Genetic differences between nitrate-negative and nitrate-positive C. pseudotuberculosis strains using restriction fragment length polymorphisms

Abstract

Corynebacterium pseudotuberculosis has been classified into two biotypes according to ability to breakdown nitrate (Biberstein et al., 1971). Restriction enzyme analysis (REA) has shown to reflect this differentiation, but numerous bands generated by this technique make interpretation difficult (Songer et al., 1988). Restriction fragment length polymorphism's (RFLP's) has become an accepted genetic tool and was used in this study to determine if differences in nitrate reduction and other phenotypic characteristics could be identified genetically. Thirteen C. pseudotuberculosis isolates from four species of domestic animals from different parts of the world were investigated for phenotypic and genetic differences. Three closely related bacteria, Corynebacterium ulcerans, Actinomyces pyogenes (previously C. pyogenes), and Rhodococcus equi (previously C. equi) were included in the study to determine if the RFLP bands were unique to C. pseudotuberculosis. All C. pseudotuberculosis isolates were positive for urease production. Some differences in maltose and sucrose fermentation ability and nitrate reduction were recorded. Genetic differences were identified between the nitrate-positive group and the nitrate-negative group using non-radioactive ribosomal RNA (rRNA) probes Southern blotted to restriction digests of ApaI, PstI, and Sst I. A small number of bands were seen, with distinct differences between the nitrate-positive and the nitrate-negative strains. No genetic variations were seen between strains which reflected differences in carbohydrate fermentation. Strains isolated from different animal species and from different parts of the world could not be differentiated genetically using these three restriction enzymes.