Common Ribotypes Research Articles

Diversity of Clostridioides difficile PCR ribotypes isolated from freshwater sediments depends on the isolation method.

Clostridioides difficile is an intestinal pathogen of humans and animals. In community-associated infections, the environment is suggested to play a significant role in overall transmission routes. Although the prevalence of C. difficile in freshwater and soil has been widely studied, little is known about its presence in sediments. In this study, we tested 15 sediment samples collected from various freshwater sources. C. difficile was isolated from all sampled sites, yielding a total of 171 strains grouped into 26 ribotypes, with 001/072 and 014/020 being the most prevalent. Genome sequencing of 37 isolates from 17 PCR ribotypes confirmed the presence of highly related strains in the geographically distant and unlinked water samples. Eight divergent PCR ribotypes from clades C-II and C-III were found in six samples. In each sample, the unbound fraction (supernatant after sediment wash) and bound fraction (sonicated sediment sample) were subjected to enrichment. Sonication was only slightly better than washing in terms of sample positivity (14 positive samples with sonication and 11 with washing). However, sonication substantially increased the diversity of the PCR ribotypes obtained (23 in sonicated samples vs nine in washed samples). In conclusion, sediments are a rich source for investigating the diversity of environmental C. difficile, including isolates from divergent lineages. Selection of the isolation method can significantly impact the diversity of captured PCR ribotypes.IMPORTANCEClostridioides difficile, a pathogenic bacterium that can cause intestinal infections in humans and animals, thrives in the gut but also disperses widely through spores found in the environment. Clinical and environmental strains often overlap with common PCR ribotypes, which are consistently isolated worldwide. Environmental studies have mostly focused on water and soil, but sediments have been very poorly studied. In this study, we investigated the presence of C. difficile in various freshwater sediments and evaluated the effectiveness of two different isolation approaches on positivity rates and strain diversity. C. difficile was found to be highly prevalent in sediments, with an isolation rate of 100%. Sonication proved to be more effective than simple washing for capturing a greater diversity of PCR ribotypes. Overall, this study underscores the widespread presence of C. difficile in freshwater sediments and emphasizes the importance of continued surveillance and monitoring to understand its ecology and transmission dynamics.

Clostridioides difficile infection epidemiology during the COVID-19 pandemic in Greece.

Aim: The aim was to highlight the incidence and epidemiology of C. difficile infections (CDI) in a tertiary Greek hospital during the COVID-19 pandemic.Methods: A single-center prospective observational cohort study was conducted (October 2021 until April 2022). 125 C. difficile isolates were cultured from hospitalized patients stool samples and screened by PCR for toxin A (tcdA), toxin B (tcdB), binary toxin (cdtA and cdtB) genes and the regulating gene of tcdC.Results: The incidence of CDI increased to 13.1 infections per 10,000bed days. The most common PCR ribotypes identified included hypervirulent RT027-related RT181 (73.6%), presumably hypervirulent RT126 (8.0%) and toxin A negative RT017 (7.2%).Conclusion: Although the incidence of CDI increased significantly, the CDI epidemiology remained stable.

A US-based national surveillance study for the susceptibility and epidemiology of Clostridioides difficile isolates with special reference to ridinilazole: 2020-2021.

We have previously reported on the susceptibility and epidemiology of Clostridioides difficile isolates from six geographically dispersed medical centers in the United States. This current survey was conducted with isolates collected in 2020-2021 from six geographically dispersed medical centers in the United States, with specific attention to susceptibility to ridinilazole as well as nine comparators. C. difficile isolates or stools from patients with C. difficile antibiotic-associated diarrhea were collected and referred to a central laboratory. After species confirmation of 300 isolates at the central laboratory, antibiotic susceptibilities were determined by the agar dilution method [M11-A9, Clinical and Laboratory Standards Institute (CLSI)] against the 10 agents. Ribotyping was performed by PCR capillary gel electrophoresis on all isolates. Ridinilazole had a minimum inhibitory concentration (MIC) 90 of 0.25 mcg/mL, and no isolate had an MIC greater than 0.5 mcg/mL. In comparison, fidaxomicin had an MIC 90 of 0.5 mcg/mL. The vancomycin MIC 90 was 2 mcg/mL with a 0.7% resistance rate [both CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria]. The metronidazole MIC 90 was 1 mcg/mL, with none resistant by CLSI criteria, and a 0.3% resistance rate by EUCAST criteria. Among the 50 different ribotypes isolated in the survey, the most common ribotype was 014-020 (14.0%) followed by 106 (10.3%), 027 (10%), 002 (8%), and 078-126 (4.3%). Ridinilazole maintained activity against all ribotypes and all strains resistant to any other agent tested. Ridinilazole showed excellent in vitro activity against C. difficile isolates collected between 2020 and 2021 in the United States, independent of ribotype.

Geographic isolation and ribotypes on the example of some endemics of the genus Corydalis (Fumariaceae)

The main topic of this scientific work is the study of the characteristics of species ribotypes under conditions of long-term ecological and reproductive isolation. Three groups of endemics were selected. The first group is gypsophilic species: ^rydalis sangardanica Mikhailova, С. microphylla Mikhailova, С. gypsophyla Mikhailova. The second group is the rock cushion species: С. mira (Batalin) C. Y. Wu et H. Chuang, and С. yazgulemica Mikhailova et Sochivko. The third group is rocky, geographically isolated species: С. macrocalyx Litv, and С. rupestris Kotschy. The scientific novelty in this work is the use of genetic methods of analysis in the field of studying the possible hybridization of species of the genus Corydalis (Fumariaceae). NGS (Next-Generation Sequencing) was performed on the Illumina MiSeq platform. The first transcribed spacer (ITS1) of the nuclear ribosomal 35S rDNA gene was taken as a marker region. As a result, for each sample, a set of Zotu multicopy ribotypes (zero-radius operational taxonomic unit) was obtained, on the basis of which a qualitative and quantitative comparison of the samples was performed. It was shown that there are no common ribotypes in all three groups. The number of ribotypes in the studied species varies from one to six, which may be due to the peculiarities of hybridization in the past.

Characterization of Food Chain Clostridioides difficile Isolates in Terms of Ribotype and Antimicrobial Resistance.

The aim of this study was to characterize C. difficile isolates from the farm, abattoir, and retail outlets in Ireland in terms of ribotype and antibiotic resistance (vancomycin, erythromycin, metronidazole, moxifloxacin, clindamycin, and rifampicin) using PCR and E-test methods, respectively. The most common ribotype in all stages of the food chain (including retail foods) was 078 and a variant (RT078/4). Less commonly reported (014/0, 002/1, 049, and 205) and novel (RT530, 547, and 683) ribotypes were also detected, but at lower frequencies. Approximately 72% (26/36 tested) of the isolates tested were resistant to at least one antibiotic, with the majority of these (65%; 17/26) displaying a multi-drug (three to five antibiotics) resistant phenotype. It was concluded that ribotype 078, a hypervirulent strain commonly associated with C. difficile infection (CDI) in Ireland, was the most frequent ribotype along the food chain, resistance to clinically important antibiotics was common in C. difficile food chain isolates, and there was no relationship between ribotype and antibiotic resistance profile.

380. A molecular epidemiological exploration of reduced vancomycin susceptibility in Clostridioides difficile

Abstract Background Use of vancomycin to treat Clostridioides difficile infection (CDI) has increased following recent IDSA/SHEA treatment guideline updates, applying a selection pressure for resistance development. We previously demonstrated acquired mutations in VanSR two-component system led to constitutive vanG expression and improved in vitro C. difficile survival in physiologic vancomycin concentrations. We aim to describe the molecular epidemiology of reduced vancomycin susceptibility in clinical isolates during a period of high vancomycin use. Methods A cohort study was performed including adult patients hospitalized with CDI in two health systems (14 hospitals) in the Houston Area between 2017-2021. (Stool transport) C. difficile were ribotyped by fluorescent PCR and susceptibility tested by agar dilution in accordance with CLSI standards. Reduced vancomycin susceptibility was defined by minimum inhibitory concentrations (MICs) >2 mg/L. Sanger sequencing was conducted on a subgroup of isolates to identify VanSR mutations. Analysis using Chi square was performed using IBM SPSS Statistics (v 28.0.1.0). Results A total of 36% (165/465) of isolates exhibited reduced vancomycin susceptibility (MIC50 = 2 mg/L, MIC90 = 4 mg/L, range 0.5-16 mg/L), of which 348 were ribotyped. A significantly higher proportion of ribotype (RT) 027 isolates demonstrated reduced vancomycin susceptibility (83%) compared to other common ribotypes (30%); p< 0.001). No differences based on collection year (p=0.3) or healthcare system (p=0.08) were observed. Overall, 11% (7/56) of isolates exhibiting mutations in VanS (n=1), VanR (n=5), or both (n=1). VanSR mutations were present in 47% (7/15) of those with MICs >2mg/L vs 0% (0/41) of those with MICs ≤2 mg/L (p< 0.001). Conclusion A high proportion of clinical C. difficile isolates exhibited elevated MICs to vancomycin, which was most common in RT027 isolates. Mutations in the vanG regulator, VanSR, correlated with elevated MICs in a subgroup of isolates. Future research is needed to expand upon molecular mechanisms and clinical implications of reduced vancomycin susceptibility. Disclosures Kevin W. Garey, PharmD, MS, Acurx Pharmaceuticals: Grant/Research Support|Paratek Pharmaceuticals: Grant/Research Support|Seres Therapeutics: Grant/Research Support|Summit Pharmaceuticals: Grant/Research Support.

1669. A US Based National Surveillance Study for the Susceptibility and Epidemiology of Clostridioides difficile Associated Diarrheal Isolates with Special Reference to Ridinilazole: 2020-2021

Abstract Background We have previously reported on a US based national surveillance study from 6 geographically dispersed medical centers for the susceptibility and epidemiology of C. difficile isolates. This current survey was conducted with isolates collected in 2020-21 with specific attention to the susceptibility to ridinilazole as well as nine comparators. A summary of the susceptibilities of 300 isolates of C. difficile tested against 10 antimicrobial agents Methods in 2020-21 C. difficile isolates or stools from patients with C. difficile toxin positive antibiotic associated diarrhea were collected in 6 US medical centers and referred to a central lab. After confirmation of 300 isolates antibiotic susceptibilities were determined by agar dilution method (CLSI M11-A9) against 10 agents listed in Table below. Ribotyping was performed by PCR capillary gel electrophoresis on all isolates. Results A summary of the susceptibilities of the 300 isolates against 10 antimicrobial agents are shown in the Table. The most common ribotype was 014-020 (14.4% compared to 11.8% in 2016), followed by 106 (10%, compared to 15% in 2016), 027 (10% compared to 13.1% in 2016), 002 (8% compared to 8.5% in 2016, 078-126 (4.3% compared to 1.3% in 2016). Ridinilazole was very potent against all isolates collected in the US with an MIC 90 of 0.25 mcg/ml, including against all the hypervirulent ribotypes as well as those isolates with antibiotic resistances to other antibiotics in the test panel. Conclusion Ridinilazole showed excellent in-vitro activity against C. difficile isolates in the US, independent of ribotype. There has been a striking change in ribotype distribution compared to 2016, accompanied by a reduction in C. difficile resistance to moxifloxacin and imipenem. Disclosures Cheleste M. Thorpe, MD, Actelion: Grant/Research Support|Deinove: Advisor/Consultant|General Mills: Grant/Research Support|Merck: Grant/Research Support|Pfizer: Grant/Research Support|Summit: Advisor/Consultant|Summit: Grant/Research Support Stuart Johnson, M.D., Ferring Pharmaceuticals: Membership on Ferring Publication Steering Committee|Ferring Pharmaceuticals: Employee|Summit Plc: Advisor/Consultant Dale N. Gerding, MD, Destiny Pharma plc.: Advisor/Consultant Karen C. Carroll, MD, Abbott Diagnostics: Advisor/Consultant|BD Diagnostics: Advisor/Consultant|BD Diagnostics: Grant/Research Support|BioRad, Inc: Grant/Research Support|Meridian Biosciences: Grant/Research Support|Roche Diagnostics: Advisor/Consultant|Scanogen, Inc: Advisor/Consultant|Scanogen, Inc: Grant/Research Support|Thermo Fisher, Inc: Advisor/Consultant Kevin W. Garey, PharmD, MS, Acurx: Grant/Research Support|cidara: Advisor/Consultant|cidara: Grant/Research Support|Paratek: Grant/Research Support|Seres Health: Grant/Research Support|Summit: Grant/Research Support Esther Duperchy, PhD, Summit Plc: Employee David R. Snydman, MD, Merck: Advisor/Consultant|Merck and Company: Grant/Research Support|Prolacta: Advisor/Consultant|Prolacta: Grant/Research Support|Seres: Advisor/Consultant|Seres Health: Grant/Research Support|Summit Plc: Grant/Research Support|Takeda: Advisor/Consultant|Takeda: Grant/Research Support|Visterra: Advisor/Consultant.

Community-onset Clostridioides difficile infection in south Serbia

BackgroundData from the past decade indicates that Clostridioides difficile infection (CDI) is not only a nosocomial infection but is also increasingly recognized as a disease in the community. ObjectiveWe aimed to study community-onset (CO) CDI in the various age groups in south Serbia with its clinical characteristics, risk factors and microbiological characterization. MethodsThe study group included 93 patients with CO-CDI (median age 62). The control group consisted of 186 patients with community-onset diarrhea and stool samples negative tested for CDI. ResultsOf all CDI cases diagnosed with a community onset, 74.19% had a previous contact with a healthcare facility in the previous 12 weeks, but 34.40% have no record on hospitalization in the previous 12 months. Using a multivariate statistical regression model, the following risk factors for CO-CDI development were found; antacid usage (OR = 0.267, 95%C.I.:0.10–0.291, p < 0.01), chronic kidney disease (OR = 0.234, 95%C.I.:0.10–0.51, p < 0.01) and antibiotic use during the prior 2 months (OR = 0.061, 95%C.I.:0.02–0.17, p < 0.01), especially tetracycline's (OR = 0.146, 95% C.I.:0.07–0.22, p < 0.01) and cephalosporin's (OR = 0.110, 95%C.I.:0.14–0.42, p < 0.01). The most common ribotypes (RTs) detected in patients with CO-CDI were RT001 (32.3%) and RT027 (24.7%). All tested toxin producing C. difficile isolates were sensitive to metronidazole, vancomycin and tigecycline. A high rate of resistance to moxifloxacin (73.11%) and rifampicin (23.65%) was found. ConclusionPatients with CO-CDI had frequently contact with healthcare facility in the previous 12 weeks. Restriction of antacid usage and of high-risk antibiotics in the community may help reduce the incidence of CO-CDI.

Make It Less difficile: Understanding Genetic Evolution and Global Spread of Clostridioides difficile

Clostridioides difficile is an obligate anaerobic pathogen among the most common causes of healthcare-associated infections. It poses a global threat due to the clinical outcomes of infection and resistance to antibiotics recommended by international guidelines for its eradication. In particular, C. difficile infection can lead to fulminant colitis associated with shock, hypotension, megacolon, and, in severe cases, death. It is therefore of the utmost urgency to fully characterize this pathogen and better understand its spread, in order to reduce infection rates and improve therapy success. This review aims to provide a state-of-the-art overview of the genetic variation of C. difficile, with particular regard to pathogenic genes and the correlation with clinical issues of its infection. We also summarize the current typing techniques and, based on them, the global distribution of the most common ribotypes. Finally, we discuss genomic surveillance actions and new genetic engineering strategies as future perspectives to make it less difficile.

Molecular epidemiology and clinical characteristics of Clostridioides difficile infection in patients with inflammatory bowel disease from a teaching hospital.

Clostridioides difficile infection (CDI) in patients with inflammatory bowel disease (IBD) is of increasing concern. This study aimed to investigate the molecular epidemiology and antimicrobial susceptibilities of toxigenic C. difficile isolated from IBD patients and to evaluate the risk factors for CDI in IBD population. Loose or watery stools from IBD patients were tested for glutamate dehydrogenase, C. difficile toxins A&B and anaerobic culture. Toxigenic C. difficile isolates were characterized by multi-locus sequence typing, ribotyping and antimicrobial susceptibility testing. The prevalence of CDI in IBD patients was 13.6% (43/317). The dominant sequence types (STs) were ST35 (20.9%), ST2 (18.6%) and ST37 (16.3%). The most common ribotypes (RTs) were RT 017 (18.6%), RT 012 (14.0%), and RT 220 (14.0%), whereas RT 027 and RT 078 were not detected in this study. All the isolates were susceptible to vancomycin and metronidazole. The multidrug resistance rate of C. difficile RT 017 was higher (p < 0.01) than that of other RT strains. Recent hospitalization, use of corticosteroids and proton pump inhibitors were related to increased risk of CDI in IBD patients; of these, recent hospitalization and proton pump inhibitors use were independent risk factors. Patients with IBD have a relatively high incidence rate of CDI. C. difficile RT 017 is most frequently isolated from IBD patients in this region and warrants more attention to its high resistance rate. Clinicians should pay greater attention to CDI testing in IBD patients with diarrhea to ensure early diagnosis and initiation of effective treatment.

Characterization of Healthcare-Associated and Community-Associated Clostridioides difficile Infections among Adults, Canada, 2015-2019.

We investigated epidemiologic and molecular characteristics of healthcare-associated (HA) and community-associated (CA) Clostridioides difficile infection (CDI) among adult patients in Canadian Nosocomial Infection Surveillance Program hospitals during 2015–2019. The study encompassed 18,455 CDI cases, 13,735 (74.4%) HA and 4,720 (25.6%) CA. During 2015–2019, HA CDI rates decreased by 23.8%, whereas CA decreased by 18.8%. HA CDI was significantly associated with increased 30-day all-cause mortality as compared with CA CDI (p<0.01). Of 2,506 isolates analyzed, the most common ribotypes (RTs) were RT027, RT106, RT014, and RT020. RT027 was more often associated with CDI-attributable death than was non-RT027, regardless of acquisition type. Overall resistance C. difficile rates were similar for all drugs tested except moxifloxacin. Adult HA and CA CDI rates have declined, coinciding with changes in prevalence of RT027 and RT106. Infection prevention and control and continued national surveillance are integral to clarifying CDI epidemiology, investigation, and control.

Multi-country surveillance of Clostridioides difficile demonstrates high prevalence of spores in non-healthcare environmental settings

BackgroundC. difficile spores are frequently isolated from hospital and non-healthcare settings but a worldwide analysis has not been done. The study objectives were to assess C. difficile spore contamination in the hospital and non-healthcare environments across a variety of countries. MethodsField studies assessed hospital vs. non-healthcare C. difficile spore contamination in hospitals, non-healthcare buildings, outdoor environments, and shoes. Swabs were cultured anaerobically for C. difficile and typed using PCR-fluorescent ribotyping. C. difficile contamination by swabbing area and geographic locations were compared. FindingsA total of 7,857 unique samples were collected primarily from the USA (89%) in addition to 9 other countries. The global prevalence of C difficile from environmental samples was 25.3% and did not differ between countries. In USA based studies, C. difficile contamination rates were similar for healthcare buildings (23.2%), non-healthcare buildings (23.4%), and outdoor spaces (24.7%). Floor samples had significantly higher (p < 0.001) C. difficile contamination rate (46.5%) followed by non-floor samples (21.1%), and bathrooms (15.3%). In a comparison of USA to other country samples, C. difficile contamination rates were similar for USA samples (21.5%) compared to rest of world samples (22.3%; p = 0.61). The most common ribotypes included F014-020 (15.7%), F106 (12.6%), F010 (8.9%), F027 (8.8%), and F002 (8.1%) and did not differ significantly between USA and non-USA samples. Finally, 546 of 1,218 (44.8%) shoe soles swabbed from the USA were contaminated with C. difficile spores. InterpretationThis large surveillance study of several countries demonstrated high prevalence of toxigenic C. difficile in non-healthcare environments with high contamination rates from floors and shoe soles.

Molecular epidemiology and clinical risk factors for rifaximin-non-susceptible Clostridioides difficile infection in South Korea: a prospective, multicentre, observational study

This study was designed to investigate the molecular epidemiology of Clostridioides difficile isolates in South Korea and to evaluate risk factors for rifaximin-non-susceptible C. difficile infection (CDI). A total of 413 patients with CDI from two sentinel hospitals in South Korea were enrolled in this study. Putative clinical risk factors for CDI were identified using digital medical records of the patients. Pathogen profiles, including antimicrobial susceptibility, toxin production and ribotype, were evaluated for each of the causative C. difficile isolates. Of the 413 C. difficile isolates, 81 (19.6%) were shown to be rifaximin-non-susceptible, with the most common ribotypes being 018 (56.8%; 46/81), 017 (16.0%; 13/81) and 027 (6.2%; 5/81). Rifaximin-non-susceptible C. difficile isolates exhibited higher non-susceptibility rates to most of the other drugs tested in this study compared with rifaximin-susceptible isolates. Previous history of pulmonary tuberculosis and prior rifaximin treatment were shown to be associated with the occurrence of rifaximin-non-susceptible CDI compared with susceptible CDI. Non-susceptibility rates to rifaximin for the C. difficile isolates identified in this study were reasonably high with most of the resistant strains belonging to either ribotype 018 or 017. Widespread dissemination of these clones may be the result of antimicrobial selection pressure introduced by the widespread use of rifaximin. These results suggest that a sustainable surveillance programme for CDI and C. difficile resistance is needed in order to better control CDIs and to improve therapeutic efficacy.

Molecular Characterization of, and Antimicrobial Resistance in, Clostridioides difficile from Thailand, 2017-2018.

Antimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of Clostridioides difficile infection (CDI). Many antimicrobials, such as fluoroquinolones, have been associated with outbreaks of CDI globally. This study characterized AMR among clinical C. difficile strains in Thailand, where antimicrobial use remains inadequately regulated. Stool samples were screened for tcdB and positives were cultured. C. difficile isolates were characterized by toxin profiling and PCR ribotyping. Antimicrobial susceptibility testing was performed by agar incorporation, and whole-genome sequencing and AMR genotyping were performed on a subset of strains. There were 321 C. difficile strains isolated from 326 stool samples. The most common toxigenic ribotype (RT) was RT 017 (18%), followed by RTs 014 (12%) and 020 (7%). Resistance to clindamycin, erythromycin, moxifloxacin, and rifaximin was common, especially among RT 017 strains. AMR genotyping revealed a strong correlation between resistance genotype and phenotype for moxifloxacin and rifaximin. The presence of erm-class genes was associated with high-level clindamycin and erythromycin resistance. Point substitutions in the penicillin-binding proteins were not sufficient to confer meropenem resistance, but a Y721S substitution in PBP3 was associated with a 4.37-fold increase in meropenem minimal inhibitory concentration. No resistance to metronidazole, vancomycin, or fidaxomicin was observed.

Phylogenetic Relationships of Oxytropis Section Arctobia of Northeast Asia according to Sequencing of the Intergenic Spacers of Chloroplast and ITS of Nuclear Genomes

Three intergenic spacers (psbA–trnH, trnL–trnF, trnS–trnG) of chloroplast DNA (cpDNA) and an internal transcribed spacer of nuclear ribosomal DNA (ITS rDNA) were used to study the genetic diversity and phylogenetic relationships of the species Oxytropis czukotica, O. exserta, O. gorodkovii, O. kamtschatica, O. mertensiana, O. nigrescens, O. pumilio, O. revoluta, and O. susumanica of the section Arctobia of the genus Oxytropis. According to cpDNA data, most populations are characterized by a low and medium haplotype (h varies from 0.154 to 0.583) and low nucleotide (π varies from 0.0002 to 0.0050) diversity. An analysis of the genealogical relationships of chlorotypes showed a clear separation of the studied taxa and the genetic proximity of O. nigrescens to O. susumanica and O. kamtschatica to O. exserta, the last two being the most diverged from all the others. In O. czukotica, three ITS rDNA ribotypes were detected; in O. nigrescens and O. susumanica, one common ribotype was found; and in all the others, one individual ribotype was identified for each species. According to the data of nucleotide polymorphism of markers of two genomes, the status of O. czukotica, O. gorodkovii, and O. pumilio taxa as a three separate species was confirmed. We suggest that O. susumanica is an intraspecific taxon of O. nigrescens. The revealed genetic similarities and differences of O. revoluta, O. exserta, and O. kamtschatica and their phylogenetic relationships do not correspond to the division of the Arctobia section into subsections; therefore, additional comprehensive studies are needed.

Investigation of community-associated Clostridium (Clostridioides) difficile infections in the Asia-Pacific region

Background: Clostridium (Clostridioides) difficile is the most common cause of nosocomial diarrhoeal infections in high-income countries, however, community-associated (CA)-C. difficile infections (CDIs) now make up about 30% of all CDIs. Antimicrobial use and advanced age are the most common risk factors for CDI, although CA-CDI often affects younger patients. Little is currently known about the epidemiology of CA-CDI in the Asia-Pacific region. Methods and materials: A prospective study of CDI in hospital inpatients was conducted from March 2014 to January 2015 at 70 sites in 13 Asia-Pacific countries. Recruited cases (n = 600) had ≥3 episodes of diarrhoea in a 24 h period and diagnosis of CDI by toxin enzyme immunoassay, cell culture cytotoxicity assay, real-time PCR or toxigenic culture. Demographic and clinical data were collected, stool samples were cultured for C. difficile, and PCR ribotyping of isolates was performed. Proportions were compared by Chi-square test for CA-CDI (no hospitalisation in previous 12 w) vs hospital-associated (HA)-CDI (discharged from hospital in previous 4 w) cases identified in the study. A generalised linear mixed model regression was performed to find risk factors for CA-CDI. Results: HA-CDI was determined in 350 (65.4%) and CA-CDI in 74/535 (13.8%) cases aged ≥2 y. The median age of CA-CDI cases was lower at 53.5 y vs 66.0 y in HA-CDI cases (p < 0.01). Recent antimicrobial use was less frequent among CA-CDI cases (63.5% vs 80.0%, p < 0.01) while death and recurrent CDI were rarer outcomes (1.7% vs 5.8% and 5.0% vs 9.8%, p > 0.05). The most common ribotypes (RTs) causing CA-CDI were RT 017 (17.0%) followed by RTs 012 (14.6%, primarily in China), 014/020 (9.8%, predominantly in Australia and China), and RTs 002, 018 and 070 (all 7.3%). Inflammatory bowel disease (odds ratio [OR] 5.26, p < 0.05) and infection with RTs 012 (OR 5.44, p < 0.05) and 070 (OR 6.19, p < 0.05) were significantly associated with CA-CDI. Conclusion: CA-CDI patients in the Asia-Pacific region were significantly younger and had less frequent history of antimicrobial use compared to HA-CDI patients. RTs 012 and 070 were associated with CA-CDI, highlighting a need for investigation of community-based reservoirs of C. difficile including animals and outdoor environments.

Burden of Clostridium difficile Infection (CDI) Across Whole Healthcare Economies and European Borders; COMBACTE-CDI Results

Background: The burden of C. difficile infection (CDI) on healthcare facilities is well recognized. However, studies focusing on inpatient settings, in addition to ascertainment bias in general, have led to a paucity of data on the true burden of CDI across whole healthcare economies. Methods: Sites testing both inpatient and community samples were recruited from 12 European countries (1 site per 3 million population). On 2 selected days, all diarrheal fecal samples (regardless of tests requested) were sent to the European Coordinating Laboratory (ECL) for C. difficile toxin testing and culture. The CDI results and tests not requested at each submitting site were compared with the ECL results to determine the number of missed CDIs. Contemporaneous C. difficile isolates from food and animal sources were collected. All isolates underwent PCR ribotyping and toxinotyping; prevalences of ribotypes among regions of Europe and reservoir settings were compared. Results: Overall, 3,163 diarrheal fecal samples were received from 119 sites. The burden of CDI varied by country (positivity rates, 0–15.8%) and by European region; the highest positivity rate in Eastern Europe was 13.1%. The testing and positivity rates in community samples were 29.6% and 1.4% vs 74.9% and 5.0% in hospital samples; 16% and 55% of samples positive for CDI at ECL were not diagnosed in hospitals and the community. The most common C. difficile ribotypes from hospital samples were 027 (11%), 181 (12%), and 014 (8%), although prevalence varied by country. The highest prevalence of toxinotype IIIb (ribotypes 027, 181, and 176) was seen in Eastern Europe (55% of all isolates), which also had the lowest testing rate. For hospital samples, the proportion of toxinotype IIIb was inversely related to the testing rate (r = −0.79) (Fig. 1). The most common ribotypes from food sources were 078 (23%) and 126 (13%) (toxinotype V), and most common ribotypes from community samples were 078 (9%) and 039 (9%). Overall, 106 different ribotypes were identified: 25 in both the hospital and community and 16 in the hospital, community, and food chain. Conclusions: The diagnosed burden of CDI varies markedly among countries in both hospital and community settings. Reduced sampling/testing in Eastern Europe is inversely related to the proportion of toxinotype IIIb strains identified, suggesting that lack of suspicion leads to underdiagnosis and outbreaks of infection. The proportion of missed CDIs in the community was ~3.5× higher than in hospitals, indicating major underrecognition in the former setting. There were marked differences in ribotypes in different reservoir settings, emphasizing the complex epidemiology of C. difficile.Funding: Proprietary organization: COMBACTE-CDI is an EU funded (Horizon2020) consortium of academic and EFPIA partners (bioMerieux, GSK, Sanofi Pasteur, Astra Zeneca, Pfizer, Da Volterra) with additional Funding: from the EFPIA partners.Disclosures: Submitter: Kerrie Davies; the work presented is funded via the EU and EFPIA (commercial) partners in a consortium.

In vitro activity of eravacycline against common ribotypes of Clostridioides difficile.

BackgroundEravacycline is a novel synthetic fluorocycline antibacterial approved for complicated intra-abdominal infections.ObjectivesThe purpose of this study was to assess the in vitro activities of eravacycline and comparator antibiotics against contemporary clinical isolates of Clostridioides difficile representing common ribotypes, including isolates with decreased susceptibility to metronidazole and vancomycin.MethodsClinical C. difficile strains from six common or emerging ribotypes were used to test the in vitro activities of eravacycline and comparator antibiotics (fidaxomicin, vancomycin and metronidazole) by broth microdilution. In addition, MBC experiments, time–kill kinetic studies and WGS experiments were performed.ResultsA total of 234 isolates were tested, including ribotypes RT001 (n = 37), RT002 (n = 41), RT014-020 (n = 39), RT027 (n = 42), RT106 (n = 38) and RT255 (n = 37). MIC50/90 values were lowest for eravacycline (≤0.0078/0.016 mg/L), followed by fidaxomicin (0.016/0.063 mg/L), metronidazole (0.25/1.0 mg/L) and vancomycin (2.0/4.0 mg/L). MBCs were lower for eravacycline compared with vancomycin for all ribotypes tested. Both vancomycin and eravacycline demonstrated bactericidal killing, including for epidemic RT027. The presence of the tetM or tetW resistance genes did not affect the MIC of eravacycline.ConclusionsThis study demonstrated potent in vitro activity of eravacycline against a large collection of clinical C. difficile strains that was not affected by ribotype, susceptibility to vancomycin or the presence of certain tet resistance genes. Further development of eravacycline as an antibiotic to be used in patients with Clostridioides difficile infection is warranted.

Antimicrobial susceptibility of Clostridioides difficile isolated from diarrhoeal stool specimens of Canadian patients: summary of results from the Canadian Clostridioides difficile (CAN-DIFF) surveillance study from 2013 to 2017.

To summarize data generated by the Canadian Clostridioides difficile (CAN-DIFF) surveillance study from 2013 to 2017. Isolates of C. difficile (n = 2158) were cultured from toxin-positive diarrhoeal stool specimens submitted by eight hospital laboratories to a coordinating laboratory. Antimicrobial susceptibility testing was performed according to the CLSI agar dilution method (M11, 2018). Isolate ribotypes were determined using an international, standardized, high-resolution capillary gel-based electrophoresis protocol. Of the 2158 isolates of C. difficile, 2133 (98.8%) had vancomycin MICs ≤2 mg/L [i.e. were vancomycin susceptible (EUCAST breakpoint tables, v 9.0, 2019) or WT (CLSI M100, 29th edition, 2019)]. Fidaxomicin MICs were lower than those of all other agents tested (MIC90, 0.5 mg/L); however, one isolate with a fidaxomicin MIC of >8 mg/L was identified. Metronidazole MICs ranged from 0.12 to 4 mg/L; all isolates were metronidazole susceptible by the CLSI breakpoint (≤8 mg/L) compared with 96.8% susceptible by the EUCAST breakpoint (≤2 mg/L). In total, 182 different ribotypes were identified from 2013 to 2017. The most common ribotypes identified were 027 (19.3% of isolates) and 106 (8.2%). Ribotype 027 isolates were frequently moxifloxacin resistant (87.3% of isolates) and MDR (48.6%), associated with vancomycin (10/25, 40.0%) and metronidazole (58/69, 84.1%) resistance and from patients aged ≥80 years. The prevalence of ribotype 027 decreased significantly (P < 0.0001) from 2013 (27.5%) to 2017 (9.0%) and was replaced by increases in ribotype 106 (P = 0.0003) and multiple less common ribotypes. Periodic surveillance is required to monitor clinical isolates of C. difficile for changes to in vitro susceptibility testing profiles and ribotype evolution.

Molecular Phylogenetic Analysis of the Endemic Far Eastern Closely Related Oxytropis Species of Section Orobia (Fabaceae)

The questions about the taxonomic status and phylogenetic relationships of the Far Eastern closely related species Oxytropis ochotensis, O. litoralis, O. erecta, O. ruthenica, and O. kunashiriensis of the section Orobia of the genus Oxytropis still remain unresolved. The study of the polymorphism of nucleotide sequences of the psbA–trnH, trnL–trnF, and trnS–trnG cpDNA intergenic spacers showed that populations of O. ochotensis and O. erecta are characterized by a low (0.378–0.495) haplotype (h) and a low (0.0006–0.0009) nucleotide (π) diversity, and in populations of O. ruthenica, h varies from 0.154 to 0.872 and π varies from 0.0002 to 0.0016. One O. ochotensis population from Magadan oblast and one O. ruthenica population from Russky Island (Primorsky krai) are monomorphic. Low nucleotide divergence of cpDNA between species O. ochotensis, O. erecta, and O. litoralis and also statistically insignificant genetic differentiation between them, the formation of a single haplogroup in the phylogenetic network, and the absence of species-specific molecular markers indicate the unity of their gene pool. A study of the ITS rDNA polymorphism revealed private ribotypes in O. ruthenica and O. kunashiriensis, the presence of a common ribotype in O. ochotensis, O. erecta, and O. litoralis, and the intraspecific polymorphism in O. ochotensis and O. erecta. The differences revealed in the chloroplast and nuclear genomes confirm the independence of O. ruthenica and O. kunashiriensis and suggest that O. erecta and O. litoralis are local phenotypes of the widespread polyploid species O. ochotensis. An analysis of the phylogenetic relationships of the cpDNA haplotypes showed a clear separation of O. ruthenica populations into two evolutionary lineages, but with a single ITS ribotype.