Drug Resistance and R Plasmids in Escherichia coli Strains Isolated from Imported Pet Birds

Transcription

1 Microbiol. Immunol. Vol. 24 (12), , 1980 Drug Resistance and R Plasmids in Escherichia coli Strains Isolated from Imported Pet Birds Masayuki NAKAMURA, * Mitsuo FUKAZAWA,1 Haruo YOSHIMURA, and Tetsuo KOEDA Second Assay Division, National Veterinary Assay Laboratory, Tokura, Kokubunji, Tokyo 185, and Animal Quarantine Service, Hara-machi, Isogo-ku, Yokohama 235 (Accepted for publication, September 6, 1980) Abstract Drug resistance in Escherichia coli strains isolated from pet birds (mynahs, macaws, finches, common bengals, parrots, and flamingos) imported into Japan from 10 foreign countries in 1977 and 1978 was investigated. Of the 309 strains isolated from 127 pet birds in the Animal Quarantine Service, 232 (75.1 %) were drug resistant. Furthermore, strains resistant to oxytetracycline hydrochloride, dihydrostreptomycin, and sulfadimethoxine were relatively common. Resistance patterns varied from single to sextuple resistance, and 148 (63.8%) of the resistant strains had conjugative R plasmids. These results suggest that the high incidence of drug resistance and R plasmids in E. coli strains isolated from these pet birds may be a reflection of the prophylactic use of antibiotics for the prevention of diseases which increasingly occur with importation of the birds. Furthermore, the results suggest that the birds may be potential reservoirs of drug-resistant E. coli for families who raise and have intimate contact with such birds. Many investigators in Japan have reported a high frequency of drug-resistant Escherichia coli strains in livestock and poultry and that about half of the strains have conjugative R plasmids (12, 14, 22). It has been suggested that this livestock and poultry may be an important source of intestinal infection in human beings. The spread of resistant E. coli strains from chickens to human beings (15), and from calves to human beings (9) has been demonstrated. In addition, it was also shown that resistant E. coli present in a commercially prepared chicken carcass colonized the intestines of a human volunteer handling the raw meat (16). However, while drug-resistant E. coli strains were isolated at a high frequency from dogs (28), domestic and feral pigeons and crows (24), all of which are in close contact with the human environment, there has been no report of extensive surveys on the antibiogram of E. coli strains isolated from pet birds. This paper deals with the high incidence of drug resistance and conjugative R plasmids in E. coli strains isolated from imported pet birds and discusses the pos- 1 Present address: Naha Airport Subbranch, Okinawa Branch, Animal Quarantine Service, Aza-kazamizu, Naha, Okinawa Prefecture

2 1132 M. NAKAMURA ET AL sibility that these pet birds may be potential reservoirs of drug-resistant E. coli for humans. MATERIALS AND METHODS Isolation of fecal E. coli strains from imported pet birds. As shown in Table 1, 127 pet birds of six types imported from 10 foreign countries in 1977 and 1978 were examined. Most of the birds in the study had little or no contact with human beings until their capture from forests, their natural habitat. It was difficult to obtain precise information on antibiotics administered to the birds for either therapeutic or prophylactic purposes after their capture. E. coli strains were isolated from fecal samples of the carcasses of the 127 birds within five days after importation at the Animal Quarantine Service of Narita and Haneda airports. Two or three strains of E. coli were isolated from each of the birds. The strains were isolated on deoxycholate-hydrogen sulfide-lactose agar (DHL, Eiken) plates, identified biochemically, and kept at 4 C in stock culture agar (Difco) until use. Drugs. The following drugs were used: oxytetracycline hydrochloride (TC), chloramphenicol (CP), dihydrostreptomycin (SM), sulfadimethoxine (SA), kanamycin (KM), aminobenzyl-penicillin (APC), and nalidixic acid (NA). NA was dissolved in 1 N NaOH and SM in phosphate buffer (ph 8.0). The others were dissolved in sterile distilled water at concentrations of 4 to 16 mg per ml. These stock solutions were further diluted before use. Test for drug resistance. A plate dilution method was used (11). Heart infusion agar (Difco) plates containing serial twofold concentrations of each drug Table 1. Isolation of Escherichia coli strains from pet birds imported into Japan

3 DRUG-RESISTANT BACTERIA IN IMPORTED PET BIRDS 1133 were prepared. For the test with SA, Mueller-Hinton agar (Difco) was used. Six-hour shaking cultures of the test strains in heart infusion broth (HI broth, Difco) were diluted about 100 times with sterile saline and inoculated into media with a multiple inoculater. Results were read after 18 hr of incubation at 37 C. The minimal inhibitory concentration (MIC) was defined as the lowest concentration of drug that prevented growth, except for SA, for which the MIC was defined as the lowest concentration of drug inhibiting confluent growth of large colonies. Strains that grew in media containing 12.5 jug of SM per ml, 25,ug of TC, CP, KM, and APC per ml and 400,ug of SA per ml were considered resistant to these drugs. Transfer of drug resistance. The recipient strain used was E. coli ML1410 (a nalidixic acid-resistant, and methionine-requiring F- derivative of E. coli K-12). Donor and recipient strains were cultured in HI broth for 6 hr at 37 C with gentle shaking. One ml of donor culture and 0.1 ml of recipient culture were mixed and conjugated for 18 hr at 37 C and then spread on DHL agar plates containing 25 iug of NA per ml and one of the selecting drugs (12.5,ag of SM per ml or 25,u,g of TC, CP, KM, or APC per ml). After incubation for 18 hr at 37 C, the plates were inspected for transconjugants. Three colonies per donor were purified and tested for their resistance patterns with TC, CP, SM, SA, KM, or APC to which the donor strain was resistant. When the transconjugant was not found on DHL agar plates, the mixed cultures of donor and recipient which had been left overnight at 25 C were spread on DHL agar plates to detect thermosensitive R plasmids. RESULTS MIC of Drugs and Incidence of Strains Resistant to Individual Drugs The MIC of six drugs for the 309 strains of E. coli are shown in Fig. 1 as distribution curves. All curves had two distribution peaks. In TC, CP, SM, KM, and APC, one peak was observed at a concentration of Đg per ml and the other at over 100 Đg per ml. For SA, one peak was observed at a concentration of 200 Đg per ml and the other at over 1,600 Đg per ml. The frequency of isolation of E. coli strains resistant to each of six drugs is shown in Table 2. Resistance was demonstrated most frequently to TC, followed by SM, SA, CP, APC, and KM resistance, although the order varied somewhat depending on the type of bird. Incidence of Drug Resistance and Conjugative R Plasmids in E. coli Strains As shown in Table 3, the incidence of resistance was high in E. coli strains isolated from various birds. Of the 309 strains, 232 (75.1 %) were resistant. Con- jugative R plasmids were found in 148 of these resistant strains (63.8%) but thermosensitive R plasmids were not evident.

4 1134 M. NAKAMURA ET AL Fig. 1. Distribution curves of MIC for Escherichia coli strains isolated from imported pet birds. Table 2. Frequencies of drug resistance in Escherichia coli strains isolated from imported pet birds Table 3. Incidence of drug resistance and conjugative R plasmids in Escherichia coli strains isolated from imported pet birds

5 DRUG-RESISTANT BACTERIA IN IMPORTED PET BIRDS 1135 Table 4. Resistance patterns and conjugative R plasmids of Escherichia coli strains isolated from mynahs Drug-Resistance Patterns of Original Strains and Conjugative R Plasmids Resistance patterns of 139 E. coli strains from mynahs are shown in Table 4. Sixteen different resistance patterns were found. Single and double resistant strains were found frequently (34 and 33 strains, respectively). TC E SM, TC, and TC E SM E SA resistant strains were relatively frequent. Conjugative R plasmids which were found in the E. coli strains showed 14 different resistance patterns. Transferred resistance patterns varied from single to sextuple resistance. In the E. coli strains isolated from macaws and finches, 10 different resistance

6 1136 M. NAKAMURA ET AL patterns from single to sextuple resistance were found. TC and TC E CP E E SA E KM E APC resistance, and TC resistance were demonstrated relatively frequently in strains isolated from macaws and finches, respectively. Resistance patterns of conjugative R plasmids varied from single to sextuple resistance in both types of birds. In strains isolated from common bengals, parrots, and flamingos, four different resistance patterns from single to quadruple resistance were found in the original strains and conjugative R plasmids. DISCUSSION Since Moore et al (21) reported that a small amount of streptomycin added to the diet caused chickens to grow more rapidly, many other growth-promoting agents, i.e., antibiotics and synthetic chemotherapeutics, have been used as feed additives to increase animal protein production in livestock and poultry. The prolonged use of these drugs for growth promotion, however, led to a high incidence of enteric bacteria exhibiting drug resistance. The effect of the use of antimicrobial agents at a nutritional level on the emergence of resistant E. coli strains in the alimentary tract of livestock and poultry has been investigated (5, 6, 8, 25, 26), and resistance frequencies reaching 70-90% have been found in E. coli strains isolated in recent years (12, 22). In contrast, there have been few reports concerning the antibiogram of E. coli strains isolated from wild animals. Sato et al (24) reported. that crows and feral pigeons which live close to humans exhibited a highfre- quency of drug-resistant E. coli strains. Mare (20) and Huber et al (10), however, investigated wild animals in Rhodesia and the U.S., respectively, and reported low frequencies of drug-resistant E. coli strains. It can be said, therefore, that antibioticresistant bacteria have been isolated only infrequently from wild animals that have little or no contact with humans. Since most pet birds have little or no contact with humans until their capture, it is probable that they had few or no drug-resistant E. coli strains when first caught. Therefore, the high incidence of drug resistance and R plasmids in E. coli strains isolated from pet birds may reflect the prophylactic use of antibiotics for the prevention of diseases which increasingly occur with the importation of the birds. There have been reports in the last decade of the presence of drug-resistant and R plasmid-carrying E. coli strains in normal healthy humans (1, 2, 13, 19, 23, 27). One of the sources of these drug-resistant bacteria in humans is thought to be livestock and poultry which carry a large number of drug-resistant bacteria in the intestinal tract. It is believed that drug-resistant bacteria may reach humans mainly through contact with animals (7, 27) and animal food (1, 4, 17, 18). The spread of resistant E. coli strains from livestock and poultry to human beings has been demonstrated for both animal and animal food contact (9, 15, 16). Since the pet birds examined carried drug-resistant and R plasmid-carrying E. coli strains at a high frequency and even three mynahs reared for 6 months after importation carried such strains to a lesser degree (unpublished data), such birds may be poten-

7 DRUG-RESISTANT BACTERIA IN IMPORTED PET BIRDS 1137 tial reservoirs of drug-resistant E. coli strains for families who raise and have intimate contact with them. Davies and Stewart (3) examined genetic similarities between R plasmids in enterobacteria from humans and domestic pets (cats and dogs) in Canberra, Australia, and pointed out that identification of the precise relationship between a population of R plasmids in humans and domestic pets might be clarified through closer study of the relationship between the R plasmid population in individual owners and their pets. In the case of the pet birds described here, a closer investigation, for example the typing of O-antigens or the genetic study of R plasmids, of drug-resistant E. coli strains in individual owners and pet birds is necessary to demonstrate the precise cross-infection of drug-resistant E. coli strains between human beings and pet birds. REFERENCES 1) Cooke, E.M., Shooter, R.A., Breaden, A.L., and O' Farrell, S.M Antibiotic sensitivity of Escherichia coli isolated from animals, food, hospital patients, and normal people. Lancet 2: ) Datta, N Drug resistance and R-factors in the bowel of London patients before and after admission to hospital. Br. Med. J. 2: ) Davies, M., and Stewart, P.R Transferable drug resistance in man and animals: Genetic relationship between R-plasmids in enteric bacteria from man and domestic pets. Aust. Vet. J. 54: ) Dorn, C.R., Tsutakawa, R.K., Fein, D., Burton, G.C., and Blenden, D.C Antibiotic resistance patterns of Escherichia coli isolated from farm families consuming home-raised meat. Am. J. Epidemiol. 102: ) Edwards, S.J Effect of streptomycin on the growth rate and intestinal flora (Escherichia coli) of piglets. J. Comp. Pathol. 71: ) Edwards, S.J Effect of antibiotics on the growth rate and intestinal flora (Escherichia coli) of calves. J. Comp. Pathol. 72: ) Fein, D., Burton, G., Tsutakawa, R., and Blenden, D Matching of antibiotic resistance patterns of Escherichia coli of farm families and their animals. J. Infect. Dis. 130: ) Harry, E.G The ability of low concentrations of chemotherapeutic substances to induce resistance in E. coli. Res. Vet. Sci. 3: ) Hirsch, D.C., and Wiger, N Effect of tetracycline upon transfer of R plasmid from calves to human beings. Am. J. Vet. Res. 38: ) Huber, W.G., Korica, D., Neal, T.P., Schnurrenberger, P.R., and Martin, R.J Antibiotic sensitivity patterns and R factors in domestic and wild animals. Arch. Environ. Health 22: ) Japan Society of Chemotherapy Method for determination of minimal inhibitory concentration of chemotherapeutic agents. Chemotherapy 16: (in Japanese). 12) Kinjo, T Drug resistant strains of bacteria isolated from domestic animals in Okinawa. II. Distribution of R factors in fecal E. coli strains isolated from pigs and chickens. Sci. Bull. Coll. Agric. Univ. Ryukyus 21: ) Kinjo, T Drug resistance and R plasmids in Escherichia coli isolated from feces of various animals and man in Okinawa. Japan. J. Zootech. Sci. 50: ) Kinjo, T., Nakachi, H., and Akamine, Y Drug resistant strains of bacteria isolated from domestic animals in Okinawa. III. Drug resistance and distribution of R factors among fecal E. coli strains isolated from cattle, goats and horses. Sci. Bull. Agric. Univ. Ryukyus 22: ) Levy, S.B., FitzGerald, G.B., and Macone, A.B Spread of antibiotic-resistant plasmids from chicken to chicken and from chicken to man. Nature 260:

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