Multi-drug-resistant coliform bacteria were isolated from feces of cattle exposed to antimicrobial ... 15, 21), and drug-resistant, intestinal E. coli of animal origin.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2001, p. 3732–3734 0099-2240/01/$04.00⫹0 DOI: 10.1128/AEM.67.8.3732–3734.2001 Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Vol. 67, No. 8
Horizontal Transfer of a Multi-Drug Resistance Plasmid between Coliform Bacteria of Human and Bovine Origin in a Farm Environment HANNE OPPEGAARD,* TERJE M. STEINUM,
AND
YNGVILD WASTESON
Department of Pharmacology, Microbiology, and Food Hygiene, The Norwegian School of Veterinary Science, N-0033 Oslo, Norway Received 12 February 2001/Accepted 3 June 2001
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.
sulfonamides (SUL), 60 g of chloramphenicol, and 10 g of enrofloxacin. Agar plates were incubated at 37°C for 24 h. A total of six cattle and five human samples exhibited multipledrug-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 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⬘-TGATATTATGGAGCAGCAGCAACGATG3⬘) (EMBL accession no. M73819), and an internal region of the dfrI gene cassette (5⬘-GTATCTACTTGATCGATCAGG C-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 g/ml) as a recipient. Transconjugants were selected on Mueller-Hinton agar containing 20 g of NAL/ml and 40 g of sulfadiazine/ml. 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 g/ml. Table 2 shows
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 g of ampicillin (AMP), 80 g of tetracycline (TET), 100 g of streptomycin (STR), 5.2 g of trimethoprim (TMP), 240 g of * Corresponding author. Mailing address: Department of Pharmacology, Microbiology, and Food Hygiene, The Norwegian School of Veterinary Science, P.O. Box 8146 Dep, N-0033 Oslo, Norway. Phone: 47 22 96 47 68. Fax: 47 22 96 48 18. E-mail: hanne.oppegaard@veths .no. 3732
VOL. 67, 2001
R PLASMIDS OF COLIFORM BACTERIA IN CATTLE AND HUMANS
TABLE 1. Resistance gene probes used in hybridization experiments Resistance determinanta
Probe descriptionb
sulI sulII dfrI dfrIV dfrXII
Reference
660-bp SacII-BglII fragment of plasmid pGS72 780-bp HincII fragment of plasmid pSUL204 500-bp HpaI fragment of plasmid pFE872 1,600-bp ClaI fragment of plasmid pUK1148 500-bp BamHI-HindIII fragment of plasmid pBEM155 tetA 750-bp SmaI fragment of plasmid RP4 tetB 2,800-bp BglII fragment of plasmid R222 tetC 1,400-bp AvaI-HindIII fragment of plasmid pBR322 strA-strB 538 bp PCR product of plasmid RSF1010
27 22 6 28 25 4 9 2 26
a
tetA, -B, and -C are classes of tetracycline resistance genes; sulI and sulII are classes of sulfonamide-resistant dihydropteroate synthases; dfrI, dfrIV, and dfrXII are different types of trimethoprim-resistant dihydrofolate reductases. b All plasmids were propagated in E. coli host strains.
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. Multidrug-resistant commensal strains within the Citrobacter species are only occasionally described (21). 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 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
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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 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-drugresistant 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. 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
TABLE 2. Characteristics of selected multi-drug-resistant coliform bacteria isolated from cattle and humans in a farm environment Strain and isolate
E. coli F5-96 F10-96 F37-96 F1-96 F2-97 F14-96 F15-97 F60-96 F14-97 F18-97 F22-97 C. freundii F20-96 a b c
Origin (date)a
Cow 129 Cow 125 Cow 124 Human 1 Human 1 Human 3 Human 3 Human 4 Human 4 Human 5 Human 6 Human 3
(1996) (1997) (1996) (1997) (vet 1) (vet 1) (vet 2) (vet 3) (1996)
(1996) (1997) (1997) (1997)
Serotypeb
O100:H⫺ O100:H⫺ O100:H⫺ NDc Oru:H⫹ Oru:K1:H⫺ ND O20:H⫹ ND ND ND
Resistance pattern3
Ampr Tetr Ampr Tetr Ampr Tetr Tetr Strr Ampr Tetr Ampr Tetr Ampr Tetr Ampr Tetr Ampr Tetr Ampr Tetr Ampr Tetr Ampr Tetr
Strr Tmpr Sulr Strr Tmpr Sulr Strr Tmpr Sulr Strr Strr Strr Strr Strr Strr Strr Strr
Tmpr Tmpr Tmpr Tmpr Tmpr Tmpr Tmpr Tmpr
Sulr Sulr Sulr Sulr Sulr Sulr Sulr Sulr
The year of isolation is indicated when relevant. vet, veterinarian. O, carbohydrate antigen; H, flagellar antigen; K, capsular antigen; Oru, O antigen could not be determined. ND, not done.
Plasmid size (kb)
Resistance genotype
65 65 65 ND 65 65 65 65 65 62, 6.5 60, 8 65
tetB strAB dfrI sulI sulII tetB strAB dfrI sulI sulII tetB strAB dfrI sulI sulII ND tetB strAB dfrI sulI sulII tetB strAB dfrI sulI sulII tetB strAB dfrI sulI sulII tetB strAB dfrIsulI sulII tetB strAB dfrI sulI sulII strAB sulII strAB sulII tetB strAB dfrI sulI sulII
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