Detection of R+ Bacteria in Cultured Marine Fish, Yellowtails (Seriola quinqueradiata)

Transcription

1 Japan. J. Microbiol. Vol. 17(1), 7-12, 1973 Detection of R+ Bacteria in Cultured Marine Fish, Yellowtails (Seriola quinqueradiata) Takashi AOKI, Syuzo EGUSA, and Tsutomu WATANABE Department of Fisheries, Faculty of Agriculture, University of Tokyo, Tokyo 113, and Department of Microbiology, Keio University School of Medicine, Tokyo 160 (Received for publication, April 28, 1972) ABSTRACT Many drug-resistant gram-negative bacilli were isolated from the intestinal tracts of yellowtails (Seriola quinqueradiata) cultured on farms in various parts of Kochi and Ehime Prefectures, Shikoku Island. They were Vibrio, Pseudomonas, Proteus, Citrobacter and some unidentified Enterobacteriaceae. Of these drug-resistant strains, Vibrio and Pseudomonas were found to carry R factors in high frequencies. These R factors had four types of resistance markers, SA, SA. CM, SM. CM. ABP. and SA. SM. CM. TC. All R factors were found to belong to the fi- type. In contrast, only one drug-resistant gram-negative bacillus was detected in a cultured yellowtail on farms near Numazu City, Shizuoka Prefecture. It had been shown in previous reports [3-6, 17, 18] that a large majority of the strains of fish-pathogenic Aeromonas liquefaciens and Aeromonas salmonicida isolated from intestinal tracts of cultured freshwater fishes such as carp, eel, ayu, and amago as well as from the waters of their ponds were multiple-drug-resistant and that a majority of these drug-resistant bacteria carried R factors. In Japan, not only freshwater fishes but also various kinds of marine fishes are cultivated on a large commercial scale. Among them, yellowtail (Seriola quinqueradiata) is the most widely cultivated species. It is generally cultured in a high density population by using net-cages of about 10 ~ 10 ~ 10m, 1000m3. As is usual with freshwater cultivated fishes, cultured yellowtails are frequently attacked by bacterial infections. Various antibiotics and synthetic chemotherapeutics agents have been widely em- ployed in feed and either bathing or injecting to prevent infectious or treating the infected individuals. Accordingly, it was Please address requests of reprints to Dr. Takashi Aoki, Department of Fisheries, Faculty of Agriculture, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan. believed that drug-resistant bacteria might have increased in the intestinal tracts of cultured yellowtails. The present study was initiated to obtain information on this problem with special attention directed to whether or not drug-resistant marine bacteria carry R factors. MATERIALS AND METHODS Yellowtails for bacterial isolation were obtained from 4 culturing farms in Uchinoura Bay near Numazu City, Shizuoka Prefecture in September, 1970 and from 20 farms situated in several bays along the southeast coast of Kochi and Ehime Pre- fectures, Shikoku Island in July and August, Water temperatures of the farms at the time of collection of the fishes in Shikoku Island and Shizuoka Prefecture were about 29 C and 25.5C, respectively. Intestinal tracts of 5 fishes from each farm were pooled and homogenized in a homogenizer (Nippon Seiki Co.) at 2000 rpm for 3 min. The homogenates were properly diluted with physiological saline (ph 7.2) and varying dilutions were plated on nutrient agar medium containing 25 Đg/ml of chloram- phenicol (Sankyo) (CM), the same medium containing the same concentration of tetra- 7

2 8 T. AOKI, S. EGUSA AND T. WATANABE cycline hydrochloride (Lederle) (TC) and a drug-free nutrient agar. All these media contained 3% NaCl. In the 1970 study, nutrient agar medium containing 0.5% NaCl was also used. The inoculated plates were incubated at 28C overnight and 3-4 developed colonies were picked at random and inoculated on nutrient agar slants for further studies, where each colony was regarded as a strain. Strains of gram-negative bacilli which grew on drug-containing media were identified following the methods of Bergey [9] and Cowan and Steel [10] and tested for their sensitivities to sulfisoxazole (SA), streptomycin (SM), CM, TC, aminobenzyl penicillin (ABP), fradiomycin (FRM) (identical to neomycin), kanamycin (KM), nitrofurantoin (NF) with sensitivity disks (Showa). All drug-resistant strains grown at 30C were cultured at 37C together with Escherichia coli K12 grown at 37C to determine whether or not they possessed R factors. The detected R factor-carrying strains were tested for their sensitivities to various drugs employing sensitivity disks. The R factors were transferred from isolated R+ strains to E. coli W2252 (methionine-requiring stable Hfr derivative of K12). If W2252 (R+) was found sensitive to male-specific bacteriophages fl and (Y, the R factor was regarded as otherwise as fi+ [19, 20]. The procedures and methods for these tests were the same as those described in detail in preceding papers published earlier [5, 16-18]. RESULTS The mean viable counts of bacteria from the intestinal tracts of cultured yellowtails obtained from 4 farms in Shizuoka Prefecture, which grew on nutrient agar media containing 3% NaCl and 0.5%NaCl, were 3.1 ~ 104/g and 2.0 ~ 104/g, respectively, as shown in Table 1. Neither CM-resistant nor TC-resistant bacteria were isolated on 3% NaCl nutrient agar medium containing the respective drug. A TC-resistant strain was isolated from only one out of 4 farms on 0.5%NaC1 nutrient agar medium containing TC (25ƒÊg/ml) (Table 1). R factor could not be detected in this strain. The mean viable count of bacteria cultured from the intestinal tracts of yellowtails from 20 culturing farms of Shikoku Island on drug-free nutrient agar was 6.7 x 105/g (Table 2). CM- and TC-resistant bacteria were obtained from 6 and 14 out of the 20 farms, respectively. The mean viable counts of the resistant bacteria were 2.2 ~ 103 and 5.5 ~ 104/g, respectively. Twenty-one strains of CM-resistant gramnegative bacilli from samples obtained from Table 1. Drug-resistant bacteria isolated from intestinal tracts of cultured yellowtails (Seriola quinqueradiata) in Numazu City, Shizuoka Prefecture CM, chloramphenicol; TC, tetracycline. Mean body weight, g; mean body length, 24.8 cm for yellowtails.

3 R+ BACTERIA IN CULTURED MARINE FISH 9 Table 2. Drug-resistant bacteria isolated from intestinal tracts of cultured yellowtails (Seriola quinqueradiata) in Kochi and Ehime Prefectures, Shikoku Island Mean body weight, 208 g; mean body length, 21.5 cm for yellowtails. Table 3. Resistance patterns and R factors of bacteria isolated from intestinal tracts of cultured yellowtails (Seriola quivueradiata) Bacteria were isolated on nutrient agar containing CM (25 Đg/ml). Shikoku Island were studied. Among them, six were identified as Vibrio, six as Pseudomonas, five as Proteus, one as a Citrobacter and three not identified. The drug-resistance markers of these strains are shown in Table 3. Of these drug-resistant strains two Vibrio strains and two Pseudomonas strains were found to carry R factors. Drug-resistance markers of the detected R factors were SA. CM. and SM. CM. ABP, respectively (Table 3). Forty-six TC-resistant strains were isolated on nutrient agar containing TC (25 Đg/ml) from the intestinal tracts of fishes from the Shikoku Island. Results of their identifica-

4 10 T. AOKI, S. EGUSA AND T. WATANABE Table 4. Resistance patterns and R factors of bacteria isolated from intestinal tracts of cultured yellowtails (Seriola quinqueradiata) Bacteria were isolated on nutrient agar containing TC (25 Đg/ml). Table 5. Resistance patterns and R factor of bacteria isolated from intestinal tracts of cultured yellowtails (Seriola quinqueradiata) Bacteria were isolated on drug-free nutrient agar.

5 R+ BACTERIA IN CULTURED:MARINE FISH 11 tion and their drug-resistance patterns are given in Table 4. R factors were detected in 25 out of 31 Vibrio strains. Drug-resistance markers of these R factors were all SA. SM. CM. TC. R factors were not detected in any other species of bacteria. Seventy-four gram-negative bacilli were isolated on drug-free nutrient agar from the intestinal tracts of fishes from the Shikoku Island. Out of the 74 strains, sixty-seven were identified as Vibrio, three as Pseudomonas, three as Citrobacter and one not identified. R factor was detected in only one of the 67 Vibrio strains and in no other species. This R factor had a drug-resistance marker SA (Table 5). DISCUSSION It was shown previously that the majority of drug-resistant bacteria which were isolated from the intestinal tracts of cultured freshwater fishes and the water of their ponds carry R factors [5, 6, 17, 18]. In the present study, many drug-resistant strains of gram-negative bacilli, particularly of Vibrio were isolated from the intestinal tracts of cultured yellowtails obtained from farms along the southeast coast of the Shikoku Island. Furthermore, R factors were detected in drug-resistant Vibrio and Pseudomonas strains. In contrast, only one TC-resistant strain, which had no R factor, was isolated from the intestinal tract of a cultured yellowtail from farms near Numazu City in Shizuoka Prefecture in our investigation. The reason for our failure to isolate R+ bacteria in the investigation involving the Shizuoka Prefecture is not clear. It might reflect the difference in the ways that the chemotherapeutic agents were employed. Those Vibrio strains were identified following Bergey's Manual [9]. Out of the 104 strains, six were not sensitive to Vibrio-static agent 0/129 [7]. Although it had been reported that R factor could be transferred in vitro from Salmonella typhosa to Vibrio comma [8], from Shigella to non-agglutinable Vibrio [1] and from E. coli to Vibrio parahaemolyticus [12], there had been no report that R factor-carrying halophilic Vibrio strains were isolated from animals. Vibrio strains isolated from the intestinal tracts of yellowtails in the present study were not identical to the species mentioned above but were identified as related to halophilic bacteria, which were reported as the causative agents of ulcer of marine fish [14]. Development of drug resistance of fish-pathogenic halophilic Vibrio in vitro in subculture in drug-containing culture media had been reported but no results on the transmission of R factor to them had been reported [13, 15]. Drugresistance markers of the 28 R factors detected in Vibrio strains isolated from the intestinal tracts of yellowtails were as follows: one, SA, two, SM. CM. ABP; and twenty-five, SA. SM. CM. TC. R factor with resistance markers SA. SM. CM. TC were detected in Vibrio strains isolated on nutrient agar containing TC (25 Đg/ml). The fact that this type of R factor could not be detected by selection on nutrient agar containing CM in a concentration of 25 Đg/ml seemed to indicate that Vibrio strains harboring this R factor could phenotypically express the resistance to CM only in levels lower than 25 Datta et al [11] reported the detection of an R factor in Pseudomonas aeruginosa. Our discovery of Pseudomonas strains carrying R factors from the intestinal tracts of marine fish in this study seemed to be the first report on halophilic Pseudomonas. Resistance markers of these R factors were SA. CM. It was of particular interest that all the R factors detected in Vibrio and Pseudomonas strains from yellowtails were found to belong to the fi- type, unlike most R factors from Salmonella and Shigella [19-21]. Our new finding should be of great importance from the standpoint of public health, because yellowtails are frequently consumed raw in Japan. Accordingly, there is a possibility that the R factors may be transferred to human enteric bacteria as is the case with domestic animals [2, 16]. ACKNOWLEDGEMENT We are grateful to the staff members of Ehime Prefectural Fisheries Experimental Station and of Shizuoka Prefectural Fisheries Experimental Station, Numazu Branch, for their kindness giving us the oppotunities to collect t he samples. We would also like to express our appreciation to Dr. R. Kusuda of Kochi University and Mr. T. Saito and Miss. C. Yada of Keio University for their kind assistance in collecting the samples. This investigation was supported by

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