Bacillus popilliae and B. lentimorbus, causative agents of milky disease in Japanese beetles and related scarab larvae, have been differentiated based upon a small number of phenotypic characteristics, but they have not previously been examined at the molecular level. Thirty-four isolates of these bacteria were examined for DNA similarity. Three distinct but related similarity groups were identified; the first contained strains of B. popilliae, the second contained strains of B. lentimorbus, and the third contained two strains distinct from but related to B. popilliae. Some strains received as B. popilliae were found to be most closely related to B. lentimorbus and some received as B. lentimorbus were found to be most closely related to B. popilliae." Geographically distinct strains of B. popilliae and B. lentimorbus were analyzed using RAPD. Eight decamer primers were tested against nineteen new and seventeen isolates previously described by randomly amplified polymorphic DNA (RAPD) analysis (M. Tran). Of the new isolates, ten were found to be B. popilliae while nine isolates were more related to the B. lentimorbus species. Paraspore formation, believed to be a characteristic unique to B. popilliae, was found to occur among a subgroup of B. lentimorbus strains. Using a combination of two PCR primer pairs, the cry18Aa1 gene was detected in 31 of 35 B. popilliae isolates and in 1 of 18 B. lentimorbus isolates. When hemolymph smears were examined microscopically, a parasporal crystal was seen in three of the four B. popilliae strains where the PCR primers could not amplify the paraspore gene. The fourth strain was not tested due to the unavailability of infected hemolymph. A paraspore was also detected by microscopic examination in a subgroup of 14 B. lentimorbus strains. In combination, the primer pairs CryBp1 and CryBp2 are effective at detecting the paraspore gene in B. popilliae isolates, but not in the B. lentimorbus isolates. Growth in media supplemented with 2% NaCl was found to be less reliable in distinguishing the species than was vancomycin resistance, the latter present only in B. popilliae. The basis for vancomycin resistance in all isolates was investigated using a polymerase chain reaction assay designed to amplify the vanB gene in enterococci. An amplicon was identified and sequenced. The amplified portion of the putative ligase gene in B. popilliae had 77% and 68-69% nucleotide identity to the sequences of the vanA gene and the vanB genes, respectively. There was 75% and 69-70% identity between the deduced amino acid sequence of the putative ligase gene in B. popilliae and the deduced amino acid sequence of the vanA gene and the vanB genes, respectively. It has been determined that the vanE gene is located either on a plasmid greater than 16 kb in size or on the chromosome. The gene in B. popilliae may have had an ancestral gene in common with vancomycin resistance genes in enterococci. Bacillus sphaericus strains isolated on the basis of pathogenicity for mosquito larvae and strains isolated on the basis of a reaction with a B. sphaericus DNA homology group IIA 16S rRNA probe were analyzed for DNA similarity. All of the pathogens belonged to homology group IIA, but this group also contained nonpathogens. It appears inappropriate to designate this homology group a species based solely upon pathogenicity. / Ph. D.

The rate at which bacterial pathogens are becoming resistant to antibiotics is quite alarming, and therefore much attention has been focussed on this area. The mechanism whereby the bacterial cells acquire resistance is studied in order to determine how this process works as well as to determine if any future resistance mechanisms can be circumvented. In this study three different genera and the antibiotics that are resistant to them were used, namely, penicillin resistant Streptococcus, vancomycin resistant Enterococcus and methicillin resistant Staphylococcus. The results prove that the active sites SXXK, SXN and KT(S) G in the penicillin resistance Streptococcus plays a major role in resistance. It is seen in this study that the SXXK active site is found in all the resistant and most of the intermediate strains, therefore proving to be an important component of the cell wall resistance. It was subsequently noticed the greater the number of mutations found in the sequences the higher the resistance. Three dimensional structures showed the actives sites and their binding pockets. The results also show the change in conformation with a mutation in the active site. The results also proved that the Penicillin Binding Protein (PBP) genes essential for resistance are PBP Ia, PBP 2b and PBP 2x. The results obtained, for the vancomycin resistance in Enterococcus study, proved that the VanC and VanE cluster are very much alike and VanE could have evolved from VanC. There is also close similarity between the different ligase genes. The VanX 3D structure shows the position of the critical amino acids responsible for the breakdown of the D-Ala-D-Ala precursors, and the VanA ligase 3D structure shows the amino acids responsible the ligation of the D-Ala-D-Lac precursors. The analysis performed on the methicillin resistance in Staphylococcus study showed that the genes used to confer resistance are very similar between different strains as well as different species. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.