ABSTRACT
Salmonella spp. are important food-borne pathogens that are demonstrating increasing antimicrobial resistance rates in isolates obtained from food animals and humans. In this study, 10 multidrug-resistant, cephalosporin-resistant Salmonellaisolates from bovine, porcine, and human sources from a single geographic region were identified. All isolates demonstrated resistance to cephamycins and extended-spectrum cephalosporins as well as tetracycline, chloramphenicol, streptomycin, and sulfisoxazole. Molecular epidemiological analyses revealed eight distinct chromosomal DNA patterns, suggesting that clonal spread could not entirely explain the distribution of this antimicrobial resistance phenotype. However, all isolates encoded an AmpC-like β-lactamase, CMY-2. Eight isolates contained a large nonconjugative plasmid that could transformEscherichia coli. Transformants coexpressed cephalosporin, tetracycline, chloramphenicol, streptomycin, and sulfisoxazole resistances. Plasmid DNA revealed highly related restriction fragments though plasmids appeared to have undergone some evolution over time. Multidrug-resistant, cephalosporin-resistant Salmonellaspp. present significant therapeutic problems in animal and human health care and raise further questions about the association between antimicrobial resistance, antibiotic use in animals, and transfer of multidrug-resistant Salmonella spp. between animals and man.
Salmonella spp. are important zoonotic pathogens in humans and animals. In the United States it is estimated that over 1.4 million cases of salmonellosis occur each year, 95% of which are the result of food-borne transmission (28). Large outbreaks have been associated with ingestion of poultry, meat, and milk and other dairy products (6). Although the majority of infections result in asymptomatic or self-limited diarrheal illness, severe, life-threatening bacteremias and other deep-seated infections do occur, particularly in immunocompromised hosts, neonates, and the elderly (7, 14).
Increasing rates of antimicrobial resistance in Salmonellaisolates have been reported from a number of developing and developed countries. In the United States, resistance to tetracycline increased from 9% in 1980 to 24% in 1990 and resistance to ampicillin increased from 10 to 14% (25). In Britain, rates of antimicrobial resistance for Salmonella enterica serovar Typhimurium were higher, with 45% of isolates resistant to tetracyclines, 40% of isolates resistant to sulfonamides, and 17% of isolates resistant to ampicillin (42). Much of this multidrug resistance has been linked to the spread of a single strain of Salmonellaserovar Typhimurium, definitive phage type 104 (DT104), through food animals and humans (16). Most of these multidrug-resistant DT104 isolates have a chromosomal gene cluster that codes for resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (11, 37). Of increasing concern is the fact that animal- and human-associated multidrug-resistant DT104 isolates resistant to quinolones have been reported (29, 41).
In this study, we report the identification and molecular characterization of bovine, porcine, and human multidrug-resistantSalmonella isolates that are resistant to extended-spectrum cephalosporins and cephamycins. A plasmid-mediated CMY-2ampC-like gene was identified in all animal and human isolates. Plasmid-mediated AmpC-type β-lactamases have been identified in Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Enterobacter aerogenes clinical isolates from humans in the United States, Europe, and other regions (5, 9, 44). Reports of humanSalmonella isolates expressing an AmpC-like β-lactamase have been quite rare (15, 23), though the prevalence may be increasing (E. F. Dunne, P. F. Fey, P. Shillam, P. Kludt, W. Keene, E. Harvey, K. Stamey, T. Barrett, N. Marano, and F. J. Angulo, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 716, 1999). Additionally, animal Salmonella isolates expressing an AmpC-like enzyme have not been reported. In this study, the molecular characterization of cephalosporin-resistantSalmonella isolates from food animals and humans residing in a single geographic region is reported. These results underscore concern for increasing antimicrobial resistance inSalmonella spp. and suggest that careful epidemiological and antimicrobial surveillance studies are needed to assess the selective conditions associated with cephalosporin resistance amongSalmonella isolates from farm animals and the association of these isolates with human disease.
MATERIALS AND METHODS
Organisms.A total of 158 isolates of Salmonellaspp. were recovered from various animals between November 1998 and May 1999 at the Iowa State University Veterinary Diagnostic Microbiology Laboratory. Organisms were obtained from liver, stool, intestine, lung, and lymph node samples of diseased animals. A total of 320 human isolates of Salmonella spp. were analyzed. These isolates had been referred to the Iowa State Hygienic Laboratory from numerous microbiology laboratories throughout the state of Iowa. All isolates were transferred to the Medical Microbiology Division of the Department of Pathology at the University of Iowa College of Medicine for further characterization. Isolates were stored at −70°C on porous beads (ProLab, Austin, Tex.) until further use.
Antimicrobial susceptibility testing.MICs of selected antimicrobials were determined by broth microdilution as described by the National Committee for Clinical Laboratory Standards. Custom-designed, cation-adjusted Mueller-Hinton broth microdilution trays purchased from TREK Diagnostic Systems Inc., Westlake, Ohio, were inoculated and incubated at 35°C for 16 to 20 h (21, 31). Sulfadiazine and sulfisoxazole susceptibility tests were performed by disk diffusion (21, 31). Cefoxitin and streptomycin MICs were determined using the E test methodology as described by the manufacturer (AB Biodisk, Solna, Sweden). Briefly, organisms were diluted to a McFarland standard of 0.5 and streaked onto Mueller-Hinton agar plates. After E-test strip application, the plates were incubated at 35°C for 16 to 20 h.
Isoelectric focus analysis.Crude β-lactamase extracts were prepared by freeze-thaw lysis of bacterial cultures grown to exponential-growth stage in tryptic soy broth as previously described (8). Analytical isoelectric focusing was performed using a Multiphore II electrophoresis system with commercially prepared ampholine-polyacrylamide plates (pI 3.5 to 9.5; Amersham Pharmacia Biotech, Piscataway, N.J.). β-lactamase activity was detected with 0.5 mg of nitrocephin (Becton-Dickinson, Franklin Lakes, N.J.) per ml. TEM-1, TEM-4, SHV-1, SHV-3, and SHV-5 β-lactamases expressed inE. coli C600 were used as isoelectric focus standards. These enzymes are known to migrate at pIs of 5.4, 5.9, 7.6, 7.0, and 8.2, respectively (22). Known pIs of each standard were plotted against the distance from the cathode, and a regression analysis was performed (using Microsoft Excel 98 software). Unknown β-lactamase pIs were calculated using the regression curve generated from each gel.
Pulsed-field gel electrophoresis.Genomic DNA was isolated and digested with XbaI (New England Biolabs, Beverly, Mass.) as previously described (32). Electrophoresis was performed on the CHEF-DRII (Bio-Rad Laboratories, Richmond, Calif.) with the following conditions: 0.5× Tris-borate-EDTA, 1% agarose, 13°C, 6 V/cm for 23 h (with switch times ranging from 5 to 60 s). Molecular weight standards were a λ ladder that contained concatamers of the 48.5-kb phage DNA (FMC BioProduct, Rockland, Maine). Gels were stained with ethidium bromide and photographed using a Gel Doc 1000 system (Bio-Rad Laboratories). Strains which contained restriction fragment patterns that differed by more than three bands were considered unique (2).
Molecular techniques.Plasmid DNA was isolated using the Concert Mini-Prep system (Gibco BRL). Alternatively, large plasmids were isolated using a protocol described previously for isolation of large bacterial artificial chromosome plasmid DNA (36). Large plasmid DNA was then digested using PlasmidSafe DNase (Epicentre Technologies, Madison, Wis.) according to the manufacturer's recommendations. Conjugation experiments were performed as previously described (35). Briefly, cultures of the E. colirecipient HB101 containing pEm7/Zeo (a Zeocin resistance plasmid; Invitrogen, Carlsbad, Calif.) and the donor Salmonellaisolate were grown overnight in Luria broth (LB). A 10:1 suspension of donor-recipient culture was diluted in fresh LB. Aliquots were spotted onto sterile filters placed on LB plates and incubated overnight. Filters were eluted in sterile saline, and serial dilutions were plated on LB plus Zeocin (50 μg/ml) and cefoxitin (50 μg/ml). Transformation of large plasmid DNA from the Salmonellaisolates was performed using standard electroporation techniques with DH10B electrocompetent E. coli (Gibco BRL, Grand Island, N.Y.). Transformants were selected on LB agar containing 50 μg of cefoxitin (Sigma, St. Louis Mo.) per ml. Plasmid DNA restriction fragment length polymorphisms were analyzed by agarose gel electrophoresis of plasmid DNA cleaved with various restriction endonucleases (New England Biolabs).
PCR analysis was performed on total DNA as prepared using the CTAB protocol described previously (4) or plasmid DNA treated with PlasmidSafe DNase. Amplification was performed with consensus primers for the bla genes encoding BIL-1, LAT-1, LAT-2, and CMY-2 and the ampC gene of Citrobacter freundiiOS60 (ampC1, 5′-ATGATGAAAAAATCGTTATGC-3′;ampC2, 5′-TTGCAGCTTTTCAAGAATGCGC-3′ [23]) or TEM-1 (5′-CCCGAATTCGGAAGAGTATGAGTATTC-3′ and 5′-CCCGGATCCCAGTTACCAATGCTTAATC-3′). PCR fragments were isolated using Qiaquick PCR cleanup columns (Qiagen, Valencia, Calif.). DNA sequence analysis was performed using Big Dye terminator cycle sequencing chemistry with AmpliTaq polymerase FS enzyme (Applied Biosystems, Foster City, Calif.). The reactions were performed and analyzed with an Applied Biosystems model 373A stretch fluorescent automated sequencer at the University of Iowa DNA Core Facility.
RESULTS
Characterization of animal and human Salmonellaisolates.Eight of 158 (5.1%) Salmonella isolates recovered from symptomatic large animals were determined to be resistant to extended-spectrum cephalosporins (ceftazidime, cefotaxime, and ceftiofur), a monobactam (aztreonam), and a cephamycin (cefoxitin), as well as to ticarcillin and piperacillin (Tables1 and 2). The β-lactamase inhibitor clavulanic acid (fixed concentration of 2 μg/ml) had no effect on ticarcillin or ampicillin MICs (data not shown). As seen in other CMY studies, tazobactam reduced piperacillin MICs by fourfold or more in most isolates, though in this study the majority of isolates remained resistant to this antimicrobial combination (23, 44). All eight isolates were resistant to tetracycline, sulfamethoxazole, streptomycin, and chloramphenicol. In addition, six of eight isolates were resistant to gentamicin and three of eight isolates were resistant to trimethoprim-sulfamethoxazole. One isolate (isolate 613) was intermediate to ciprofloxacin and resistant to nalidixic acid.
Cephalosporin-resistant Salmonella isolates
Antimicrobial susceptibilities
All eight isolates had been obtained from bovine or porcine sources in geographically distinct areas of Iowa. The majority of isolates were obtained from deep-seated sites, including lung, intestine, and liver or lymph node, with one isolate recovered from stool. Organisms represented a variety of serotypes though five isolates wereSalmonella serovar Typhimurium or Salmonellaserovar Typhimurium subsp. copenhagen. None of these isolates were DT104.
Two of 320 (0.6%) human isolates of Salmonella spp. submitted to the Iowa State Hygienic Laboratory during 1998 were found to have antibiograms similar to the animal isolates. Both had been recovered from stool specimens in two geographically distinct cities in Iowa. The serotypes were Salmonella serovar Typhimurium andSalmonella serovar Newport. The Salmonellaserovar Typhimurium isolate, 1339, was DT104. Both human isolates were resistant to cephalosporins as well as tetracycline, streptomycin, chloramphenicol, and sulfamethoxazole.
Molecular epidemiology.The clonal relatedness of the 10 multidrug-resistant isolates of Salmonella was assessed by pulsed-field gel electrophoresis (PFGE) analysis of restriction endonuclease-digested chromosomal DNA (Fig.1 and Table 1). Overall, eight distinct PFGE patterns were identified in the 10 organisms. Two bovine isolates demonstrated identical chromosomal DNA patterns, and two porcine isolates shared a different restriction fragment pattern. There was no obvious geographic link between isolates demonstrating similar PFGE patterns. Additionally, the PFGE patterns of the human isolates differed from those of the animal isolates.
PFGE of XbaI-digested chromosomal DNA ofSalmonella isolates. Isolates 912993, 12386, and 16033 are previously characterized Salmonella serovar Typhimurium DT104 isolates (13). MWM, molecular weight standard (a λ DNA ladder containing concatamers of the 48.5-kb phage DNA).
Molecular analysis of cephalosporin resistance.The antibiogram demonstrated by these organisms was consistent with the expression of a cephalosporinase similar to the chromosomally encoded inducible AmpC enzymes found in many members ofEnterobacteriaceae. However, Salmonella species are not known to encode an inducible chromosomal AmpC enzyme (27). To determine whether these organisms expressed a β-lactamase, crude bacterial isolates were analyzed by isoelectric gel electrophoresis. A β-lactamase with a pI of ≥8.7 was detected in all 10 isolates. Additionally, five isolates demonstrated a second β-lactamase that comigrated with the TEM-1 control, with a pI of 5.4.
Conjugation studies were performed to determine whether cephalosporin resistance could be transferred to E. coli. Despite multiple attempts, resistance could not be transferred by conjugation to a recipient E. coli strain carrying a Zeocin resistance plasmid (HB101pE7zeo). Cephalosporin resistance, however, could be transferred through bacterial transformation. DH10B electrocompetent bacteria were transformed with Salmonella plasmid DNA. Eight of 10 Salmonella isolates transferred cephalosporin resistance. Additionally, resistances to tetracycline, sulfamethoxazole, and chloramphenicol were transferred to E. coli. Since DH10B E. coli is streptomycin resistant, this experiment was unable to determine whether streptomycin resistance was also present on plasmid DNA. Transfer of gentamicin resistance was less consistent. Only two of four Salmonella isolates carrying gentamicin resistance transferred this resistance to E. coli. Trimethoprim-sulfamethoxazole resistance cosegregated in one of two isolates while quinolone resistance did not cotransfer from the one isolate that expressed this resistance. All transformants expressed plasmid DNA that migrated at approximately 75 kb on agarose gels. Additionally, all transformants demonstrated β-lactamase with a pI of ≥8.7. Two of three Salmonella isolates that originally expressed a β-lactamase with a pI of 5.4 were able to transfer expression of this enzyme to E. coli (data not shown).
The sequences, hydrolysis patterns, and isoelectric points of a number of β-lactamases have been identified over the past decade (12). Review of the literature revealed a chromosomal AmpC enzyme identified in C. freundii that migrated at a pI of approximately 8.6 to 8.7 in isoelectric focus analysis (26, 39). Additionally, a single Salmonella serovar Seftenberg clinical isolate has been reported to carry a plasmid-mediated cephalosporinase that showed significant homology to the C. freundii ampC gene (23). PCR analysis of total bacterial DNA was performed using consensus primers for theCitrobacter family of ampC genes (23). A 1,143-bp fragment was amplified from all 10 Salmonellaisolates while an unrelated Enterobacter cloacae isolate known to express a different AmpC enzyme and a wild-typeSalmonella isolate remained negative (data not shown).
Each PCR fragment was isolated and the entire nucleotide sequence of the ampC-like gene was determined from both strands. All animal and human isolates contained an identical DNA sequence that has been designated CMY-2, an enzyme identified in a K. pneumoniae isolate found in Greece (5). A highly related though distinct enzyme, which differs only by two amino acids in the signal peptide sequence, has been identified in a singleSalmonella serovar Seftenberg organism isolated from an Algerian child (23). TEM-1 PCR primers amplified the TEM gene from isolates 263, 272, 274, 370, 532, and 613, all of which expressed a β-lactamase with a pI of 5.4.
Plasmid restriction fragment length polymorphisms.Plasmid DNA from the E. coli transformants was isolated and digested with PstI, EcoRI (Fig.2) or BamHI (data not shown). Complete identity between any plasmids was not observed. However, many fragments were shared among the transformants, suggesting that the plasmids may share a highly related plasmid backbone.
PstI- or EcoRI-digested plasmid DNA isolated from the DH10B E. coli transformants.
DISCUSSION
Antibiotic resistance in Salmonella has intensified substantially worldwide (19, 25, 33, 42, 46, 47). For years, ampicillin, trimethoprim-sulfamethoxazole, and chloramphenicol were the recommended antimicrobial agents for severe Salmonellainfections. Rising rates of resistance to these agents have significantly reduced the efficacy of these agents. Consequently, fluoroquinolones and expanded-spectrum cephalosporins have become the recommended antimicrobial agents for invasive Salmonellainfections. Multidrug-resistant, quinolone-resistantSalmonella strains are now being reported (29). The present study describes multidrug-resistant, cephalosporin-resistant Salmonella isolates from both food animals and humans.
Salmonella isolates resistant to broad-spectrum cephalosporins were first reported in the 1980s, and since that time additional isolates have been identified (10, 30). The majority of cephalosporin-resistant Salmonella isolates express an extended-spectrum β-lactamase able to hydrolyze oxyimino cephalosporins and monobactams but not the cephamycins. Recently, a second mechanism of cephalosporin resistance in Salmonellahas been observed (4, 23). These isolates express plasmid-mediated AmpC-like β-lactamases that hydrolyze the cephamycins as well as the extended-spectrum cephalosporins and monobactams. The isolates described in the present study express a plasmid-mediated CMY-2 AmpC-like enzyme that has been identified previously in a single Salmonella isolate from Algeria (23). This enzyme belongs to a small family of plasmid-mediated AmpC-like enzymes (LAT-1, LAT-2, BIL-1, CMY-2 and -2b, CMY-3, CMY-4, and CMY-5) that share homology with the chromosomalampC from C. freundii (5, 23, 44, 45). Further analysis has shown that the plasmid-encoded CMY-5 gene is followed by the blc and sugE genes of C. freundii and this genetic organization is identical to that found on the C. freundii chromosome, providing strong evidence that the CMY genes have been translocated from Citrobacterto other bacterial species (45).
AmpC-mediated cephalosporin-resistance in Salmonella appears to be more widespread in the United States than previously thought. The prevalence of cefoxitin and cefotaxime resistance in human isolates from this study was 0.6%, which is quite similar to recent (1998) data from the CDC National Antimicrobial Resistance Monitoring System, where 0.7% of over 1,400 U.S. isolates were resistant to ceftriaxone (Dunne et al., 39th ICAAC). The majority of the CDC isolates expressed a CMY-2-, LAT-1-, BIL-1-like enzyme (Dunne et al., 39th ICAAC). There are four additional isolates reported from the United States, though three of the four may have been acquired outside the United States (18, 20).
Salmonella is widespread in nature and can colonize or infect a variety of domesticated and wild animals ranging from mammals to birds and reptiles. Most human nontyphoidal Salmonellainfections in the United States are related to ingestion of contaminated food products rather than person-to-person transmission or direct fecal-oral transmission. Many outbreaks have been traced to ingestion of contaminated animal products, in some cases, derived from specific farms, flocks, or herds of animals (1, 29, 38). A recent outbreak in Denmark of quinolone-resistant Salmonellaserovar Typhimurium DT104 infections was traced to contaminated pork products (29). Additional investigation identified two infected swine herds responsible for the outbreak (29). Sporadic cases have also been associated with consumption of animal products such as raw eggs and unpasteurized milk (17).
The extended-spectrum cephalosporin ceftiofur has been approved for therapeutic veterinary use in the United States. This agent is used commonly for respiratory tract infections, metritis, foot rot, and abscess prophylaxis in day-old chicks. However, very little is known regarding the frequency of use of this agent. Nearly 6% of the veterinary Salmonella isolates examined in this study were resistant to extended-spectrum cephalosporins. This percentage does not reflect the overall carriage rate in asymptomatic animals or symptomatic animals that may have responded to empiric therapy, since all isolates were obtained from symptomatic animals. Nonetheless, the prevalence of resistance is striking. Also noteworthy is the fact that all isolates demonstrated resistance to streptomycin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. ThoughSalmonella serovar Typhimurium, DT104 is the most common multidrug-resistant serotype, with 32% of recent isolates carrying the ampicillin-chloramphenicol-streptomycin-sulfamethoxazole-tetracycline resistance phenotype, other serotypes express a similar resistance phenotype (1998 annual report of the National Antimicrobial Resistance Monitoring System, Centers for Disease Control and Prevention [http://www.cdc.gov/ncidod/dbmd/narms/98toc.htm]).
Antibiotic resistance can be spread throughout populations by epidemic spread of a particular isolate or through exchange of genetic material. In the present study, two pairs of isolates expressed identical or highly related PFGE patterns, possibly revealing a clonal relationship between some of the isolates. However, the fact that eight different PFGE patterns were identified suggests that the distribution of these isolates was not entirely due to clonal spread. Despite the fact that human salmonellosis is strongly linked to food from animal sources, there was no clonal relationship between the two human and eight animal isolates.
A nonconjugative but transferable plasmid encoding the CMY-2 gene was identified in 80% of isolates in this study. All E. colitransformants demonstrated a plasmid of approximately 75 kb. Though the plasmids were not identical, in that cotransfer of gentamicin or trimethoprim-sulfamethoxazole occurred in only a subset of isolates and RFLP patterns were different, the cotransfer of CMY-2, chloramphenicol, sulfamethoxazole, tetracycline, and possibly streptomycin in all isolates suggests that a highly related gene cluster may reside on each plasmid from the human and animal isolates. Additionally, the RFLP patterns identified many highly conserved restriction fragments among each of the plasmids, suggesting that the plasmids may be genetically similar but that they may have evolved over time. Previous studies have shown that plasmid DNA can change rapidly (24, 40).
DT104 isolates of Salmonella typically carry a chromosomal integron that encodes all or a subset of the antimicrobial resistance genes (11, 37). More recently, multidrug-resistantSalmonella serovar Typhimurium isolates that contain a similar integron-associated gene cluster encoded on a transferable plasmid have been identified (43). Transformants in this study were analyzed for type 1 integrons (34). All animal transformants demonstrated one or more integrons though preliminary evidence did not find the CMY-2 gene to be encoded within an integron (P. L. Winokur, unpublished data). It is tempting to speculate that acquisition of cephalosporin resistance may relate to therapeutic ceftiofur use. However, persistence of these multidrug-resistant strains of Salmonella spp. in farm animals may be further encouraged by the use of other antimicrobials as growth promotants, a common practice in the veterinary industry.
The results of this study do not definitively prove spread of multidrug-resistant Salmonella from an animal source to humans. However, the genetic relatedness of the plasmids identified and their prevalence in the animal isolates is suggestive. Additional studies will be required to further explore the association of resistance with various antibiotic use practices in food animals carrying a cephalosporin-resistant Salmonella and the possible transfer of multidrug-resistant, cephalosporin-resistantSalmonella spp. between animals and humans.
ACKNOWLEDGMENTS
We thank M. Loeffeholz at the Iowa State Hygienics Laboratory for providing human Salmonella isolates; B. D. Jones, University of Iowa, for providing the Salmonella serovar Typhimurium DT104 isolates; and M. Cormican and C. O'Hare, University of Ireland, Galway, United Kingdom, for phage type analysis. P.L.W. was supported in part by a VA Merit Review award.
ADDENDUM
In a recent study, Fey et al. (14a) identified two multidrug-resistant, cephalosporin-resistant Salmonellaisolates that each carry a 160-kb plasmid encoding the CMY-2 gene. These isolates, one human and one bovine, were epidemiologically linked and shared similar PFGE patterns. These data suggest that ceftriaxone-resistant Salmonella has been transmitted from food animals to humans.
FOOTNOTES
- Received 18 February 2000.
- Returned for modification 21 June 2000.
- Accepted 25 July 2000.
- Copyright © 2000 American Society for Microbiology
