Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents.

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.

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.