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Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3′)-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven "hypervirulent" RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential. [ABSTRACT FROM AUTHOR]

• Data on molecular epidemiology and antimicrobial resistance of Clostridium difficile are scarce. • The zoonotic potential of C. difficile is currently poorly understood. • In Zimbabwe a large strain diversity was present. • Low rates of antimicrobial resistance were observed (except for moxifloxacin). • Clusters of isolates from humans and chicken/soil could indicate for ongoing transmission. Clostridioides difficile is the major cause of infectious nosocomial diarrhoea in industrialized nations. Data on the occurrence of C. difficile in Africa, ribotype (RT) distribution, antimicrobial susceptibility patterns and potential zoonotic transmission are scarce. 80 Zimbabwean C. difficile isolates from different sources (chicken n = 30], soil n = 21] and humans n = 29]) were investigated using ribotyping, toxin gene detection, resistance testing, multiple-locus variable-number tandem repeat analysis (MLVA), and whole genome sequencing (WGS). Among chicken isolates, the most common RTs were RT103 (6/30), RT025 (5/30) and RT070 (4/30). Within soil samples, RT025 and RT056 were most common (3/21 each). In contrast, the non-toxigenic RT084 was most frequently found in human isolates (4/29). Toxin genes were detected in only 19/29 human isolates. Susceptibility testing showed no resistance against metronidazole and vancomycin, and resistance against macrolides and rifampicin was scarce (3/80 and 2/80, respectively); however, 26/80 isolates showed moxifloxacin resistance. MLVA and WGS of strains with identical RTs stemming from different sources revealed clustering of RT025 and RT084 isolates from human und non-human samples. No "hypervirulent" strains were found. The detected clusters between human, chicken and soil isolates indicate ongoing transmission between humans and environmental sources and might point towards a zoonotic potential. [ABSTRACT FROM AUTHOR]

Clostridium ( Clostridioides ) difficile is the main cause for nosocomial diarrhoea in industrialised nations. Epidemiologic data on the pathogen’s occurrence in other world regions are still scarce. In this context we characterized with phenotypic and molecular genetic methods C. difficile isolates stemming from hospitalised patients with diarrhoea in Lebanon. From 129 stool samples of symptomatic patients at a tertiary care University hospital in Lebanon, a total of 107 C. difficile strains were cultivated and underwent ribotyping, toxin gene detection and antibiotic resistance testing. Ribotype 014 (RT014, 16.8%) predominated, followed by RT002 (9.3%), RT106 (8.4%) and RT070 (6.5%). Binary toxin gene-positive isolates (RT023, RT078 and RT126) were rarely detected and RT027 was absent. Interestingly, within one isolate only the toxin A gene ( tcdA ) was detected. Multiple-locus variable-number tandem repeat analysis (MLVA) revealed strong strain diversity in most RTs. The isolates were sensitive to metronidazole and vancomycin, and only a small proportion of strains displayed resistance against moxifloxacin, rifampicin, and clarithromycin (5.6%, 1.9%, and 2.8%), respectively. The data indicate that the genetic strain composition of Lebanese strains differs markedly from the situation seen in Europe and North America. Especially the epidemic RTs seen in the latter regions were almost absent in Lebanon. Interestingly, most strains showed almost no resistance to commonly used antibiotics that are suspected to play a major role in the development of C. difficile infection, despite frequent use of these antibiotics in Lebanon. Thus, the role of antimicrobial resistance as a major driving force for infection development remains uncertain in this area. [ABSTRACT FROM AUTHOR]

• C. difficile RT027 prevalence increased in Germany when compared to 2011–2013. • Resistance to metronidazole is scarce, except for RT027. • Resistance to vancomycin was not encountered yet. • Rifampicin resistance reached >40% among German RT027 isolates. • Molecular and antimicrobial surveillance are crucial. Clostridioides difficile is a Gram positive spore-forming rod and mainly responsible for nosocomial diarrhea in developed nations. Molecular and antimicrobial surveillance is important for monitoring the strain composition including genotypes of high epidemiological importance such as ribotype 027 (RT027) and corresponding resistance patterns. 1535 isolates obtained from samples sent between 2014 and 2019 to the German National Reference Center (NRC) for diagnostic reasons (NRC strain set), and 1143 isolates from a Tertiary Care University Center in Saarland, Germany (non-NRC strain set), were evaluated using antibiotic susceptibility testing and ribotyping. In the NRC strain set, RT027 overtook RT001, the main RT found in the preceding studies, and dominated with 36.2%, followed by RT001 (13.3%), and RT014 (8.5%). Of note, since 2016 a constant decrease of RT027 could be noticed. In the non-NRC strain set a large strain diversity was present with RT014 (18%) and RT001 (8.9%) being most prevalent. In NRC samples, resistance towards metronidazole, vancomycin, moxifloxacin, clarithromycin and rifampicin was 2.7%, 0%, 57.1%, 53.2% and 19.2%, respectively. Metronidazole resistance was almost exclusively found in RT027 isolates. Rifampicin resistance was also observed predominantly in isolates of RT027, constituting an almost four-fold increase, when compared to preceeding studies in this region. In conclusion these data demonstrate that RT027 is a driver for rifampicin and metronidazole resistance, underlining the importance of continuous surveillance efforts. [ABSTRACT FROM AUTHOR]

Clostridioides difficile is a major cause of nosocomial diarrhea. Several "hypervirulent" lineages such as ribotype 027 (RT027) and RT078 are of high epidemiological importance, leading to outbreaks and more severe courses of disease. An active surveillance system targeting molecular epidemiology and corresponding antimicrobial resistance has not been established in Germany. Since October 2019, University Hospitals throughout Germany collected by two dates every year (1st April and October, respectively) their first ten unselected samples being tested positive for C. difficile. Out of 1026 samples received from 29 sites, 876 toxigenic C. difficile strains could be cultivated. PCR ribotyping of these strains revealed a large strain diversity with RT014 (17.5%) dominating, followed by isolates of the major nosocomial lineage RT001 (7.1%) and the "hypervirulent" lineage RT078 (5.9%). Notably, prevalence of RT027 was low with ∼3.5% at all time points analyzed, while the abundance of RT001 isolates significantly declined from 12.3% to 3.7% during the sampling period (P < 0.001). Antimicrobial resistance against clarithromycin, moxifloxacin, and rifampicin was detected in 18%, 15%, and 4% of the tested isolates, respectively. Highest resistance rates were found among RT027 isolates (83%, 87% and 63% for clarithromycin, moxifloxacin, and rifampicin, respectively). Vancomycin resistance was not detected, and metronidazole resistance was observed only for a single RT027 isolate. This Germany-wide continuing surveillance effort with a standardized mode of isolate acquisition indicates that isolates of RT027 were only sporadically detected under these strain acquisition conditions, and RT001 seems to become less important in the hospital setting, being replaced by other RTs. • Surveillance is crucial for detecting emerging strains and antimicrobial resistance. • For Germany no standardized surveillance scheme is established. • Several study sites send continuously samples on two time points a year. • Antimicrobial resistance towards metronidazole was scarce and absent for vancomycin. • RT027 was of minor importance and RT001 declined over time. [ABSTRACT FROM AUTHOR]