J. Med. Microbiol. - Vol. 46 (1997), 1007-1011 0 1997 The Pathological Society of Great Britain and Ireland BACTERIAL STRUCTURE AND PATHOGENICITY Screening of TnphoA mutants of Vibrio cholerae 0139 for identification of antigens involved in colonisation V. P. BONDRE, R. SRIVASTAVA, V. B. SINHA and B. S. SRIVASTAVA Division of Microbiology, Central Drug Research Institute, Lucknow 22600 I, India A new serogroup of Vibrio cholerae non-01, designated as 0139, has emerged causing cholera-like disease among adults. Laboratory and field studies clearly show that there is no cross-protection between 01 and 0139 pathogenic strains. Since colonisation of the intestine is a most important step in the pathogenesis of cholera caused by 01 strains and colonising antigens are known to be protective, investigation of the colonising antigens of 0139 strain was initiated. By TnphoA mutagenesis, mutants were generated with insertions in the genome encoding membrane spanning or secretory proteins. Screening of the mutants for adherence to rabbit intestinal surface and colonisation in 5-day-old mice resulted in the identification of mutant clones, which were less adhesive than was the wild-type parent strain and which could not efficiently colonise the gut. Such non-colonising strains were attenuated in virulence. Analysis of the proteins by SDS-PAGE revealed that the non-colonising membrane protein. Introduction In late 1992, a cholera-like disease caused by a new non-0 1 organism designated as Yibrio cholerae 0 139 spread across India, Bangladesh [l, 21 and Thailand [3]. The disease caused by 0139 strains was reported to be indistinguishable from cholera in clinical features and response to treatment [4]. As most of the affected patients were adults who were not primed previously with the 0139 serogroup and prior exposure to l? cholerae 0 1 gave no cross-protection, it may be argued that the 0139 strains differ from 0 1 strains in critical antigens involved in protection and the vaccine being developed against V cholerae 0 1 is not likely to be effective against V cholerae 0139. Hence, the virulence factors of serogroup 0139 will have to be investigated before formulating strategies to develop vaccine for the control of cholera caused by 0139 strains. Studies on the pathogenesis of F! cholerae 01 suggest that adherence of vibrios to the mucosal cell surface is an important step leading to the colonisation and subsequent production of the cholera toxin that causes Received 4 Feb. 1997; revised version accepted 23 April 1997. Corresponding author: Dr B. S. Srivastava. mutants did not express a 40-kDa outer- diarrhoea [5, 61. It is also known that the antigens involved in colonisation induced protection in experi- mental cholera [7-91. Therefore, the colonising antigens of the serogroup 0139 were investigated. A panel of mutants was generated by TnphoA mutagen- esis and screened to identify the adhesive and colonising antigens of F! cholerae 0139. Materials and methods Bacteria and media Bacteria, plasmids and TnphoA mutants are described in Table 1. The rifampicin-resistant spontaneous mutant, VO15-rif, was isolated by plating 2 X lo* cells of strain V015 on L-agar plates containing rifampicin 100 pglml. Nutrient broth and L-broth (Difco) were routinely used for growing the bacteria and were prepared according to the manufacturer’s instructions. Plates were prepared by adding Difco agar 1.2%. L-broth, brain heart infusion broth (BHI) and casamino yeast extract broth (CYE) have been described previously [ lo]. Bacterial dilutions were made in phosphate-buffered saline (PBS), pH 7.3 [ 111. Membrane-filtered solutions of tetracycline (12 mg/L), kanamycin (45 mg/L), gentamicin (30 mg/L), rifampicin (50 mg/L) and 5-bromo,4- chloro,3-indolylphosphate (BCIP, 40 mg/L) were added to the sterilised medium.