Clostridium difficile ribotype 017 – characterization, evolution and epidemiology of the dominant strain in Asia

Clostridium difficile ribotype (RT) 017 is an important toxigenic C. difficile RT which, due to a deletion in the repetitive region of the tcdA gene, only produces functional toxin B. Strains belonging to this RT were initially dismissed as nonpathogenic and circulated largely undetected for almost two decades until they rose to prominence following a series of outbreaks in the early 2000s. Despite lacking a functional toxin A, C. difficile RT 017 strains have been shown subsequently to be capable of causing disease as severe as that caused by strains producing both toxins A and B. While C. difficile RT 017 strains can be found in almost every continent today, epidemiological studies suggest that the RT is endemic in Asia and that the global spread of this MLST clade 4 lineage member is a relatively recent event. C. difficile RT 017 transmission appears to be mostly from human to human with only a handful of reports of isolations from animals. An important feature of C. difficile RT 017 strains is their resistance to several antimicrobials and this has been documented as a possible factor driving multiple outbreaks in different parts of the world. This review summarizes what is currently known regarding the emergence and evolution of strains belonging to C. difficile RT 017 as well as features that have allowed it to become an RT of global importance

In Silico Characterisation of Putative Prophages in Lactobacillaceae Used in Probiotics for Vaginal Health

While live biotherapeutics offer a promising approach to optimizing vaginal microbiota, the presence of functional prophages within introduced Lactobacillaceae strains could impact their safety and efficacy. We evaluated the presence of prophages in 895 publicly available Lactobacillaceae genomes using Phaster, Phigaro, Phispy, Prophet and Virsorter. Prophages were identified according to stringent (detected by ≥4 methods) or lenient criteria (detected by ≥2 methods), both with >80% reciprocal sequence overlap. The stringent approach identified 448 prophages within 359 genomes, with 40.1% genomes harbouring at least one prophage, while the lenient approach identified 1671 prophages within 83.7% of the genomes. To confirm our in silico estimates in vitro, we tested for inducible prophages in 57 vaginally-derived and commercial Lactobacillaceae isolates and found inducible prophages in 61.4% of the isolates. We characterised the in silico predicted prophages based on weighted gene repertoire relatedness and found that most belonged to the Siphoviridae or Myoviridae families. ResFam and eggNOG identified four potential antimicrobial resistance genes within the predicted prophages. Our results suggest that while Lactobacillaceae prophages seldomly carry clinically concerning genes and thus unlikely a pose a direct risk to human vaginal microbiomes, their high prevalence warrants the characterisation of Lactobacillaceae prophages in live biotherapeutics

Characterization of recombinant β-fructofuranosidase from Bifidobacterium adolescentis G1

<p>Abstract</p> <p>Background</p> <p>We have previously reported on purification and characterization of β-fructofuranosidase (β-FFase) from <it>Bifidobacterium adolescentis </it>G1. This enzyme showed high activity of hydrolysis on fructo-oligosaccharides with a low degree of polymerization. Recently, genome sequences of <it>B. longum </it>NCC2705 and <it>B. adolescentis </it>ATCC 15703 were determined, and <it>cscA </it>gene in the both genome sequences encoding β-FFase was predicted. Here, cloning of <it>cscA </it>gene encoding putative β-FFase from <it>B. adolescentis </it>G1, its expression in <it>E. coli </it>and properties of the recombinant protein are described.</p> <p>Results</p> <p>Using the information of <it>cscA </it>gene from <it>Bifidobacterium adolescentis </it>ATCC 15703, <it>cscA </it>gene from <it>B. adolescentis </it>G1 was cloned and sequenced. The N-terminal amino acid sequence of purified β-FFase from <it>B. adolescentis </it>G1 was identical to the deduced amino acid sequences of <it>cscA </it>gene from <it>B. adolescentis </it>G1. To confirm the translated product of the <it>cscA </it>gene, the recombinant protein was expressed in <it>Escherichia coli</it>. Molecular mass of the purified recombinant enzyme was estimated to be about 66,000 by SDS-PAGE and 60,300 by MALDI TOF-MS. The optimum pH of the enzyme was 5.7 and the enzyme was stable at pH 5.0-8.6. The thermostability of the enzyme was up to 50°C. The <it>K</it>_m(mM), <it>V</it>_max(μmol/mg of protein/min), <it>k</it>₀(sec^-1) and <it>k</it>₀/<it>K</it>_m(mM^-1sec^-1) for 1-kestose, neokestose, nystose, fructosylnystose, sucrose and inulin were 1.7, 107, 107.5, 63.2, and 1.7, 142, 142.7, 83.9, and 3.9, 152, 152.8, 39.2, and 2.2, 75, 75.4, 34.3, and 38, 79, 79.4, 2.1, and 25.9, 77, 77.4, 3.0, respectively. The hydrolytic activity was strongly inhibited by AgNO₃, SDS, and HgCl₂.</p> <p>Conclusion</p> <p>The recombinant enzyme had similar specificity to the native enzyme, high affinity for 1-kestose, and low affinity for sucrose and inulin, although properties of the recombinant enzyme showed slight difference from those of the native one previously described.</p

Clostridioides difficile infection in Africa: A narrative review

Clostridioides (Clostridium) difficile infection (CDI) places a burden on healthcare facilities worldwide. Most research studies have been concentrated in high-income countries in North America, Europe, Asia and Australia, where C. difficile is the leading cause of diarrhoea associated with antimicrobial use. This narrative review summarises African CDI studies, focussing on reports published in the last 20 years. Although relatively sparse, the data suggest that CDI is an important cause of diarrhoea on the continent. African CDI patient populations are often younger than in European and North American settings, probably due to the high prevalence of co-morbid conditions such as tuberculosis, particularly in sub-Saharan Africa. Strain typing data are rare and where reported generally limited to single sites and institutions. Despite challenges, including a lack of facilities and awareness, there is a need for further investigation to more accurately determine the true burden of disease caused by C. difficile in Africa

Evolutionary and genomic insights into Clostridioides difficile sequence Type 11: a diverse zoonotic and antimicrobial-resistant lineage of global one health importance

Clostridioides difficile (Clostridium difficile) sequence type 11 (ST11) is well established in production animal populations worldwide and contributes considerably to the global burden of C. difficile infection (CDI) in humans. Increasing evidence of shared ancestry and genetic overlap of PCR ribotype 078 (RT078), the most common ST11 sublineage, between human and animal populations suggests that CDI may be a zoonosis. We performed whole-genome sequencing (WGS) on a collection of 207 ST11 and closely related ST258 isolates of human and veterinary/environmental origin, comprising 16 RTs collected from Australia, Asia, Europe, and North America. Core genome single nucleotide variant (SNV) analysis identified multiple intraspecies and interspecies clonal groups (isolates separated by ≤2 core genome SNVs) in all the major RT sublineages: 078, 126, 127, 033, and 288. Clonal groups comprised isolates spread across different states, countries, and continents, indicative of reciprocal long-range dissemination and possible zoonotic/anthroponotic transmission. Antimicrobial resistance genotypes and phenotypes varied across host species, geographic regions, and RTs and included macrolide/lincosamide resistance (Tn6194 [ermB]), tetracycline resistance (Tn6190 [tetM] and Tn6164 [tet44]), and fluoroquinolone resistance (gyrA/B mutations), as well as numerous aminoglycoside resistance cassettes. The population was defined by a large “open” pan-genome (10,378 genes), a remarkably small core genome of 2,058 genes (only 19.8% of the gene pool), and an accessory genome containing a large and diverse collection of important prophages of the Siphoviridae and Myoviridae. This study provides novel insights into strain relatedness and genetic variability of C. difficile ST11, a lineage of global One Health importance

Antimicrobial resistance in Clostridium difficile ribotype 017

Introduction: Antimicrobial resistance (AMR) played an important role in the initial outbreaks of Clostridium difficile infection (CDI) in the 1970s. C. difficile ribotype (RT) 017 has emerged as the major strain of C. difficile in Asia, where antimicrobial use is poorly regulated. This strain has also caused CDI outbreaks around the world for almost 30 years. Many of these outbreaks were associated with clindamycin and fluoroquinolone resistance. AMR and selective pressure is likely to be responsible for the success of this RT and may drive future outbreaks. Areas covered: This narrative review summarizes the prevalence and mechanisms of AMR in C. difficile RT 017 and transmission of these AMR mechanisms. To address these topics, reports of outbreaks due to C. difficile RT 017, epidemiologic studies with antimicrobial susceptibility results, studies on resistance mechanisms found in C. difficile and related publications available through Pubmed until September 2019 were collated and the findings discussed. Expert opinion: Primary prevention is the key to control CDI. This should be achieved by developing antimicrobial stewardship in medical, veterinary and agricultural practices. AMR is the key factor that drives CDI outbreaks, and methods for the early detection of AMR can facilitate the control of outbreaks

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

Correction for Cairns et al., "Comparative Genome Analysis and Global Phylogeny of the Toxin Variant Clostridium difficile PCR Ribotype 017 Reveals the Evolution of Two Independent Sublineages".

Page 868, Table 2: The entry corresponding to sublineage 1 in the "Country of origin" column should read "Argentina, Australia, Bulgaria, Canada, China, Czech Republic, Greece, Hong Kong, Ireland, Japan, South Korea, Kuwait, Poland, Portugal, Romania, Singapore, Slovenia, South Africa, The Netherlands, UK, USA." Page 868, Table 2: The entry corresponding to sublineage 2 in the "Country of origin" column should read "Australia, Hong Kong, Indonesia, Japan, South Korea, Poland, Singapore, South Africa, Taiwan, The Netherlands, UK, USA." Page 869, Fig. 1: The labels SL1 and SL2 are incorrectly placed. The upper portion should be labelled "SL2," and the lower portion should be labelled "SL1." Page 869, Fig. 1 legend, lines 3 and 4: The sentence beginning "The SL1 and SL2 sublineages" should read "The SL1 and SL2 sublineages were differentiated by four SNPs (Table 3), with the reference strain M68 falling into SL1." Page 871, Figure 3 legend, line 2: The sentence beginning "The analysis indicates a split" should read "The analysis indicates a split from SL2 (lower) into SL1 (upper) c1990, with the M68 reference in SL1.