Ribotypes Research Articles

Clostridioides (Clostridium) difficile in children with diarrhoea in Vietnam

BackgroundClostridioides (Clostridium) difficile commonly causes hospital-acquired infection which can range from mild diarrhoea to life-threatening toxic megacolon and even death. Reports on C. difficile infection (CDI) in Vietnam are limited, so this study was designed to evaluate the prevalence, molecular epidemiology and antimicrobial susceptibility of C. difficile isolated from children with diarrhoea in Vietnam. Infants are often colonised with C. difficile and it was hypothesised that those colonising strains would represent strains of C. difficile circulating in the hospital/region at the time, however, this was not an attempt to determine if C. difficile was the cause of the diarrhoea. MethodsDiarrhoeal stool samples collected at two children's hospitals in northern Vietnam from October 1, 2020 to February 28, 2021 were transported to Perth, Western Australia, for culture of C. difficile and further investigations on isolates; PCR ribotyping, toxin gene profiling and antimicrobial susceptibility testing. ResultsFrom these hospitals, 370 diarrhoeal stool samples were collected, most from children aged 1–15 months (71.9%; 266/370). The overall prevalence of C. difficile in stool samples from children aged ≤16 years was 37.8% (140/370) and the highest prevalence was in the 2–12 months age group (52.9%; 74/140). In total, 151 isolates of C. difficile were recovered; the proportion of toxigenic isolates was 16.6% (25/151). Of the 25 toxigenic C. difficile isolates, the toxin gene profiles A+B+CDT− and A−B+CDT− comprised 72% and 28%, respectively. The four most prevalent C. difficile ribotypes (RTs) were QX 011 (25/151), RT 010 (25/151), QX 107 (12/151) and RT 012 (11/151). All isolates were susceptible to vancomycin, metronidazole and fidaxomicin, while there was significant resistance to clindamycin (90.1%), and some to moxifloxacin (6.6%) and rifaximin (3.3%). ConclusionThe prevalence of C. difficile in children with diarrhoea was high (37.8%) although the proportion of toxigenic strains was comparatively low. The clinical significance of any isolate needs to be determined.

Characterization of a Clostridioides difficile outbreak caused by PCR ribotype 046, associated with increased mortality

ABSTRACT This study describes a large nosocomial outbreak of Clostridioides difficile infections (CDI) dominated by ribotype (RT) 046 in a Swedish hospital. The present study aimed to examine the pathogenicity of this RT, explore epidemiological links by whole genome sequencing (WGS), and evaluate different interventions implemented to stop the outbreak. Clinical isolates (n = 366) collected during and after the outbreak were ribotyped and 246 isolates were subjected to WGS. Medical records of patients infected with the seven most common RTs were evaluated. RT046 was spread effectively throughout the hospital and was the most common among the 44 different RTs found (114/366 isolates). Infection with RT046 was associated with higher mortality compared to other strains (20.2% to 7.8%), although there were no differences in concomitant disease, age or antibiotic treatment. To control the outbreak, several measures were successfully implemented.

Implementation of a Clostridioides difficile sentinel surveillance system in Germany: First insights for 2019–2021

ObjectivesClostridioides 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. MethodsSince 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. ResultsOut 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. ConclusionsThis 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.

Global evolutionary dynamics and resistome analysis of Clostridioides difficile ribotype 017.

Clostridioides difficile PCR ribotype (RT) 017 ranks among the most successful strains of C. difficile in the world. In the past three decades, it has caused outbreaks on four continents, more than other ‘epidemic’ strains, but our understanding of the genomic epidemiology underpinning the spread of C. difficile RT 017 is limited. Here, we performed high-resolution phylogenomic and Bayesian evolutionary analyses on an updated and more representative dataset of 282 non-clonal C. difficile RT 017 isolates collected worldwide between 1981 and 2019. These analyses place an estimated time of global dissemination between 1953 and 1983 and identified the acquisition of the ermB-positive transposon Tn6194 as a key factor behind global emergence. This coincided with the introduction of clindamycin, a key inciter of C. difficile infection, into clinical practice in the 1960s. Based on the genomic data alone, the origin of C. difficile RT 017 could not be determined; however, geographical data and records of population movement suggest that C. difficile RT 017 had been moving between Asia and Europe since the Middle Ages and was later transported to North America around 1860 (95 % confidence interval: 1622–1954). A focused epidemiological study of 45 clinical C. difficile RT 017 genomes from a cluster in a tertiary hospital in Thailand revealed that the population consisted of two groups of multidrug-resistant (MDR) C. difficile RT 017 and a group of early, non-MDR C. difficile RT 017. The significant genomic diversity within each MDR group suggests that although they were all isolated from hospitalized patients, there was probably a reservoir of C. difficile RT 017 in the community that contributed to the spread of this pathogen.

Clostridioides (Clostridium) difficile isolated from paediatric patients in Western Australia 2019–2020

Less is understood about the epidemiology of Clostridioides difficile infection (CDI) in children compared to adults, and its impact is complicated by variations in the natural development of infection in paediatric patients. The interplay of rising CDI incidence in hospitalised paediatric patients, emergence of hypervirulent strains and community associated CDI (CA-CDI) in the past decade is a potential threat in both hospital and community settings. Research in Australia regarding paediatric CDI is limited. Here, we report the molecular characterisation of C.difficile isolated from paediatric patients at a tertiary hospital in Perth, Western Australia. A total of 427 stool samples was collected from patients aged from <1 to 17 years being investigated for diarrhoea from July 2019 to June 2020. Stool specimens were cultured and isolates of C.difficile characterised by ribotyping and toxin gene profiling. Clostridioides difficile was recovered from 84/427 (19.7%) samples tested. The most prevalent PCR ribotypes (RTs) were RT 002 (12.4%), a toxigenic strain, and RT 009 (15.7%), a non-toxigenic strain. Interestingly, C.difficile RT 078 and RT 017, strains that are not endemic in Australia, were isolated from a 1- and 4-year-old child, respectively. Clostridioides difficile RT 106, a strain of emerging importance in Australia, was recovered from two cases (5.3%). Resistance to metronidazole, fidaxomicin, amoxicillin, rifaximin and meropenem was not detected, however, 45 isolates (50.6%) showed resistance to at least one agent, and multidrug resistance was observed in 13.3% of the resistant isolates (6/45). This study provides a baseline for future surveillance of paediatric CDI in Australia. Given that young children can be asymptomatically colonised with toxigenic C.difficile strains, they represent a potential reservoir of strains causing CDI in adults.

A Novel Bacteriophage with Broad Host Range against Clostridioides difficile Ribotype 078 Supports SlpA as the Likely Phage Receptor.

ABSTRACTBacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI.IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal “one size fits all” phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.

Linkage study of surveillance and hospital admission data to investigate Clostridium difficile infection in hospital patients in Perth, Western Australia

ObjectivesIncreasing incidence rates of Clostridium difficile infection (CDI) and outbreaks of emerging strains have highlighted the need for continuous monitoring and surveillance of CDI in Australia. Active surveillance captures all hospital-identified CDI cases in Western Australia (WA), where all C. difficile isolates recovered are routinely PCR ribotyped. The aim of this study was to determine incidence rates and descriptive and molecular epidemiology of CDI among patients in Perth, WA using linkage of surveillance and hospital administrative records. MethodsAll CDI cases (confirmed by tcdB PCR) from July 2012 to June 2014 captured in the Hospital Infection Surveillance WA dataset for three hospitals were linked with hospital admission records from the Patient Administration System and ribotyping data to calculate incidence rates of CDI and the distribution of various ribotypes (RTs). ResultsThere were 381 individual cases of CDI identified among 354 hospital patients (including outpatients and ED) who experienced ≥1 CDI episode during the study period. CDI was hospital-associated in 62.7% of cases and community-associated (CA)-CDI in 31.2%. The overall incidence rate was 4.40/10,000 patient days (PD, 95% CI 3.98–4.86), females across all age groups experienced higher incidence (risk ratio 1.29, p < 0.05). The risk ratio for CA-CDI was highest (7.76, p < 0.01) for females vs males aged 15–29 years. Overall, 10.8% of cases were admitted to ICU, 15.2% had a recurrent infection and the mortality rate was 7.2%. C. difficile RT 014/020 predominated (34.9%) among 339 isolates of 71 different RTs. ConclusionsThe incidence of CDI in WA is high and RT 014/020 continues to be the dominant molecular type in an otherwise diverse array of strains. High strain diversity suggests CDI cases arise from exposure to many different reservoirs.

Motility and the genotype diversity of the flagellin genes fliC and fliD among Clostridioides difficile ribotypes

ObjectiveThe motility and genotype of the flagellin fliC and fliD genes were investigated in 82 Clostridioides difficile isolates belonging to the ribotypes (RTs): 027 (n = 41), 176 (n = 17), 023 (n = 8), 017 (n = 6) and 046 (n = 10). The reference C. difficile strains 630 and M120 were included as controls for the motility assay. MethodsA Multiple Locus Variable-number Tandem Repeat Analysis (MLVA) was used to exclude the genetic relatedness of C. difficile isolates belonging to the same RT. The variability of the fliC and fliD genes was determined by PCR-restriction fragment length polymorphism (RFLP) analysis and Sanger sequencing. The motility assay was carried out with 0.175% BHI agar tubes and BHI solid media plates with 0.4% agar. ResultsThe highest motility was observed in C. difficile RT023 isolates (p < 0.01), followed by RTs 027 and 176. C. difficile isolates of RTs 017 and 046 were less motile than RTs 027, 176 and 023 (p < 0.01). The fliC and fliD genes were present in all clinical isolates irrespective of the motility results. In the fliC gene analysis, four different RFLP groups were identified (I, II, VII, X). The fliC group VII was identified in two RTs (027 and 176), whereas the remaining three groups (I, II and X) belonged to a single RT 046, 017 and 023, respectively. The fliD gene analysis identified four new RFLP groups (a, b, c and d). ConclusionsC. difficile RT023 is highly motile and its motility is comparable to the hypervirulent RT027 and its genetic relative RT176.

Genetically related Clostridium difficile from water sources and human CDI cases revealed by whole-genome sequencing.

Clostridium difficile isolates from the environment are closely related to those from humans, indicating a possible environmental transmission route for C. difficile infection (CDI). In this study, C. difficile was isolated from 47.3% (53/112) of lake/pond, 23.0% (14/61) of river, 20.0% (3/15) of estuary and 0.0% (0/89) of seawater samples. The most common toxigenic strain isolated was C. difficile PCR ribotype (RT) 014/020 (10.5%, 8/76). All water isolates were susceptible to fidaxomicin, metronidazole, rifaximin, amoxicillin/clavulanic acid, moxifloxacin and tetracycline. Resistance to vancomycin, clindamycin, erythromycin and meropenem was detected in 5.3% (4/76), 26.3% (20/76), 1.3% (1/76) and 6.6% (5/76) of isolates, respectively. High-resolution core-genome analysis was performed on RT 014/020 isolates of water origin and 26 clinical RT 014/020 isolates from the same year and geographical location. Notably, both human and water strains were intermixed across three sequence types (STs), 2, 13 and 49. Six closely related groups with ≤10 core-genome single nucleotide polymorphisms were identified, five of which comprised human and water strains. Overall, 19.2% (5/26) of human strains shared a recent genomic relationship with one or more water strains. This study supports the growing hypothesis that environmental contamination by C. difficile plays a role in CDI transmission.

Comparative clinical outcomes evaluation of hospitalized patients infected with Clostridioides difficile ribotype 106 vs. other toxigenic strains

BackgroundAlthough Clostridioides difficile surveillance often identifies emerging strains, clinical outcome evaluations are rarely performed. Ribotype (RT) 106 is a commonly isolated C. difficile strain worldwide; however, studies investigating RT 106 clinical outcomes are limited. The purpose of this study was to investigate clinical outcomes of RT 106 infections compared with two other endemic strains of varying virulence. MethodsThis multicenter study evaluated adults hospitalized with C. difficile infection (CDI). C. difficile samples underwent PCR ribotyping and patients infected with RT 106 were compared to patients infected with a known hypervirulent strain (RT 027) and a strain associated with less virulence (RT 014–020). Electronic medical records were reviewed by blinded investigators to assess the primary outcome of poor clinical outcome (composite of initial clinical failure, discharge to a higher level of care, 90-day CDI recurrence, and CDI-contributable mortality). ResultsA total of 396 patients with CDI were identified (RT 106, 32.3%; RT 027, 29.3%; RT 014–020, 38.3%). Patients infected with RT 014–020 less often experienced a poor clinical outcome (40%) compared with RT 106 (56%) and RT 027 (65%) infection (P < 0.0001). After controlling for covariates and using RT 014–020 as a comparator, patients infected with RT 106 (OR, 2.25; 95% CI, 1.36–3.73) or RT 027 (OR, 2.56; 95% CI, 1.52–4.31) had higher odds of poor clinical outcome. Using RT 027 as the comparator, only RT 014–020 was associated with lower odds of poor clinical outcome (OR, 0.42; 95% CI, 0.27–0.65). ConclusionThis study demonstrated that the emergent C. difficile RT 106 was associated with increased rates of poor clinical outcomes compared to RT 014–020 and comparable poor clinical outcomes to RT 027. These findings can help to better understand the clinical significance of this and future emerging ribotypes.

Genetic diversity and phylogenetic analysis of the surface layer protein A gene (slpA) among Clostridioides difficile clinical isolates from Tehran, Iran

Clostridioides difficile is the most common causative agent of healthcare-associated diarrhea. C. difficile strains produce a crystalline surface layer protein (SlpA), encoded by the slpA gene. Previous studies have shown that SlpA varies among C. difficile strains. In this study, we used the SlpA sequence-based typing system (SlpAST) for the molecular genotyping of C. difficile clinical isolates identified in Iran; the PCR ribotypes (RTs) and toxin profiles of the isolates were also characterized. Forty-eight C. difficile isolates were obtained from diarrheal patients, and characterized by capillary electrophoresis (CE) PCR ribotyping and the detection of toxin genes. In addition, the genetic diversity of the slpA gene was investigated by Sanger sequencing. The most common RTs were RT126 (20.8%), followed by RT001 (12.5%) and RT084 (10.4%). The intact PaLoc arrangement representing cdu2+/tcdR+/tcdB+/tcdE+/tcdA+/tcdC+/cdd3+ profile was the predominant pattern and cdtA and cdtB genes were found in one-third of the isolates. Using the SlpA genotyping, 12 main genotypes and 16 subtypes were identified. The SlpA type 078–1 was the most prevalent genotype (20.8%), and identified within the isolates of RT126. The yok-1, gr-1, cr-1 and kr-3 genotypes were detected in 14.5%, 12.5%, 12.5% and 8.3% of isolates, respectively. Almost all the isolates with the same RT were clustered in similar SlpA sequence types. In comparison to PCR ribotyping, SlpAST, as a simple and highly reproducible sequenced-based technique, can discriminate well between C. difficile isolates. This typing method appears to be a valuable tool for the epidemiological study of C. difficile isolates worldwide.

Diversity of Circulating Clostridioides difficile Ribotypes in Mexico and Susceptibility to Fidaxomicin, Vancomycin, and Metronidazole.

In this study, we report the results of the epidemiological analysis of Clostridioides difficile ribotypes (RTs) and antimicrobial susceptibility testing. Most isolates were RT027, representing 73% (84/115) of isolates. No isolates with reduced susceptibility to fidaxomicin were found; however, 38 (33.04%) isolates had reduced susceptibility to metronidazole, and 7 isolates (6.1%) had reduced susceptibility to vancomycin. These findings highlight the need for continuous surveillance of C. difficile RTs and antimicrobial susceptibility testing.

Antimicrobial resistance progression in the United Kingdom: A temporal comparison of Clostridioides difficile antimicrobial susceptibilities

ObjectivesClostridioides difficile (CD) is widely reported as one of the most prevalent multi-drug resistant (MDR) organisms. Assessment of temporally disparate isolate collections can give valuable epidemiological data to further the understanding of antimicrobial resistance progression. MethodsA collection of 75 CD isolates (1980–86) was characterised by PCR ribotyping, cell cytotoxicity assay and susceptibility testing with a panel of 16 antimicrobials and compared to a modern surveillance collection consisting of 416 UK isolates (2012–2016). Agar-incorporation was performed to ascertain susceptibility data for vancomycin, metronidazole, rifampicin, fidaxomicin, moxifloxacin, clindamycin, imipenem, chloramphenicol, tigecycline, linezolid, ciprofloxacin, piperacillin/tazobactam, ceftriaxone, amoxicillin, tetracycline and erythromycin. Genomes were obtained using Illumina HiSeq3000 sequencing and assembled using CLC Genomics Workbench. Resistance genes were identified using the Comprehensive Antibiotic Research Database's Resistance Gene Identifier and ResFinder3.0. ResultsTwenty-six known and one previously unobserved ribotype (RT) were detected. RT015 and RT020 dominated; 21.3% and 17.3%, respectively. Three moxifloxacin resistant (16–32 mg/L) RT027 isolates were recovered, pre-dating the earliest reports of this phenotype/genotype. Phenotypic resistance was observed to moxifloxacin (9.3% of isolates), ciprofloxacin (100%), erythromycin (17.3%), tetracycline (9.3%), linezolid and chloramphenicol (4.0%). Phenotypic comparisons with modern strains revealed increasing minimum inhibitory concentrations (MIC), with MIC50 elevations of one doubling-dilution for the majority of compounds, excluding clindamycin and imipenem. Moxifloxacin MIC90 comparisons revealed a two doubling-dilution increase between temporal isolate collections. Historical genomes revealed twenty different resistance determinants, including ermB (8.0% of isolates), tetM (9.3%), cfr (5.3%) and gyrA substitution Thr-82→Ile (9.3%). Seventeen isolates (22.7%) were resistant to ≥3 compounds (MDR), demonstrating ten different combinations. Intra-RT diversity was observed. ConclusionsAntibiotic resistance in CD has increased since the early 1980s, across the majority of classes. Moxifloxacin resistance determinants may pre-date its introduction.

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.

Molecular epidemiology and antimicrobial resistance of Clostridioides difficile in Germany, 2014–2019

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.

Clostridioides difficile colonization and infection in a cohort of Australian adults with cystic fibrosis

Little is known about Clostridioides difficile infection (CDI) in patients with cystic fibrosis (CF). The aim of this study was to investigate the prevalence, molecular epidemiology and risk factors for CDI in asymptomatic and symptomatic adults with CF in Western Australia. Faecal samples from symptomatic and asymptomatic patients were prospectively collected and tested for the presence of C.difficile by toxigenic culture. Ribotyping was performed by established protocols. Logistic regression analysis was performed to analyse the risk factors for C.difficile colonization and infection. Extensive environmental sampling was performed within the CF clinic in Perth. The prevalence rates of asymptomatic toxigenic and non-toxigenic C.difficile colonization were 30% (14/46 patients) and 24% (11/46 patients), respectively. Fifteen ribotypes (RTs) of C.difficile were identified, of which non-toxigenic RT 039 was the most common. Among the symptomatic patients, the prevalence of toxigenic CDI was 33% (11/33 patients). Impaired glucose tolerance/diabetes mellitus and duration of intravenous antibiotic use in the past 12 months were significantly associated with increased risk of asymptomatic toxigenic C.difficile carriage and CDI. A trend towards higher CF transmembrane conductance regulator modulator treatment was observed in the CDI group. Extensive environmental sampling showed no evidence of toxigenic C.difficile contamination within the CF clinic. A high prevalence of asymptomatic carriage of toxigenic C.difficile was observed in adults with CF, comparable with that observed in the symptomatic CF population. There was no evidence of direct person-to-person transmission.

Molecular characterization of Clostridioides difficile ribotype 027 in a major Chinese hospital

The rapid spread of C. difficile 027 has become one of the leading threats of healthcare-associated infections wordwild. However, C. difficile 027 infections have rarely been reported in China. The objective of this study was to strengthen the understanding of the molecular characterizations of C. difficile 027 in China. In this study, stool specimens from 176 suspected CDI cases were collected from 1 Jan 2018 to 30 Jun 2019. These specimens were measured by GeneXpert test and C.difficile colonies were identified and analyzed. There were five samples positive for tcdA, tcdB, binary toxin genes and had deletions in tcdC gene. These five Clostridioides difficile isolates belonged to ST1 and confirmed as Clostridioides difficile 027 strains by PCR ribotyping. Through using whole genome sequencing, , we found that these five strains were closely clustered into the same predominant evolutionary branch and were highly similar to C. difficile 027 strain R20291. Antimicrobial susceptibility testing result showed they were highly resistant to fluoroquinolones. In Our study, five C. difficile 027 isolates were identified and characterized using MLST, PCR ribotyping and whole genome sequencing. We proposed that C. difficile 027 infections are probably neglected in China. Further epidemiological studies across the country together with the introduction of routine diagnostic testing and multi-center or national level surveillance are needed to ascertain the size of this potentially significant problem.

784. A Novel Method to Assess Virulence of Clostridioides difficile: Focus on C. difficile Ribotype 106

Background Clostridioides difficile ribotype (RT) 106 has emerged as one of the most commonly isolated strains in the USA and worldwide. However, studies investigating clinical outcomes associated with this strain are lacking. The purpose of this study was to compare disease severity, clinical cure, and recurrence rates associated with CDI caused by RT106 vs two other comparator strains.MethodsThis multicenter study (20 hospitals) assessed hospitalized patients infected with C. difficile RT106 compared to patients infected with a known hypervirulent strain (RT027) and a strain associated with less virulence (RT014-020). Electronic medical records were reviewed by investigators blinded to RT. Disease severity was calculated using the 2017 IDSA/SHEA guidelines, initial clinical cure was defined as resolution of symptoms by day 6 of treatment, and recurrence assessed 90-days after the initial positive toxin test. All isolates were ribotyped using PCR fluorescent ribotyping.ResultsA total of 380 patients with CDI aged 66 ± 17 years (Female: 59.5%; White: 70.5%) infected with RT 106 (115/380; 30.3%), RT027 (116/380; 30.5%), and RT014-020 (149/380; 39.2%) were included. Approximately half of the patients had severe CDI (47.6%). Disease severity was highest for RT027 (59.3%) followed by RT014-020 (45%), and RT106 (41.2%). Clinical cure rates were lowest for RT027 (74.8%) followed by RT106 (77.8%), and RT014-020 (85.5%). 90-day recurrence rates were highest for RT027 (20.7%) followed by RT106 (13.3%), and RT014-020 (8.7%). Compared to RT014-020, virulence increased with RT106 (OR:1.10; 95% CI: 0.67-1.8) and RT027 (OR: 2.0: 95% CI: 1.2-3.5) was noted.ConclusionOur novel analysis method established RT106 as a moderately virulent C. difficile strain vs. comparator ribotypes. This study presents a novel method for comparing clinical outcomes for emerging ribotypes.Disclosures Kevin W. Garey, PharmD, MS, FASHP, Merck & Co. (Grant/Research Support, Scientific Research Study Investigator)

Evaluation of the antimicrobial activity of ridinilazole and six comparators against Chinese, Japanese and South Korean strains of Clostridioides difficile.

Background Clostridioides difficile is the most common cause of antimicrobial-associated diarrhoea in high-income countries. Fluoroquinolone resistance enabled the emergence and intercontinental spread of the epidemic ribotype (RT) 027 strain of C. difficile in the early 2000s. Despite frequent inappropriate antimicrobial use in Asia, RT 027 is rarely isolated in the region, but the often fluoroquinolone- and clindamycin-resistant RT 017 strain predominates.ObjectivesThis study evaluated the antimicrobial activity of ridinilazole, a novel antimicrobial agent with highly specific activity for C. difficile, against clinical strains of C. difficile from Asia.Methods C. difficile strains from Japan (n = 64), South Korea (n = 32) and China (n = 44) were tested by the agar dilution method for susceptibility to ridinilazole, metronidazole, vancomycin, clindamycin, moxifloxacin, rifaximin and fidaxomicin.ResultsAll strains were susceptible to ridinilazole, with low MICs (0.03–0.25 mg/L). Several strains showed multiresistance profiles, particularly RT 017 (100% clindamycin resistant, 91.3% moxifloxacin resistant, 82.6% rifaximin resistant) and RT 369 (94.4% clindamycin resistant, 100% moxifloxacin resistant). Rifaximin resistance was absent in all strains from Japan. Multiresistance to clindamycin, moxifloxacin and rifaximin was found in 19 RT 017 strains (from China and South Korea), 2 RT 001 strains (South Korea) and 1 RT 046 strain (South Korea).ConclusionsRidinilazole showed potent activity against a range of Asian C. difficile strains, which otherwise frequently displayed resistance to several comparator antimicrobial agents. Ongoing surveillance of antimicrobial resistance profiles is required to monitor and control the spread of resistant strains.

Whole genome sequencing of Clostridioides difficile PCR ribotype 046 suggests transmission between pigs and humans.

A zoonotic association has been suggested for several PCR ribotypes (RTs) of Clostridioides difficile. In central parts of Sweden, RT046 was found dominant in neonatal pigs at the same time as a RT046 hospital C. difficile infection (CDI) outbreak occurred in the southern parts of the country. To detect possible transmission of RT046 between pig farms and human CDI cases in Sweden and investigate the diversity of RT046 in the pig population using whole genome sequencing (WGS). WGS was performed on 47 C. difficile isolates from pigs (n = 22), the farm environment (n = 7) and human cases of CDI (n = 18). Two different core genome multilocus sequencing typing (cgMLST) schemes were used together with a single nucleotide polymorphisms (SNP) analysis and the results were related to time and location of isolation of the isolates. The pig isolates were closely related (≤6 cgMLST alleles differing in both cgMLST schemes) and conserved over time and were clearly separated from isolates from the human hospital outbreak (≥76 and ≥90 cgMLST alleles differing in the two cgMLST schemes). However, two human isolates were closely related to the pig isolates, suggesting possible transmission. The SNP analysis was not more discriminate than cgMLST. No general pattern suggesting zoonotic transmission was apparent between pigs and humans, although contrasting results from two isolates still make transmission possible. Our results support the need for high resolution WGS typing when investigating hospital and environmental transmission of C. difficile.