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

Abstract

Ampicillin-resistant (Amp^r) 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 (bla_CMY, bla_TEM, bla_SHV, bla_OXA, and bla_CTX-M) and class 1 integrons. Among the Amp^r 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 bla_CMY, 47% carried bla_TEM-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 bla_CMY, bla_TEM, and class 1 integrons were transferable through conjugation and/or transformation. Our findings indicate that a varied spectrum of coresistance traits is present in Amp^r Salmonella strains in the meat supply of the United States, with a continued predominance of bla_CMY and bla_TEM 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 bla_CMY 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. Tio^r 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 bla_TEM, bla_SHV, bla_CTX-M, bla_OXA, bla_PER, bla_PSE, and bla_CMY, 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 bla_CTX-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 (bla_SHV-12) and another from a 3-month-old child (bla_CTX-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 bla_KPC-2 gene (18). In contrast to the low prevalence of ESBL-carrying Salmonella strains in the United States, AmpC resistance mediated by bla_CMY has been emerging in both humans and food animals. The bla_CMY 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 Amp^r Salmonella isolates obtained from retail meats. We screened for the presence of five beta-lactam resistance gene families (bla_CMY, bla_TEM, bla_SHV, bla_OXA, and bla_CTX-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 Amp^r 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 Amp^r 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 Amp^r 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 bla_CMY, bla_TEM, bla_SHV, bla_OXA, bla_CTX-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 bla_CMY-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 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 experiments^a

Isolate no.	Salmonella serotype	Source	Year of isolation	Resistance profileb
21380	Heidelberg	Ground turkey	2002	FOX/TET/AUG/GEN/TIO/FIS/KAN/AMP/STR
23742	Dublin	Ground beef	2003	FOX/CHL/TET/AUG/TIO/FIS/AMP/STR
29440	Enteritidis	Ground beef	2003	FOX/CHL/TET/AXO/AUG/TIO/FIS/AMP/STR
30654	Agona	Ground turkey	2003	FOX/TET/AUG/TIO/FIS/AMP
30661	Enteritidis	Chicken breast	2003	FOX/AUG/TIO/AMP
32413	Kentucky	Chicken breast	2003	FOX/AUG/TIO/AMP/STR
N187	Bredeney	Ground turkey	2004	FOX/TET/AUG/GEN/TIO/FIS/AMP/STR
N418	Heidelberg	Ground turkey	2004	FOX/CHL/TET/AUG/GEN/TIO/FIS/KAN/AMP/STR
N499	Typhimurium var. Copenhagen	Chicken breast	2004	FOX/TET/AUG/TIO/FIS/KAN/AMP
N518	Kentucky	Chicken breast	2004	FOX/TET/AUG/TIO/FIS/KAN/AMP/STR
N635	Newport	Ground beef	2004	FOX/CHL/TET/AUG/TIO/FIS/AMP/STR
N1571	Derby	Ground turkey	2004	FOX/TET/AUG/TIO/AMP
N4534	I 4,5,12: nonmotile	Chicken breast	2005	FOX/TET/AUG/TIO/FIS/AMP
N4646	Typhimurium	Ground turkey	2005	FOX/AUG/TIO/AMP
N5389	I 4,12:r:−	Ground turkey	2005	FOX/CHL/TET/AUG/TIO/FIS/AMP/STR
N5396	Anatum	Chicken breast	2005	FOX/TET/AUG/TIO/AMP/STR
N6312	I 3,10: nonmotile	Ground turkey	2005	FOX/TET/AUG/TIO/FIS/KAN/AMP/STR
N6328	Bredeney	Ground turkey	2005	FOX/TET/AXO/AUG/TIO/FIS/AMP/STR
N6430	Typhimurium	Chicken breast	2005	FOX/TET/AUG/TIO/FIS/KAN/AMP

^aThe recipient strain was E. coli DH5α (Nal^r) (Invitrogen). All 19 isolates showed decreased susceptibility (MIC, 8 to 32 μg/ml) or resistance (MIC ≥ 64 μg/ml) to AXO.

^bFIS, sulfisoxazole.

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 Amp^r 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 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 Amp^r Salmonella isolates from NARMS retail meats from 2002 to 2006

Class and/or antimicrobial	Resistance breakpoint (μg/ml)a	% of isolates showing resistance (n = 344)
β-Lactams
AUG	≥32/16c	55.5
AMP	≥32	100
ATM	≥32	1.2
FEP	≥32	0
FOT	≥64	3.5
FOX	≥32	50
CQNb	≥32	0
TAZ	≥32	24.7
TIO	≥8	50
AXO	≥64	2
IMP	≥16	0
P/T	≥128/4d	4.9
Quinolones and fluoroquinolones
CIP	≥4	0
NAL	≥32	3.3
Aminoglycosides
AMI	≥64	0
GEN	≥16	24.4
KAN	≥64	30.8
SRTb	≥64	55.5
Sulfonamide or potentiated sulfonamide
SMX	≥512	55.5
COT	≥4/76e	1.7
TET	≥16	57.8
CHL	≥32	10.8

^aMIC (μg/ml) determined via microdilution broth methods in accordance with CLSI standards.

^bNo CLSI breakpoint.

^cValues are for amoxicillin-clavulanic acid, respectively.

^dValues are for piperacillin-tazobactam, respectively.

^eValues are for trimethoprim-SMX, respectively.

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 Tio^r isolates are listed in Table 3. Some serotypes such as S. Heidelberg showed very diverse resistance patterns such that 20 resistance patterns were observed among 56 Amp^r 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 meats^a

Resistance profile	No. of isolates
AMP/AUG/AXO/FOX/TIO	37
AMP/AUG/AXO/FOX/TIO/STR/TET	35
AMP/AUG/AXO/FOX/TIO/SMX/TET	27
AMP/AUG/AXO/FOX/TIO/KAN/SMX/TET	19
AMP/AUG/AXO/FOX/TIO/CHL/STR/SMX/TET	11
AMP/AUG/AXO/FOX/TIO/CHL/STR/SMX/TET/COT	7
AMP/AUG/AXO/FOX/TIO/CHL/GEN/KAN/STR/
SMX/TET	6

^aAll Tio^r isolates showed decreased susceptibility (MIC, 8 to 32 μg/ml) or resistance to AXO.

Beta-lactam resistance genes and class 1 integrons.

For the beta-lactam resistance genes, 50% (n = 163) of the Amp^r isolates carried bla_CMY, 47% (n = 151) carried bla_TEM-1, and 2.6% (n = 9) carried both genes. No strains were found to carry bla_SHV, bla_OXA, or bla_CTX-M. All bla_CMY-positive isolates were Tio^r with decreased susceptibility or resistance to AXO, whereas all isolates carrying bla_TEM-1 and not bla_CMY were susceptible to TIO. All isolates carrying both bla_TEM-1 and bla_CMY 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 Amp^r 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 bla_CMY-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 1). The results showed that the bla_CMY 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 bla_CMY 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 bla_CMY 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 bla_CMY by either technique. PCR confirmed that all 13 transconjugates carried bla_CMY; one also carried a class 1 integron, and another carried both bla_TEM-1 and a class 1 integron. Seven transformants showed the presence of only bla_CMY.

PFGE profiles.

Genomic DNA digestion with XbaI and Bln1 generated a total of 249 PFGE profiles among the 344 Amp^r 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. 1). Most clones were seen in only one type of meat product (Fig. 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. 1). Similarly, in clone C1 (Fig. 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. 2). For example, 21 S. Typhimurium isolates formed three clusters, each containing strains sharing 90 to 100% PFGE pattern similarity (Fig. 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. 2, clusters B, C, and D).

FIG. 1.

XbaI/Bln1 PFGE profiles of selected of Amp^r clones from Salmonella serotypes Heidelberg, Kentucky, Saintpaul, and Typhimurium.

FIG. 2.

PFGE and antimicrobial resistance profiles of selected Amp^r 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 Amp^r 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 Tio^r (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 Tio^r 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 Tio^r in these two commodities. However, there were no Tio^r 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), Tio^r 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 Tio^r 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 Tio^r 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 Tio^r phenotype, were often isolated from sick cattle, the prevalence of Tio^r 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 Tio^r 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 bla_CMY, 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 bla_CMY, 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 Tio^r 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 Tio^r Salmonella isolated from varieties of animal species (16).

Our study showed that the bla_CMY and bla_TEM-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 bla_SHV and bla_OXA in addition to bla_CMY and bla_TEM-1 (31). In our study, 21 Amp^r 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 bla_CMY-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 bla_CMY and bla_TEM-1 are almost mutually exclusive, with only 2.6% isolates carrying both genes. We observed a 100% correlation between the presence of bla_CMY 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 bla_CMY-positive strains could be due to allelic differences. Among the 43 bla_CMY 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 bla_CMY 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 Amp^r isolates examined. Several clones were identified from chicken breasts (Fig. 1, clones A1, B, and C; Fig. 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. 1, clone A2, and Fig. 2, clone D) and from ground beef (Fig. 2, clone C2). Additionally, two S. Saintpaul clones, one from ground turkey and one from ground beef (Fig. 1, clone D), as well as a clone of S. Newport in ground turkey and pork chops (Fig. 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.