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.

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.

Assessment of independent comorbidities and comorbidity measures in predicting healthcare facility-onset Clostridioides difficile infection in Kenya.

INTRODUCTION: Clostridioides difficile is primarily associated with hospital-acquired diarrhoea. The disease burden is aggravated in patients with comorbidities due to increased likelihood of polypharmacy, extended hospital stays and compromised immunity. The study aimed to investigate comorbidity predictors of healthcare facility-onset C. difficile infection (HO-CDI) in hospitalized patients. METHODOLOGY: We performed a cross sectional study of 333 patients who developed diarrhoea during hospitalization. The patients were tested for CDI. Data on demographics, admission information, medication exposure and comorbidities were collected. The comorbidities were also categorised according to Charlson Comorbidity Index (CCI) and Elixhauser Comorbidity Index (ECI). Comorbidity predictors of HO-CDI were identified using multiple logistic regression analysis. RESULTS: Overall, 230/333 (69%) patients had comorbidities, with the highest proportion being in patients aged over 60 years. Among the patients diagnosed with HO-CDI, 63/71(88.7%) reported comorbidities. Pairwise comparison between HO-CDI patients and comparison group revealed significant differences in hypertension, anemia, tuberculosis, diabetes, chronic kidney disease and chronic obstructive pulmonary disease. In the multiple logistic regression model significant predictors were chronic obstructive pulmonary disease (odds ratio [OR], 9.51; 95% confidence interval [CI], 1.8-50.1), diabetes (OR, 3.56; 95% CI, 1.11-11.38), chronic kidney disease (OR, 3.88; 95% CI, 1.57-9.62), anemia (OR, 3.67; 95% CI, 1.61-8.34) and hypertension (OR, 2.47; 95% CI, 1.-6.07). Among the comorbidity scores, CCI score of 2 (OR 6.67; 95% CI, 2.07-21.48), and ECI scores of 1 (OR, 4.07; 95% CI, 1.72-9.65), 2 (OR 2.86; 95% CI, 1.03-7.89), and ≥ 3 (OR, 4.87; 95% CI, 1.40-16.92) were significantly associated with higher odds of developing HO-CDI. CONCLUSION: Chronic obstructive pulmonary disease, chronic kidney disease, anemia, diabetes, and hypertension were associated with an increased risk of developing HO-CDI. Besides, ECI proved to be a better predictor for HO-CDI. Therefore, it is imperative that hospitals should capitalize on targeted preventive approaches in patients with these underlying conditions to reduce the risk of developing HO-CDI and limit potential exposure to other patients.

Major genetic discontinuity and novel toxigenic species in Clostridioides difficile taxonomy.

Clostridioides difficile infection (CDI) remains an urgent global One Health threat. The genetic heterogeneity seen across C. difficile underscores its wide ecological versatility and has driven the significant changes in CDI epidemiology seen in the last 20 years. We analysed an international collection of over 12,000 C. difficile genomes spanning the eight currently defined phylogenetic clades. Through whole-genome average nucleotide identity, and pangenomic and Bayesian analyses, we identified major taxonomic incoherence with clear species boundaries for each of the recently described cryptic clades CI-III. The emergence of these three novel genomospecies predates clades C1-5 by millions of years, rewriting the global population structure of C. difficile specifically and taxonomy of the Peptostreptococcaceae in general. These genomospecies all show unique and highly divergent toxin gene architecture, advancing our understanding of the evolution of C. difficile and close relatives. Beyond the taxonomic ramifications, this work may impact the diagnosis of CDI.

Exploring potential of vaginal Lactobacillus isolates from South African women for enhancing treatment for bacterial vaginosis.

Antibiotics continue to be the standard-of-care for bacterial vaginosis (BV), although recurrence rates are high. Vaginal probiotics may improve durability of BV treatment, although few probiotics for vaginal health contain Lactobacillus spp. that commonly colonize the lower female genital tract. Characteristics of vaginal Lactobacillus strains from South African women were evaluated for their probiotic potential in vitro compared to strains from commercial vaginal products, including growth at varying pHs, ability to lower pH, produce D-/L-lactate and H2O2, influence growth of BV-associated Gardnerella vaginalis and Prevotella bivia, adherence to cervical cells and susceptibility to antibiotics. Fifty-seven Lactobacillus strains were purified from cervico-vaginal fluid, including L. crispatus, L. jensenii, L. gasseri, L. mucosae, and L. vaginalis. L crispatus strains grew better at pHs below 4.5 and lowered pH more effectively than other strains. Production of D-/L-lactate and H2O2 varied between Lactobacillus species and strains. Lactobacillus strains generally inhibited P. bivia more uniformly than G. vaginalis isolates. All vaginal Lactobacillus isolates were resistant to metronidazole while susceptibility to clindamycin varied. Furthermore, vaginal Lactobacillus strains tended to be broadly susceptible to penicillin, amoxicillin, rifampicin and rifabutin. Whole-genome-sequencing of five of the best-performing vaginal Lactobacillus strains confirmed their likely safety, due to antimicrobial resistance elements being largely absent, while putative intact prophages were present in the genomes of two of the five strains. Overall, vaginal Lactobacillus strains largely performed better in these in vitro assays than probiotic strains currently used in probiotics for vaginal health. Including the best-performing vaginal Lactobacillus isolates in a region-specific probiotic for vaginal health may result in improved BV treatment options.

High Prevalence of Multidrug-Resistant Clostridioides difficile Following Extensive Use of Antimicrobials in Hospitalized Patients in Kenya.

Introduction: Clostridioides difficile is a neglected pathogen in many African countries as it is generally not regarded as one of the major contributors toward the diarrheal disease burden in the continent. However, several studies have suggested that C. difficile infection (CDI) may be underreported in many African settings. The aim of this study was to determine the prevalence of CDI in hospitalized patients, evaluate antimicrobial exposure, and detect toxin and antimicrobial resistance profiles of the isolated C. difficile strains. Methods: In this cross-sectional study, 333 hospitalized patients with hospital-onset diarrhoea were selected. The stool samples were collected and cultured on cycloserine-cefoxitin egg yolk agar (CCEY). Isolates were presumptively identified by phenotypic characteristics and Gram stain and confirmed by singleplex real-time PCR (qPCR) assays detecting the species-specific tpi gene, toxin A (tcdA) gene, toxin B (tcdB) gene, and the binary toxin (cdtA/cdtB) genes. Confirmed C. difficile isolates were tested against a panel of eight antimicrobials (vancomycin, metronidazole, rifampicin, ciprofloxacin, tetracycline, clindamycin, erythromycin, and ceftriaxone) using E-test strips. Results: C. difficile was detected in 57 (25%) of diarrheal patients over the age of two, 56 (98.2%) of whom received antimicrobials before the diarrheal episode. Amongst the 71 confirmed isolates, 69 (97.1%) harbored at least one toxin gene. More than half of the toxigenic isolates harbored a truncated tcdA gene. All isolates were sensitive to vancomycin, while three isolates (2.1%) were resistant to metronidazole (MIC >32 mg/L). High levels of resistance were observed to rifampicin (65/71, 91.5%), erythromycin (63/71, 88.7%), ciprofloxacin (59/71, 83.1%), clindamycin (57/71, 80.3%), and ceftriaxone (36/71, 50.7.8%). Among the resistant isolates, 61 (85.9%) were multidrug-resistant. Conclusion: Multidrug-resistant C. difficile strains were a significant cause of healthcare facility-onset C. difficile infections in patients with prior antimicrobial exposure in this Kenyan hospital.

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.

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.

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.IMPORTANCE Historically, Clostridioides difficile (Clostridium difficile) has been associated with life-threatening diarrhea in hospitalized patients. Increasing rates of C. difficile infection (CDI) in the community suggest exposure to C. difficile reservoirs outside the hospital, including animals, the environment, or food. C. difficile sequence type 11 (ST11) is known to infect/colonize livestock worldwide and comprises multiple ribotypes, many of which cause disease in humans, suggesting CDI may be a zoonosis. Using high-resolution genomics, we investigated the evolution and zoonotic potential of ST11 and a new closely related ST258 lineage sourced from diverse origins. We found multiple intra- and interspecies clonal transmission events in all ribotype sublineages. Clones were spread across multiple continents, often without any health care association, indicative of zoonotic/anthroponotic long-range dissemination in the community. ST11 possesses a massive pan-genome and numerous clinically important antimicrobial resistance elements and prophages, which likely contribute to the success of this globally disseminated lineage of One Health importance.

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.

A comparison of Clostridium difficile diagnostic methods for identification of local strains in a South African centre.

Accurate diagnosis of Clostridium difficile infection is essential for disease management. A clinical and molecular analysis of C. difficile isolated from symptomatic patients at Groote Schuur Hospital, South Africa, was conducted to establish the most suitable clinical test for the diagnosis and characterization of locally prevalent strains. C. difficile was detected in stool samples using enzyme-based immunoassays (EIA) and nucleic acid amplification methods, and their performance was compared with that of C. difficile isolation using direct selective culture combined with specific PCR to detect the C. difficile tpi gene, toxin A and B genes and binary toxin genes. Toxigenic isolates were characterized further by ribotyping. Selective culture isolated 32 C. difficile strains from 145 patients (22 %). Of these, the most prevalent (50 %) were of ribotype 017 (toxin A- B+) while 15.6 % were ribotype 001 (toxin A+B+). No ribotype 027 strains or binary toxin genes (cdtA and cdtB) were detected. The test sensitivities and specificities, respectively, of four commercial clinical diagnostic methods were as follows: ImmunoCard Toxins A & B (40 % and 99.1 %), VIDAS C. difficile Toxin A & B (50 % and 99.1 %), GenoType CDiff (86.7 % and 88.3 %) and Xpert C. difficile (90 % and 97.3 %). Ribotype 001 and 017 strains had a 100 % detection rate by Xpert C. difficile, 100 % and 93.3 % by GenoType CDiff, 75 % and 53.3 % by ImmunoCard and 75 % and 60 % by VIDAS, respectively. The overall poor performance of EIA suggests that a change to PCR-based testing would assist diagnosis and ensure reliable detection of locally prevalent C. difficile 017 strains.

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.