Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron.

OBJECTIVES: The aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance. METHODS: The genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays. RESULTS: A metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose. CONCLUSIONS: These data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron.

Rhamnose catabolism in Bacteroides thetaiotaomicron is controlled by the positive transcriptional regulator RhaR.

Bacteroides thetaiotaomicron is an important human gut commensal, which also causes opportunistic infections outside this environment. It utilises a range of host and diet-related carbohydrates, including rhamnose. In this study, the rha gene cluster, required for rhamnose utilisation, was characterised by transcription analysis, gene targeted mutagenesis and enzyme assays. Growth in the presence of L-rhamnose induced transcription of all the genes of this cluster. The first five genes of the cluster, rhaKIPAO, were transcribed as an operon from a transcriptional start site upstream of rhaK, whereas the sixth gene, rhaR, was transcribed independently. Bioinformatic analysis and mutation of the rhaR gene identified it as encoding the positive transcriptional activator of rhaKIPAO. A rhaR mutant could not utilise rhamnose as the sole carbon source but grew normally on glucose. The rhaO gene encoded a lactaldehyde reductase, and a rhaO mutant produced reduced levels of L-1,2-propanediol during growth in rhamnose, indicating its contribution to rhamnose catabolism in Bacteroides thetaiotaomicron.

Molecular characterization of live Theileria parva sporozoite vaccine stabilates reveals extensive genotypic diversity.

The current Infection and Treatment Method of vaccination against East Coast fever comprises an inoculation of live Theileria parva sporozoites and simultaneous administration of oxytetracycline. Immunization with a combination of parasite types has been shown to provide broader protection than inoculation of individual strains. In this study, we used a high-throughput capillary electrophoresis system to determine the genotypic composition of the Muguga Cocktail, a widely used vaccine stabilate derived from three seed stabilates-Muguga, Serengeti-transformed and Kiambu 5. Five satellite markers were used to genotype the vaccine and reference stabilates from two commercial-scale preparations of the vaccine. In addition, 224 cloned cell lines established by infection of bovine lymphocytes with T. parva parasites from the component stabilates were genotyped. The results indicate that, for the recently prepared batch, there are at least eight genotypes in each of the Muguga and the Serengeti-transformed stabilates, while parasites from the Kiambu 5 stabilate showed no diversity at the five loci. The Serengeti-transformed stabilate contained parasites of the Kiambu 5 genotype and of two genotypes present in the Muguga stabilate, whereas there were no genotypes common to the Muguga and Kiambu 5 stabilates. When stabilates from the two vaccine batches were compared, no allelic variations were identified between the Muguga and Kiambu 5 parasites, while lack of sufficient clones prevented a full comparison of the Serengeti-transformed stabilates. The findings will facilitate examination of the extent to which the vaccine strains become resident in areas under vaccination, the identification of 'breakthrough' strains and the establishment of the quality assurance protocols to detect variations in the production of the vaccine. The cloned cell lines will be useful for further understanding the antigenic diversity of parasites in the vaccine.