R-FACTORS IN CALCUTA STRAINS OF VIBRIO CHOLERAE AND ENTEROBACTERIACEAE 971 R-Factors in Calcutta Strains of Vibrio cholerae and Members of the Enterobacteriaceae by L. M. PRESCOTT, Scientist, WHO Cholera Team, Calcutta, India, A. DATrA, Research Officer. Indian Council of Medical Research, Cholera Research Centre, Calcutta, and G. C. DArrA, Research Assistant, Indian Council ofMedical Research, Cholera Research Centre, Calcutta In 1959, several Japanese investigators a, 1' dis- The strains called V. cholerae, biotype El Tor covered that multiple-drug-resistance was transferred (sometimes referred to as " atypical " V. cholerae by an episomal character known as the resistance or Vibrio eltor), were non-haemolytic, chicken- transfer factor (RTF). R-factors (RTF plus specific cell-agglutination-positive, cholera-phage-IV-resis- genes for drug-resistance) have since been isolated in tant, and polymyxin-B-resistant (10 ,ug). The Shi- many countries: Germany,c the Philippines,, Eng- gella strains were kindly made available by Dr Ranjit land,e Israel f and the USA. Fragmentary references Sen, All-India Institute ofHygiene and Public Health, have been made to the experimental transfer of Calcutta, India. The other Enterobacteriaceae R-factors from Shigella and Salmonella to Vibrio strains were made available by Dr S. Gorbach, cholerae.h 4 The present study was designed to Johns Hopkins Center for Medical Research and screen Calcutta strains of V. cholerae and members Training, School of Tropical Medicine, Calcutta, of the Enterobacteriaceae for the naturally occurring India. The R-factor-containing strain of Escherichia presence of R-factors, and also to corroborate coli, CSH-2 (222), was obtained from Dr T. Wata- previous findings by transferring R-factors from nabe, Keio University, Tokyo, Japan. members of the Enterobacteriaceae to V. cholerae, Methods. When examining the phenomenon of from V. cholerae to V. cholerae, and from V. cholerae episome-mediated transfer of multiple-drug-resist- to members of the Enterobacteriaceae. ance, preliminary ranges of sensitivity for donors and recipients were defined by testing the organisms Material and methods against low, medium and high concentrations of Strains. Altogether, 225 strains of V. cholerae and antimicrobial sensitivity discs (Difco). Tetracycline V. cholerae, biotype El Tor, isolated at the Cholera (Tc), streptomycin (Sm), chloramphenicol (Cm) and Research Centre, Calcutta, from hospital cases, erythromycin (Em) were tested on nutrient agar latrine samples and carriers, were screened for the (Difco) plates, while sulfadiazine (Su) and Triple- presence of R-factors. These strains were character- sulfonamide (Ts) were tested on Mueller-Hinton ized at the time of isolation.1' k A battery of bio- medium (Difco) plates. chemical and serological tests was repeated at the Cultures of donor and recipient strains (0.1 ml of time of this study to prove that no loss of charac- 6-hour brain-heart infusion (BHI) broth (Difco)) ters had occurred during holding in stock culture. were mixed and inoculated into 1 ml of BHI broth. The cultures were incubated for 18 hours at 37°C a Ochiai, K., Yamanaka, T., Kimura, K. & Sawada, 0. and plated on antibiotic-containing medium which (1959) Nippon Iji Shinpo, 1835, 38-42. b Watanabe, T. & Fukasawa, F. (1961) J. Bact., 81, 669. was differential for the recipient; bile salt agar (BSA) 'Lebek, G. (1963) Zbl. Bakt., L Abt. Orig., 188, 444-505. with Sm, Cm or Tc for vibrios and lactose broth dKuwahara, S. Goto, S., Kimura, M. & Abe, H. (1967) agar (LBA) containing Sm, Cm or Tc for members Bull. Wld Hlth Org., 37, 763, 771. of the Enterobacteriaceae. Colonies ' Datta, N. (1962) J. Hyg. (Lond.), 60, 301-310. displaying drug- f Marberg, K., Altmann, G. & Eshkol-Bruck, A. (1958) resistance were picked and examined serologically Amer. J. trop. Med. Hyg., 7, 51-57. and biochemically. Disc sensitivity tests were U Falkow, S. (1965) Amer. J. Med., 39, 753-765. repeated to establish further the passage of R-factors A Baron, L. S. & Falkow, S. (1961) Genetics, 46, 849. 4Kuwahara, S., Akiba, T., Koyama, K. & Arai, T. (1963) from donor to recipient. Jap. J. Microbiol., 7, 61. When surveying for naturally occurring R-factors, a De, S. P., Ghosh, A. K. & Shrivastava, D. L. (1965) strains showing patterns of drug-resistance after disc Indian J. med. Res., 53, 614-622. k Sinha, R., Deb, B. C., De, S. P., Abou-Gareeb, A. H.-& sensitivity tests were examined according to the Shrivastava, D. L. (1967) Bull. Wld Hlth Org., 37, 89-100. above procedures. 2274K 972 NOTES R-FACTOR SURVEY OF CALCUTTA STRAINS OF VIBRIO CHOLERAE AND MEMBERS OF THE ENTEROBACTERIACEAE Donor | Strains Strains with Strains R-factor a strain _________________organism examined|eamine1 multiple-drug-resistance R-factorspassing Recipient Vibrio cholerei 65 3 2 Su, Ts, EM, Tc (1) Proteus vulgaris 2 Vibrio cholerae, biotype El Tor 160 5 2 Su, Ts, Em, Tc (1) Proteus vulgaris 2 Su, Ts, Tc (1) Escherichia coli 6 6 4 Su, Ts, Em, Tc (3) Vibrio cholerae NIH 41 Su, Em, Tc (1) Enterobacter aerogenes 4 3 3 Su, Ts, Em, Tc (3) Vibrio cholerae NIH 41 Enterobacter cloacae 1 1 1 Su, Em, Tc (1) Vibrio cholerae NIH 41 Klebsiella pneumoniae 2 1 1 Su, Ts, Em, Tc (1) Vibrio cho/erae NIH 41 Shigelia flexneri 14 5 2 Su, Ts, Em, Sm (2) Klebsiella pneumoniae 49a Shigella boydii 7 7 7 Su, Ts, Em, Sm (7) Klebsiella pneumoniae 49a Shigella dysenteriae 3 0 0 Shlge/la sonnei 1 1 1 Su, Ts, Em, Sm (1) K/lebsiel/a pneumonlae 49a a Su = sulfadiazine, Ts= Triplesulfonamide, Em= erythromycin, Sm = streptomycin, Tc = tetracycline. The numbers in parentheses indicate the number of strains that have R-factors as listed. Results R-factor was the same in all 10 strains (Su, Ts, Em Episome-mediated transfer of drug-resistance. The Sm). Of the other 13 strains of Enterobacteriaceae original R-factor (Su, Sm, Cm, Tc) was passed from studied, 7 contained the R-factor (Su, Ts, Em, Tc) Escherichia coli CSH-2(222) to V. cholerae. Re- while 2 had segregants of this R-factor (Su, Em, Tc) transmission was then carried out from vibrio to (see accompanying table). vibrio, vibrio to Proteus vulgaris or vibrio to E. coli. Discussion Tertiary transmission was also achieved from P. vulgaris to vibrio. In contrast to Kuwahara's It can be seen from the experimental data that received the R-factor R-factors can pass back and forth quite easily studies, all organisms which between members of the Enterobacteriaceae and retained it at the same high level of resistance as in vibrios. At the same time, the data in the table the parent E. coli strain. Vibrios held the R-factors show that some members of the Enterobacteriaceae as long as they were grown on antibiotic-containing carry the same R-factors as the vibrios. It is possible media. After 4 or 5 passages on nutrient agar, the to surmise, therefore, that the R-factor is being R-factors were lost. P. vulgaris and E. coli, after passed from one group to the other. acquiring the R-factors from V. cholerae, appear to It is also interesting to note that, although the retain them at the initial high level of resistance shigellae which did contain R-factors all carried indefinitely, regardless of the medium used. resistance to Sm, the 3 strains of S. flexneri which Survey ofnaturally occurring R factors in Calcutta did not pass R-factors did contain, however, strains of vibrios and members of the Enterobacteria- multiple-drug-resistance to Su, Ts, Em and Tc. ceae. Of 225 vibrios examined, 4 strains were found Spontaneous segregants, as described by Wata- to contain R-factors. One strain of V. cholerae nabe,' also appeared in Calcutta strains. These and 1 strain of V. cholerae, biotype El Tor, had segregants were either (Su, Ts, Tc) or (Su, Em, Tc). the R-factor (Su, Ts, Em, Tc). The other 2 organ- They, just like the complete R-factor, could transfer isms, an El Tor vibrio and a V. cholerae strain, their resistance by conjugation. contained a segregant of this R-factor (Su, Ts, Tc). The appearance of R-factors in Calcutta strains of Altogether, 25 strains of shigellae were examined and 10 strains were found to have R-factors. The Watanabe, T. (1963) Bact. Rev., 27, 87-115. PERORAL TRANSMISSION OF COXIELLA BURNETI TO SMALL RODENTS 973 V. cholerae does not pose a major problem to the for survival as sensitive strains are eradicated. treatment of cholera at present. The possibility does Public health laboratories, therefore, should be made arise, however, that, since tetracycline is the drug of aware of this possibility, so that continued sur- choice in the treatment of cholera, those strains with veillance of the changing patterns of drug sensitivity multiple-drug-resistance may be selectively chosen is maintained from year to year. Elimination of Coxiella burneti in Faeces and the Transmission of this Agent to Small Rodents by the Peroral Route by P. At, J. RiEHAMEK and R. BREZINA, Institute oJ Virology, Czechoslovak Academy of Sciences, WHO Regional Reference Centre for Human Rickettsiosis, Bratislava, Czechoslovakia In the course of investigations into foci of Q-fever dosage of 105 EID50 contained in 0.2 ml of suspen- in Slovakia, antibodies against Coxiella burneti were sion. detected in the sera of the small rodents Clethrio- From 9 to 29 days after the infection the faeces nomys glareolus and Apodemus flavicollis. were collected at 2-day intervals. From these sam- The reason for our study was to explain the ples, suspensions were prepared and centrifuged and relationship between C. burneti and infected animals 0.5 ml of the suspension was injected intraperitoneal- in the foci, and to determine mechanisms of infec- ly into 3-5-g white mice.