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Appl Environ Microbiol. Aug 2001; 67(8): 3732–3734.
PMCID: PMC93080

Horizontal Transfer of a Multi-Drug Resistance Plasmid between Coliform Bacteria of Human and Bovine Origin in a Farm Environment

Abstract

Multi-drug-resistant coliform bacteria were isolated from feces of cattle exposed to antimicrobial agents and humans associated with the animals. Isolates from both cattle and humans harbored an R plasmid of 65 kb (pTMS1) that may have been transferred between them due to selective antibiotic pressure in the farm environment.

The amount of antimicrobial agents used for therapeutic and nontherapeutic purposes in agriculture far exceeds what is used for humans in many parts of the world (11). Since exposure to antimicrobial agents is the most important factor with regard to development of antimicrobial resistance, animals and animal products could thus be significant sources of resistant bacteria for the human population (1, 5, 12, 17, 18). Nonpathogenic, multiple-drug-resistant Escherichia coli in the intestine is probably an important reservoir of resistance genes (3, 10, 13, 15, 21), and drug-resistant, intestinal E. coli of animal origin may colonize the human intestine, at least temporarily (14, 15, 20). However, the ease with which bacteria acquire new resistance genes by self-transmissible and mobilizable plasmids and conjugative transposons may represent a more significant contribution to the increasing incidence of resistant strains (19, 23, 24).

In this study, farm inhabitants were investigated for the occurrence of multi-drug-resistant intestinal E. coli. On the farm studied here, various antimicrobials had been used extensively, primarily to treat recurrent Staphylococcus aureus mastitis in dairy cattle (L. Sølverød, personal communication). One family lived on the farm, and one veterinarian had served the animals. During the spring of 1996, fecal swabs (Culturette; Becton Dickinson Europe, Meyland, France) were collected from 13 cattle, three family members, and the local veterinarian. One year later, sampling of the family members and the veterinarian was repeated, and fecal swabs from four other veterinarians operating sporadically in the area were also included. For a primary screening of the total aerobic fecal flora, each swab was plated onto blood agar (blood agar base [Difco Laboratories, Detroit, Mich.] containing 5% citrated bovine blood) and Mueller-Hinton agar (Difco) with Neo-Sensitabs (Rosco Diagnostica, Taastrup, Denmark) containing 33 μg of ampicillin (AMP), 80 μg of tetracycline (TET), 100 μg of streptomycin (STR), 5.2 μg of trimethoprim (TMP), 240 μg of sulfonamides (SUL), 60 μg of chloramphenicol, and 10 μg of enrofloxacin. Agar plates were incubated at 37°C for 24 h. A total of six cattle and five human samples exhibited multiple-drug-resistant patterns (Ampr Tetr Strr Tmpr Sulr). From these plates, three colonies were picked from the zones close to the Neo-Sensitabs containing AMP, TET, and SUL, respectively. Colonies were subcultivated on blood agar and BTB-lactose agar plates to assure pure cultures and retested for susceptibility to the above-mentioned antimicrobials. MICs of AMP, TET, STR, TMP, and SUL were determined (16). A total of 39 of 90 lactose-fermenting (coliform) bovine and human isolates expressed resistance to more than one antimicrobial and were selected for further studies. Species identification was performed by using Enterotube (Becton Dickinson). A selection of three bovine and three human E. coli isolates with the phenotype Ampr Tetr Strr Tmpr Sulr was serotyped at Statens Seruminstitut, Copenhagen, Denmark. Plasmid DNA was isolated by using the Maxi kit (Qiagen GmbH, Hilden, Germany), digested with EcoRI, HindIII, and BamHI (Gibco BRL, Gaithersburg, Md.), and hybridized with probes for various resistance determinants (Table (Table1)1) by using the AlkPhos Direct Nucleic Acid Labelling and Detection system (Amersham Life Science). Class 1 integrons, commonly carried by large conjugative plasmids, are of particular importance in development of multiple-drug resistance in gram-negative bacteria (7, 8) and were detected by using the Gene Amp PCR Reagent kit (Perkin-Elmer Cetus, Norwalk, Conn.) with plasmid DNA as a template, primers corresponding to the 5′ end of the integrase gene intI (5′-TGATATTATGGAGCAGCAGCAACGATG-3′) (EMBL accession no. M73819), and an internal region of the dfrI gene cassette (5′-GTATCTACTTGATCGATCAGGC-3′) (EMBL accession no X00926). The multi-drug-resistant human and bovine coliforms were used as donors in broth mating experiments with nalidixic acid (NAL)-resistant, plasmid-free E. coli DH5 (MICNAL > 20 μg/ml) as a recipient. Transconjugants were selected on Mueller-Hinton agar containing 20 μg of NAL/ml and 40 μg of sulfadiazine/ml.

TABLE 1
Resistance gene probes used in hybridization experiments

Of 39 multi-drug-resistant coliform isolates, 30 carried the phenotype Ampr Tetr Strr Tmpr Sulr and the MICs of AMP, TET, STR, TMP, and SUL were >256 μg/ml. Table Table22 shows characteristics of selected isolates. These were all E. coli, except one human isolate of Citrobacter freundii, and the data support the view that E. coli is a major carrier of resistance traits in the coliform flora of both humans and animals. Multi-drug-resistant commensal strains within the Citrobacter species are only occasionally described (21).

TABLE 2
Characteristics of selected multi-drug-resistant coliform bacteria isolated from cattle and humans in a farm environment

A single plasmid of approximately 65 kb, designated pTMS1, was found in eight bovine and five human multi-drug-resistant isolates that were studied in detail (Table (Table2).2). Restriction analysis of pTMS1 from both sources showed identical profiles. Hybridization studies showed that sulI, sulII, dfrI, strA-strB, and tetB resistance determinants were associated with pTMS1. The hybridizing patterns were identical for all probes, suggesting a similar organization of resistance genes, and the presence of sulI was evidence of a class 1 integron. Furthermore, PCR results indicated that pTMS1 contained the dfrI gene downstream of and adjacent to the intI gene of the class 1 integron. Despite identical restriction and hybridization patterns, no PCR product was obtained when the 65-kb plasmid of C. freundii was used as template DNA, indicating a different organization of gene cassettes in this isolate.

No identical serotypes were found when comparing bovine and human E. coli carrying pTMS1 that were O100:H− and O20:H+ or nontypable, respectively. Although only six strains were serotyped, this result indicates that resistance was disseminated through horizontal transfer of resistance genes, rather than by transfer of the resistant isolates themselves.

Transconjugants expressing resistance to NAL, AMP, TET, STR, TMP, and SUL were obtained at moderate frequencies (approximately 5 × 10−5) only when bovine E. coli strains were donors. This could be because conjugation was dependent of host factors that were not present in the human strains. Transfer of a plasmid of approximately 65 kb, with a restriction pattern identical to that of pTMS1, was confirmed by isolation of the plasmid from donor, recipient, and transconjugants.

The results indicate persistence of pTMS1 in E. coli in the intestine of one farm inhabitant and the local veterinarian (Table (Table2).2). Also, a second farm inhabitant seemed to have acquired E. coli harboring pTMS1 between the first and second sampling. The persistence of antibiotic resistance is not fully understood, but one hypothesis is that acquisition of resistance genes results in gain of fitness and colonizing ability (23). However, we cannot exclude the possibility that multi-drug-resistant fecal flora were established due to other factors before or during this study.

The penicillin- and tetracycline-resistant S. aureus causing mastitis in the herd shared a single plasmid of approximately 20 kb harboring the tetA(K) determinant, while the blaZ gene is chromosomally located (29). S. aureus and E. coli differ with regard to resistance genes, and genetic exchange between staphylococci and coliform bacteria is unlikely to occur. The results of this study do, however, demonstrate how antibacterial treatment targeted at a pathogenic organism in one organ system may affect the endogenous flora of other organ systems in the exposed individual and in its environment.

Acknowledgments

The present study was founded by the Norwegian Research Council (project number 122758/310).

We thank Liv Sølverød (TINE Norwegian Dairies) for organizing the collection of bacterial strains and the farmers and veterinarians participating in the project. We also thank Marianne Sunde and Henning Sørum (The Norwegian School of Veterinary Science) for supplying PCR primers, plasmid RSF 1010, E. coli strain Se132, and plasmids used as molecular weight markers and in the construction of resistance gene probes.

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