different stages of meat production and processing. PFGE using XbaI and BlnI digestion generated 249 patterns among the 344 Ampr isolates examined. Several clones were identified from chicken breasts (Fig. (Fig.1,1, clones A1, B, and C; Fig. Fig.2,2, clusters A1 to A3 and B1 and B2) collected from different stores in different states from 2002 to 2006, suggesting that they may come from a common source at slaughter or in a processing plant or on a farm. Similar observations were made regarding isolates from ground turkey (Fig. (Fig.1,1, clone A2, and Fig. Fig.2,2, clone D) and from ground beef (Fig. (Fig.2,2, clone C2). Additionally, two S. Saintpaul clones, one from ground turkey and one from ground beef (Fig. (Fig.1,1, clone D), as well as a clone of S. Newport in ground turkey and pork chops (Fig. (Fig.2,2, clone C1), were found in meat samples from the same store during the same sampling month, suggesting that cross-contamination occurred during processing at the retail outlet. Similar findings were seen in our previous study of S. Heidelberg (35).

In summary, resistance to AMP and extended-spectrum cephalosporins in Salmonella is not uncommon in the U.S. retail meat supply, and its occurrence is due largely to the presence of TEM and CMY enzymes. CMY continues to be the only apparent explanation for extended-spectrum cephalosporin resistance. Ongoing testing of domestically produced meat commodities will help provide information needed to better understand risks associated with antimicrobial resistance of enteric pathogens in the domestic retail meat supply, including the detection of new resistant genotypes should they arise.

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 October 2009.

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