29; D. Schneider, S. Ingram, and C. Parker,. Abstr. Annu. Meet. ... The method of Parker et al. (26) was ...... Garber, E. D., A. J. Hackett, and R. Franklin. 1952.
Vol. 22?,No. 1
INFECTION AND IMMUNITY, Oct. 1978, p. 181-188 0019-9567/78/0022-0181$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in IU.S.A.
Isolation and Phenotypic Characterization of VirulenceDeficient Mutants of Vibrio cholerae VICKIE S. BASELSKI,t SUSAN UPCHURCH,4 AND CHARLOTTE D. PARKER* Department ofMicrobiology, University of Texas, Austin, Texas 78712
Received for publication 2 August 1978
Mutants of Vibrio cholerae were isolated on the basis of reduced ability to induce diarrhea in orally challenged infant mice. Nitrosoguanidine-treated clones were screened for low fluid accumulation ratios in individual mice, and presumptive mutants were confirmed in additional mouse tests. Mutants were examined for alterations in phage type, motility, toxin production, proteolytic activity, neuraminidase production, amylase production, morphology, growth requirements, carbohydrate fermentations, in vitro growth patterns, and cell surface alterations. The types of mutants found included several with previously recognized virulence-associated markers (rough, nonmotile, toxin deficient, protease deficient); several types with pleiotropic alterations (cell morphology, decreased extracellular products); and several with no previously recognized virulencedeficient phenotype (purine requiring, cell surface altered, rapid death in vitro, no defect found). Dose-response kinetics showed that most mutants could provoke diarrhea if given in 100-fold greater numbers than the dose used for screening. Recovery of viable organisms from the gut late in infection showed reduction of survival and/or multiplication capacity for the mutants, with variation in the degree of reduction for the different classes.
The use of genetic techniques to derive isogenic mutants for studies of pathogenesis has been successfully applied to Vibrio cholerae by a number of researchers (4, 5, 12, 16, 18, 19, 28, 29; D. Schneider, S. Ingram, and C. Parker, Abstr. Annu. Meet. Am. Soc. Microbiol. 1977, D26, p. 74). The essential features of the genetic approach are, first, the application of methods for isolation and selection of altered phenotypes to the pathogen and, second, availability of a convenient, relevant animal model for measuring virulence. With V. cholerae, the primary methodology used has been selection of mutants with specific defects from mutagen-induced cultures and examination of these mutants in an animal model for alterations in disease-inducing ability. Application of this genetic approach to studies of cholera pathogenesis has indicated that toxin production (18, 19), motility (16), smooth lipopolysaccharide structure (15), neuraminidase production, and nutritional restrictions, (e.g., streptomycin dependence [5, 12]) affect the disease-inducing capacity of V. cholerae. In addition, several other, more complex mutants have been shown to be avirulent (4, 29). The genetic approach taken in the present study is novel, however, in that we have at-
tempted to isolate directly mutants which are unable to elicit disease and then to examine these mutants for deviations from wild-type behavior. A comprehensive evaluation of the mutant phenotypes and their interactions with the host environment should contribute to an understanding of V. cholerae parameters involved in the infective process. We have utilized the infant mouse model (30) as modified in this laboratory for quantitation of a diarrheal response by use of a fluid accumulation (FA) ratio (2). The FA model is ideally suited for our approach, as it allows mutagentreated clones to be screened for avirulence in individual animals. This communication describes the isolation and preliminary characterization of virulence-deficient mutants of V. cholerae, which fail to elicit a typical diarrheal response in infant mice after oral challenge with viable organisms. (Presented in part at the 77th Annual Meeting of the American Society for Microbiology, 1977, New Orleans, La.) MATERIALS AND METHODS Bacterial strains. All mutants were derived from a highly infant-mouse-virulent human isolate, CA401 (20). Parental strains were spontaneous drug-resistant mutants selected on meat extract agar (MEA) with either 25 ,g of rifampin per ml (designated Rifl01) or
t Present address: Center for Disease Control, Atlanta, GA 30333. t Present address: University of Illinois, Urbana, IL 61801.
181
BASELSKI, UPCHURCH, AND PARKER
INFECT. IMMUN.
100 ,ug of streptomycin per ml (designated Str-101, StrlO2, and StrlO3). Cultures were maintained as stocks frozen at -70'C in brain heart infusion broth (BHIB) plus 15% glycerol or on MEA at 4VC with a maximum of three transfers. Infant mice. Infant mice were taken from our CFW breeding colony in the University of Texas Animal Resources Center, Austin, Tex. Breeders were fed Wayne Lab Blocks. The mice used were 6 to 7 days old, weighing 3.0 ± 0.5 g. FA ratio model. FA ratio model has been described in detail previously (2). Basically, mice were inoculated orally, after a 6-h fast, with MEA-grown cells suspended in BHIB + 0.01% Evans blue. At the appropriate time, mice were sacrificed and the FA ratio was determined as: weight of stomach and intestines (gut)/(mouse body weight - gut weight). Mutagenesis. The method of Parker et al. (26) was used for mutagenesis with nitrosoguanidine. Mutagenized cells were aliquoted for outgrowth in BHIB and then frozen at -70'C with 15% glycerol until needed for screening. Screening procedure. Outgrown cultures were streaked to MEA plus the appropriate antibiotic. After incubation, isolated colonies were picked to MEA master plates without antibiotic. Growth from the master plates was suspended in BHIB plus dye to a cell density of approximately 2 x 108 colony-forming units (CFU) per ml. These suspensions were the inocula used for screening. The screening protocol was based on kinetics of FA previously reported (2). A 0.05-ml dose of each suspension was given orally to individual mice. This dose contained about 107 CFU, which will promote a nearmaximal FA ratio with strain CA401. At 16 to 18 h post-inoculation, the time required for a maximal FA ratio to occur at the dose level given, the FA ratios were determined. The clones which gave a negative FA ratio (
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