DNA repair in Bacteroides fragilis Bf-2

Bibliography:pages 157-175.Repair deficient mutants of Bacteroides fragilis have been isolated in order to study the responses of this organism to various DNA damaging agents at the physiological and molecular levels. Two types of mutants were isolated by ethyl methane sulphonate mutagenesis of B.fragilis followed by selection for sensitivity to mitomycin C. One mutant (UVS9) showed sensitivity to both mitomycin C and far-UV irradiation. The other (MTC25) was more sensitive to mitomycin C than UVS9, but showed wild-type resistance to UV radiation. Both mutant strains had wild-type resistance to methyl methane sulphonate

An AraC/XylS family transcriptional regulator homologue from Bacteroides fragilis is associated with cell survival following DNA damage

A putative transcriptional regulator of the AraC/XylS family was identified in a genomic genebank of Bacteroides fragilis Bf-1, which partially relieved the sensitivity of Escherichia coli DNA repair mutants to the DNA-damaging agents, metronidazole and mitomycin C. A homologue of this gene with the same phenotype was identified as BF638R3281 in B. fragilis 638R. Transcription of BF638R3281 was constitutive with respect to exposure to sublethal doses of metronidazole. BF638R3281 was interrupted by single cross-over gene-specific insertion mutation, and the gene disruption was confirmed by PCR and DNAsequencing analysis. The mutant grew more slowly than the wild type, and the mutation rendered B. fragilis more sensitive to metronidazole and mitomycin C. This indicates that the BF638R3281 gene product plays a role in the survival of B. fragilis following DNA damage by these agents

Clostridium difficile in patients attending tuberculosis hospitals in Cape Town, South Africa, 2014–2015

Background: Diarrhoea due to Clostridium difficile infection (CDI) poses a significant burden on healthcare systems around the world. However, there are few reports on the current status of the disease in sub-Saharan Africa. Objectives: This study examined the occurrence of CDI in a South African population of tuberculosis patients, as well as the molecular epidemiology and antibiotic susceptibility profiles of C. difficile strains responsible for disease. Methods: Toxigenic C. difficile in patients with suspected CDI attending two specialist tuberculosis hospitals in the Cape Town area were detected using a PCR-based diagnostic assay (Xpert® C. difficile). C. difficile strains isolated from PCR-positive specimens were characterised by ribotyping, multilocus variable-number tandem-repeat analysis and antibiotic susceptibility testing. Results: The period prevalence of CDI was approximately 70.07 cases per 1000 patient admissions. Strains belonging to ribotype 017 (RT017) made up over 95% of the patient isolates and all of them were multi-drug resistant. Multilocus variable-number tandem-repeat analysis revealed several clusters of highly related C. difficile RT017 strains present in tuberculosis patients in several wards at each hospital. Conclusion: Tuberculosis patients represent a population that may be at an increased risk of developing CDI and, in addition, may constitute a multi-drug resistant reservoir of this bacterium. This warrants further investigation and surveillance of the disease in this patient group and other high-risk patient groups in sub-Saharan Africa

Prevalence of gastrointestinal pathogenic bacteria in patients with diarrhoea attending Groote Schuur Hospital, Cape Town, South Africa

Background. Diarrhoea due to gastrointestinal infections is a significant problem facing the South African (SA) healthcare system. Infections can be acquired both from the community and from the hospital environment itself, the latter acting as a reservoir for potential pathogenic bacteria.Objectives. To examine the prevalence of a panel of potential diarrhoea-causing bacteria in patients attending a tertiary healthcare facility in Cape Town, SA.Methods. Polymerase chain reaction (PCR) primers specific for Clostridium difficile, Shigella spp., Salmonella spp., Klebsiella oxytoca, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC), Staphylococcus aureus, enterotoxigenic Bacteroides fragilis and Campylobacter spp. were used to screen total bacterial genomic DNA extracted from stool samples provided by 156 patients with diarrhoea attending Groote Schuur Hospital, Cape Town, SA.Results. C. difficile was the most frequently detected pathogen (16% of cases) in the 21 - 87-year-old patient range, but was not present in samples from the 16 - 20-year-old range. K. oxytoca (6%), EPEC/EHEC strains (9%) and S. aureus (6%) were also detected. The remaining pathogens were present at low frequencies (0 - 2.9%), and the occurrence of mixed infections was 5%. The majority of non-C. difficile-related diarrhoeas were community acquired.Conclusion. C. difficile was the main cause of infectious diarrhoea in the sampled patients, while K. oxytoca and EPEC/EHEC strains were present as relatively minor but potentially significant pathogens

Twenty-eight divergent polysaccharide loci specifying within- and amongst-strain capsule diversity in three strains of Bacteroides fragilis

Comparison of the complete genome sequence of Bacteroides fragilis 638R, originally isolated in the USA, was made with two previously sequenced strains isolated in the UK (NCTC 9343) and Japan (YCH46). The presence of 10 loci containing genes associated with polysaccharide (PS) biosynthesis, each including a putative Wzx flippase and Wzy polymerase, was confirmed in all three strains, despite a lack of cross-reactivity between NCTC 9343 and 638R surface PS-specific antibodies by immunolabelling and microscopy. Genomic comparisons revealed an exceptional level of PS biosynthesis locus diversity. Of the 10 divergent PS-associated loci apparent in each strain, none is similar between NCTC 9343 and 638R. YCH46 shares one locus with NCTC 9343, confirmed by mAb labelling, and a second different locus with 638R, making a total of 28 divergent PS biosynthesis loci amongst the three strains. The lack of expression of the phase-variable large capsule (LC) in strain 638R, observed in NCTC 9343, is likely to be due to a point mutation that generates a stop codon within a putative initiating glycosyltransferase, necessary for the expression of the LC in NCTC 9343. Other major sequence differences were observed to arise from different numbers and variety of inserted extra-chromosomal elements, in particular prophages. Extensive horizontal gene transfer has occurred within these strains, despite the presence of a significant number of divergent DNA restriction and modification systems that act to prevent acquisition of foreign DNA. The level of amongst-strain diversity in PS biosynthesis loci is unprecedented

The Bifidobacterium longum NCIMB 702259(T) ctr Gene Codes for a Novel Cholate Transporter

Preexposure of Bifidobacterium longum NCIMB 702259(T) to cholate caused increased resistance to cholate, chloramphenicol, and erythromycin. The B. longum ctr gene, encoding a cholate efflux transporter, was transformed into the efflux-negative mutant Escherichia coli KAM3, conferring resistance to bile salts and other antimicrobial compounds and causing the efflux of [(14)C]cholate

Induction of Sucrose Utilization Genes from Bifidobacterium lactis by Sucrose and Raffinose

The probiotic organism Bifidobacterium lactis was isolated from a yoghurt starter culture with the aim of analyzing its use of carbohydrates for the development of prebiotics. A sucrose utilization gene cluster of B. lactis was identified by complementation of a gene library in Escherichia coli. Three genes, encoding a sucrose phosphorylase (ScrP), a GalR-LacI-type transcriptional regulator (ScrR), and a sucrose transporter (ScrT), were identified by sequence analysis. The scrP gene was expressed constitutively from its own promoter in E. coli grown in complete medium, and the strain hydrolyzed sucrose in a reaction that was dependent on the presence of phosphates. Primer extension experiments with scrP performed by using RNA isolated from B. lactis identified the transcriptional start site 102 bp upstream of the ATG start codon, immediately adjacent to a palindromic sequence resembling a regulator binding site. In B. lactis, total sucrase activity was induced by the presence of sucrose, raffinose, or oligofructose in the culture medium and was repressed by glucose. RNA analysis of the scrP, scrR, and scrT genes in B. lactis indicated that expression of these genes was influenced by transcriptional regulation and that all three genes were similarly induced by sucrose and raffinose and repressed by glucose. Analysis of the sucrase activities of deletion constructs in heterologous E. coli indicated that ScrR functions as a positive regulator