Toxin A-negative toxin B-positive ribotype 017 Clostridium difficile is the dominant strain type in patients with diarrhoea attending tuberculosis hospitals in Cape Town, South Africa.

The molecular epidemiology of C. difficile strains causing disease in South Africa is currently unknown. Previously, multidrug resistant ribotype (RT)017 strains were those most commonly isolated from patients with diarrhoea attending Groote Schuur Hospital in Cape Town, South Africa. This larger study aimed to investigate the molecular epidemiology and antibiotic susceptibility profiles of C. difficile strains in the greater Cape Town and regional areas. C. difficile strains were isolated from patients with diarrhoea attending hospitals in the Western Cape region of South Africa that tested positive using the GeneXpert CDiff diagnostic test. Ribotyping and multilocus variable-number tandem-repeat analysis (MLVA) were used to type isolates, and their susceptibilities to several antibiotics were determined by gradient diffusion test strips. A total of 269 non-repeat C. difficile isolates were obtained. A large proportion of isolates (64.3 %) belonged to the RT017 group, many of which were clonally related when investigated by MLVA. RT017 strains were particularly prevalent in patients attending specialist tuberculosis (TB) hospitals. The majority of RT017 isolates were co-resistant to moxifloxacin and rifampicin, two antibiotics which are used intensively during anti-TB therapy. Non-RT017 strains were generally susceptible to both antibiotics. Resistance to erythromycin was observed for both groups of strains. RT017 C. difficile strains are the most commonly isolated strains from patients attending healthcare facilities in the greater Cape Town and regional areas. The presence of multidrug resistant RT017 strains in patients with diarrhoea attending local TB hospitals reflects a potential reservoir for future infections.

Characterisation of Clostridium difficile strains isolated from Groote Schuur Hospital, Cape Town, South Africa.

The C. difficile infection rate in South Africa is concerning. Many strains previously isolated from diarrhetic patients at Groote Schuur Hospital were ribotype 017. This study further characterised these strains with respect to their clonal relationships, antibiotic susceptibility, toxin production and various attributes impacting on pathogen colonisation. Multilocus variable-number tandem-repeat analysis (MLVA) was used to characterise all C. difficile isolates. Antibiotic susceptibility was determined by E-test and PCR-based analysis of the ermB, gyrA and gyrB genes. Auto-aggregation of cells was measured in broth, and biofilm formation observed in 24-well plates. Toxins were measured using the Wampole C DIFF TOX A/B II kit. Most isolates belonged to the ribotype 017 group. Identical MLVA types occurred in different wards over time, and several patients were infected with identical strains. All isolates were susceptible to vancomycin and metronidazole, but some ribotype 017 isolates showed reduced metronidazole susceptibility (≥2 mg l(-1)). Sixty-nine percent of ribotype 017 isolates were resistant to moxifloxacin, and 94 % to erythromycin, compared to 0 % and 17 % resistance, respectively, in non-ribotype 017 isolates. The ermB gene and mutations in the gyrA and/or gyrB genes were linked to erythromycin and moxifloxacin resistance, respectively. Ribotype 017 isolates auto-aggregated more strongly than other isolates and produced lower levels of the TcdB toxin than a reference strain. Certain strains produced strong biofilms. Patient-to-patient transfer and unique infection events could cause the predominance of ribotype 017 strains in the cohort. Multi-drug resistant strains are a potential reservoir for future infections.

A functional analysis of the formyl-coenzyme A (frc) gene from Lactobacillus reuteri 100-23C.

AIM: To examine the role of the Lactobacillus reuteri 100-23C frc gene product in oxalate metabolism, host colonization and the acid stress response. METHODS AND RESULTS: Genes encoding putative formyl-CoA transferase (frc) and oxalyl-CoA decarboxylase (oxc) enzymes are present in the genome sequences of Lact. reuteri strains. Two strains isolated from humans harboured an IS200 insertion sequence in the frc ORF and a group 2 intron-associated transposase downstream of the frc gene, both of which were lacking in two strains of animal origin, which contained intact frc and oxc genes. An frc(-) insertional mutant of Lact. reuteri 100-23C was compared with the parent strain with respect to oxalate degradation, colonization of an RLF-mouse host model and growth in the presence of acids. Neither parent nor mutant degraded oxalate in vitro or in vivo. However, the parent outcompeted the frc(-) mutant in the mouse intestine during co-colonization and the frc(-) mutant showed a reduced growth rate in the presence of hydrochloric acid. CONCLUSIONS: Intact oxc and frc genes do not ensure oxalate degradation under the conditions tested. The frc gene product is important during host colonization and survival of acid stress by Lact. reuteri 100-23C. SIGNIFICANCE AND IMPACT OF THE STUDY: Oxalate metabolism by oxalate-degrading intestinal bacterial strains may be important in preventing urolithiasis and might lead to the derivation of probiotic products. To produce safe and efficacious probiotics, however, an understanding of the genetic characteristics of potential oxalate degraders must be obtained, together with knowledge of their functional ramifications.

Diversity of faecal oxalate-degrading bacteria in black and white South African study groups: insights into understanding the rarity of urolithiasis in the black group.

AIM: To examine whether enhanced diversity or numbers of oxalate-degrading bacteria in the gastrointestinal tracts of black South Africans play a role in determining the rarity of urolithiasis in this group. METHODS AND RESULTS: Fresh faecal samples collected from healthy black and white South African male volunteers were analysed in terms of bacterial oxalate-degrading activity, bacterial diversity and relative species abundance. Varied bacterial populations prepared from samples from the low-risk black group showed a significantly higher level of oxalate degradation. Denaturing gradient gel electrophoresis analyses of Lactobacillus and related spp. and Bifidobacterium spp. 16S rRNA PCR products revealed a significantly higher faecal Lactobacillus diversity for the low-risk black group relative to the higher-risk white group. Quantitative real-time PCR experiments did not show any significant differences between the study groups for Lactobacillus and related spp.. However, Bifidobacterium spp. were present at a significantly higher relative abundance in the black group. Oxalobacter formigenes was present only at very low levels in either group. CONCLUSIONS: The low abundance of O. formigenes and increased diversity and abundance of oxalate-degrading Lactobacillus and Bifidobacterium spp. in the black South African population suggest that these strains rather than O. formigenes may protect this group against calcium oxalate kidney stone disease. SIGNIFICANCE AND IMPACT OF THE STUDY: The South African black population harbours a pool of potential oxalate-degrading lactic acid bacteria, which is more abundant and diverse than that of white South Africans. This may be useful in developing probiotics for calcium oxalate kidney stone prophylaxis.

Antibiotic resistance and adhesion potential of Bacteroides fragilis clinical isolates from Cape Town, South Africa.

The minimum inhibitory concentrations of 23 Bacteroides fragilis clinical isolates from Cape Town, South Africa, were established using the E-test method. Eight percent of the strains were found to be highly resistant to metronidazole (≥256 mg/L) imipenem and cefoxitin. This is an 8% increase in resistance compared to the previous metronidazole susceptibility screening performed in South Africa in 1998. Clindamycin was the most effective antibiotic with all strains showing sensitivity. Most of the strains (65%) were tetracycline resistant, while one strain, B. fragilis GSH15, showed multidrug resistance to metronidazole, imipenem, cefoxitin and tetracycline. PCR screening revealed that none of the strains contained any of the published nim genes. The particle agglutination assay was employed to determine the ability of the isolates to bind the ECM components fibronectin, laminin, mucin and collagen. This revealed that 78% of the clinical isolates adhered to all four ECM components to varying extents, with the strongest being to laminin and weakest to mucin and collagen Type I.

Crystallization of recombinant Bacteroides fragilis glutamine synthetase (GlnN) isolated using a novel and rapid purification protocol.

Glutamine synthetase enzymes (GSs) are large oligomeric enzymes that play a critical role in nitrogen metabolism in all forms of life. To date, no crystal structures exist for the family of large (∼1 MDa) type III GS enzymes, which only share 9% sequence identity with the well characterized GSI and GSII enzymes. Here we present a novel protocol for the isolation of untagged Bacteroides fragilis GlnN expressed in an auxotrophic Escherichia coli strain. The rapid and scalable two-step protocol utilized differential precipitation by divalent cations followed by affinity chromatography to produce suitable quantities of homogenous material for structural characterization. Subsequent optimizations to the sample stability and solubility led to the discovery of conditions for the production of the first diffraction quality crystals of a type III GS enzyme.

Xylose utilization and short-chain fatty acid production by selected components of the intestinal microflora of a rodent pollinator (Aethomys namaquensis).

Namaqua rock mice (Aethomys namaquensis) consume nectar xylose when visiting Protea flowers. Whole-animal metabolism studies suggest that the gastrointestinal microflora plays an important role in xylose metabolism in A. namaquensis. We collected caecal contents under anaerobic conditions, cultured caecal microflora both aerobically and anaerobically, and assessed caecal microbial xylose utilization using a (14)C-xylose incubation assay. All four mice sampled hosted culturable caecal micro-organisms that tested positive for xylose utilization. These were classified by 16S rRNA based taxonomy as: Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis, Shigella boydii, Arthrobacter sp. and members of the fungal genera Aspergillus and Penicillium. Cultures of these isolates were then analyzed by gas chromatography to determine the types and quantities of short-chain fatty acids produced by xylose fermentation. These results are discussed in the context of other studies of gut microflora in vertebrates.

A functional analysis of the Bifidobacterium longum cscA and scrP genes in sucrose utilization.

The role of genes involved in sucrose catabolism was investigated with a view to designing effective prebiotic substrates to encourage the growth of Bifidobacterium in the gut. Two gene clusters coding for sucrose utilisation in Bifidobacterium longum NCC2705 were identified in the published genome. The genes encoding putative sucrose degrading enzymes, namely, the scrP (sucrose phosphorylase) and the cscA (beta-fructofuranosidase), were cloned from B. longum NCIMB 702259(T) and expressed in Escherichia coli DH5alpha. Both complemented the sucrase negative phenotype of untransformed cells and showed specific sucrase activity. Transcriptional analysis of the expression of the genes in B. longum grown in the presence of various carbohydrate substrates showed induction of scrP gene expression in the presence of sucrose and raffinose, but not in the presence of glucose. The cscA gene showed no increased transcription in B. longum grown in the presence of any of the carbohydrates tested. Phylogenetic analysis indicates that the B. longum CscA protein belongs to a distinct phylogenetic cluster of intracellular fructosidases, which specifically cleave the shorter fructose oligosaccharides.

Use of traditional African fermented beverages as delivery vehicles for Bifidobacterium lactis DSM 10140.

A microencapsulation delivery system for Bifidobacterium lactis, a possible probiotic suited for use by the rural population of South Africa, was evaluated using two existing traditional fermented foods, amasi and mahewu. Gellan/xanthan microcapsules containing viable B. lactis, were tested under simulated physiological conditions, and added to pasteurized beverages. The capsules protected the organism under simulated low pH conditions associated with the stomach and from the biocidal activity of pancreatic and bile acids. For mahewu, microencapsulation of B. lactis with storage aerobically at 4 degrees C and 22 degrees C enhanced survival over a 21-day period as compared to free cells. In amasi, differences in viability between immobilized and free cells were less noticeable. An analytically trained taste panel was unable to detect a significant difference in texture in any of the samples fortified with microcapsules. Although flavour differences were noted for mahewu containing either free or immobilized cells, after 14 days refrigerated storage, these were not disliked. No significant flavour difference was noted between amasi containing immobilized cells and untreated controls over the period tested.

In-vitro antibiotic susceptibility and molecular analysis of anaerobic bacteria isolated in Cape Town, South Africa.

One hundred and twenty-three anaerobic isolates from Cape Town, South Africa, were tested in vitro against cefoxitin, chloramphenicol, metronidazole, co-amoxiclav, benzylpenicillin and clindamycin. Forty-five Gram-positive organisms were tested against RP59500 (a streptogramin). Resistance in the Gram-positive isolates was as follows: of the Clostridium perfringens isolates, 4% were resistant to benzylpenicillin and 4% to clindamycin; of the Peptostreptococcus anaerobius isolates, 10% were resistant to benzylpenicillin, 10% to cefoxitin and 10% to metronidazole; of the isolates of Peptostreptococcus spp., 12% were resistant to benzylpenicillin, 6% to metronidazole, 6% to chloramphenicol and 12% to RP59500. Of the Gram-negative organisms, those in the Bacteroides fragilis group were resistant to benzylpenicillin (83%), cefoxitin (5%), clindamycin (5%) and co-amoxiclav (2%). One clindamycin-resistant B. fragilis isolate carried a plasmid homologous to the ermF erythromycin resistance determinant.

Cloning of an EF-P homologue from Bacteroides fragilis that increases B. fragilis glutamine synthetase activity in Escherichia coli.

Investigations of possible regulators of Bacteroides fragilis glutamine synthetase (GS) activity were done in Escherichia coli using a compatible dual-plasmid system. The B. fragilis glnA gene, together with upstream and downstream flanking regions, was cloned onto the low copy number plasmid pACYC184 and expressed in the E. coli glnA ntrB ntrC deletion strain, YMC11. GS activity was monitored following co-transformation with a B. fragilis genomic library carried on the compatible plasmid pEcoR251. A gene was cloned that caused a twofold increase in B. fragilis GS activity but did not affect the activity of the E. coli GS enzyme or the B. fragilis sucrase (ScrL). Deletion of the B. fragilis glnA downstream region decreased basal levels of GS activity, but did not affect the ability of the cloned gene to increase the B. fragilis GS activity. Reporter gene analysis, using the B. fragilis glnA promoter region fused to the promoterless Clostridium acetobutylicum endoglucanase gene, showed no increase in reporter gene activity. This demonstrated that the increase in GS activity was not regulated at the transcriptional level, and that the cloned gene product was not affecting the copy number of the plasmid in trans. Sequence data indicated that the cloned gene had good amino acid identity to a range of elongation factor P (EF-P) proteins, the highest being to that of a Synechocystis sp (48%), and the least to Mycobacterium genitalium (27%). Amino acid identity to the E. coli EF-P was intermediate (37%). A possible role for EF-P in enhancing translation of the B. fragilis glnA mRNA is proposed.

Mode of action of metronidazole and a Bacteroides fragilis metA resistance gene in Escherichia coli.

The in-vivo mode of action of metronidazole and the MetA protein which confers resistance to metronidazole, was investigated in Escherichia coli wild type and DNA repair-deficient strains. All the E. coli strains were moderately susceptible to metronidazole under aerobic conditions but were more susceptible under anaerobic conditions, and the amount of DNA breakage was less under anaerobic than aerobic conditions. E. coli excision (uvr) and recombination (rec) mutants were more susceptible than DNA repair wild type strains. Metronidazole did not induce cell lysis in E. coli but caused single strand DNA breaks in wild type and repair-deficient E. coli strains. The MetA protein reduced DNA breakage caused by metronidazole in E. coli wild type and DNA repair-deficient strains grown under aerobic and anaerobic conditions, and inhibited the suppressor effect of the RecE protein in E. coli recBC strains. The MetA protein did not inactivate metronidazole.

A reporter gene vector to investigate the regulation of glutamine synthetase in Bacteroides fragilis Bf1.

The Clostridium acetobutylicum eglA gene, encoding a beta-1,4-endoglucanase (EG), was shown to be a useful reporter gene for the study of gene expression in Bacteroides fragilis. The eglA reporter gene has the advantages that it can be easily identified in both Escherichia coli and B. fragilis on agar media containing carboxymethylcellulose, and EG production can be rapidly quantified in liquid medium. Since the B. fragilis glutamine synthetase (GS) is inactivated in permeabilized cells and cell extracts, the eglA reporter gene was used to study the regulation of GS production in B. fragilis. Gene fusions containing the GS glnA promoter region fused to the promoterless eglA gene showed that glnA expression was regulated by nitrogen in B. fragilis at the transcriptional level. A glnA upstream region containing a near-perfect direct repeat sequence was essential for efficient GS expression and for regulation by nitrogen.

Molecular analysis of a gene from Bacteroides fragilis involved in metronidazole resistance in Escherichia coli.

The region of Bacteroides fragilis DNA on the recombinant plasmid pMT100 responsible for conferring metronidazole resistance in Escherichia coli strains was characterized. An open reading frame (ORF1) of 195 bp encoded a protein of 64 amino acids with a predicted M(r) of 7.3 kDa. Deletion analysis indicated that ORF1 conferred the metronidazole resistance phenotype and encoded a protein with an apparent M(r) of approximately 8-10 kDa.

Induction and repair of DNA strand breaks in Bacteroides fragilis.

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.

Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli.

Since reduced metronidazole causes DNA damage, resistance to metronidazole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia coli. Genes from B. fragilis Bf-2 were cloned on a recombinant plasmid pMT100 which made E. coli AB1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistance and uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fragilis Bf-2 cells.

Nucleotide sequence and expression of a cloned Thiobacillus ferrooxidans recA gene in Escherichia coli.

The nucleotide sequence of the recA gene of Thiobacillus ferrooxidans has been determined. No SOS box characteristic of LexA-regulated promoters could be identified in the 196-bp region upstream from the coding region. The cloned T. ferrooxidans recA gene was expressed in Escherichia coli from both the lambda pR and lac promoters. It was not expressed from the 2.2-kb of T. ferrooxidans DNA preceding the gene. The T. ferrooxidans recA gene specifies a protein of 346 amino acids that has 66% and 69% homology to the RecA proteins of E. coli and Pseudomonas aeruginosa, respectively. Most amino acids that have been identified as being of functional importance in the E. coli RecA protein are conserved in the T. ferrooxidans RecA protein. Although some amino acids that have been associated with proteolytic activity have been substituted, the cloned protein has retained protease activity towards the lambda and E. coli LexA repressors.

Pyrimidine dimer excision repair of DNA in Bacteroides fragilis wild-type and mitomycin C-sensitive/UV-sensitive mutants.

An enzyme preparation purified from Micrococcus luteus was shown to be specific for UV-induced pyrimidine dimers and was suitable for the detection of DNA excision repair systems. The wild-type Bacteroides fragilis Bf-2 strain and a mitomycin C-sensitive mutant (MTC25) had constitutive dimer excision systems which functioned efficiently under anaerobic and aerobic conditions. A UV-sensitive mutant (UVS9) had markedly reduced levels of the constitutive dimer excision systems under anaerobic and aerobic conditions. Since liquid holding recovery under aerobic conditions was inhibited by chloramphenicol whereas the final level of excision repair in B. fragilis Bf-2 was not affected, it is concluded that pyrimidine dimer removal is not the process responsible for increased physiological aerobic liquid holding recovery.

Isolation and physiological characterization of mitomycin C-sensitive/UV-sensitive mutants in Bacteroides fragilis.

Mutants of Bacteroides fragilis sensitive to mitomycin C were isolated after mutagenesis with ethyl methane sulphonate. One mutant (MTC25) was markedly sensitive to mitomycin C but was unaffected as regards UV sensitivity; another mutant (UVS9) was sensitive to UV radiation but was only moderately sensitive to mitomycin C. Caffeine decreased the survival after UV-irradiation of the wild-type, MTC25 and UVS9 strains by the same relative amount. Aerobic liquid holding recovery occurred in each of the three strains. The MTC25 and UVS9 mutants showed reduced host cell phage reactivation. The wild-type, MTC25 and UVS9 strains all showed UV- and H2O2-induced phage reactivation. The physiological characterization of the MTC25 and UVS9 mutants indicates that it is possible to differentiate between mechanisms for the repair of mitomycin C- and UV-induced DNA damage in B. fragilis.