Virulence and genomic diversity among clinical isolates of ST1 (BI/NAP1/027) Clostridioides difficile.

Clostridioides difficile produces toxins that damage the colonic epithelium, causing colitis. Variation in disease severity is poorly understood and has been attributed to host factors and virulence differences between C. difficile strains. We test 23 epidemic ST1 C. difficile clinical isolates for their virulence in mice. All isolates encode a complete Tcd pathogenicity locus and achieve similar colonization densities. However, disease severity varies from lethal to avirulent infections. Genomic analysis of avirulent isolates reveals a 69-bp deletion in the cdtR gene, which encodes a response regulator for binary toxin expression. Deleting the 69-bp sequence in virulent R20291 strain renders it avirulent in mice with reduced toxin gene transcription. Our study demonstrates that a natural deletion within cdtR attenuates virulence in the epidemic ST1 C. difficile isolates without reducing colonization and persistence. Distinguishing strains on the basis of cdtR may enhance the specificity of diagnostic tests for C. difficile colitis.

Non-Toxigenic Clostridioides difficile Strain E4 (NTCD-E4) Prevents Establishment of Primary C. difficile Infection by Epidemic PCR Ribotype 027 in an In Vitro Human Gut Model.

Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI.

Low-toxin Clostridioides difficile RT027 strains exhibit robust virulence.

Clostridioides difficile is a leading cause of healthcare-associated infections worldwide. Currently, there is a lack of consensus for an optimal diagnostic method for C. difficile infection (CDI). Multi-step diagnostic algorithms use enzyme immunosorbent analysis (EIA)-based detection of C. difficile toxins TcdA/TcdB in stool, premised on the rationale that EIA toxin-negative (Tox-) patients have less severe disease and shorter diarrhoea duration. The aim of this study was to characterize toxigenic (i.e. tcdA/tcdB-positive) C. difficile strains isolated from diarrheic patient stool with an EIA Tox- (i.e. "discrepant") CDI diagnostic test result. Recovered strains were DNA fingerprinted (ribotyped), subjected to multiple toxin, genome and proteome evaluations, and assessed for virulence. Overall, of 1243 C. difficile-positive patient stool specimens from Southern Arizona hospitals, 31% were discrepant. For RT027 (the most prevalent ribotype)-containing specimens, 34% were discrepant; the corresponding RT027 isolates were cytotoxic to cultured fibroblasts, but their total toxin levels were comparable to, or lower than, the historic low-toxin-producing C. difficile strain CD630. Nevertheless, these low-toxin RT027 strains (LT-027) exhibited similar lethality to a clade-matched high-toxin RT027 strain in Golden Syrian hamsters, and heightened colonization and persistence in mice. Genomics and proteomics analyses of LT-027 strains identified unique genes and altered protein abundances, respectively, relative to high-toxin RT027 strains. Collectively, our data highlight the robust virulence of LT-027 C. difficile, provide a strong argument for reconsidering the clinical significance of a Tox- EIA result, and underscore the potential limitations of current diagnostic protocols.

Clostridioides difficile Ribotype 027 (RT027) Outbreak Investigation Due to the Emergence of Rifampicin Resistance Using Multilocus Variable-Number Tandem Repeat Analysis (MLVA).

OBJECTIVE: The aim of this study was Clostridioides difficile outbreak investigation due to the emergence of rifampicin resistant ribotype 027 (RT 027) fecal isolates from patients of Polish tertiary care hospital between X. 2017 and II. 2018 using multilocus variable tandem repeat analysis (MLVA). MATERIALS AND METHODS: Twenty-nine C. difficile fecal isolates from patients of tertiary care hospital in Southern Poland were ribotyped and analyzed by MLVA. Multiplex PCR (mPCR) for genes encoding GDH (gluD), toxins A (tcdA)/ B (tcdB), 16S rDNA and binary toxin genes (ctdA and ctdB) was performed. The antibiotic susceptibility profile was determined by E-test. RESULTS: The A, B and binary toxins encoding genes were detected in all 29 C. difficile strains which were sensitive to metronidazole, vancomycin and were resistant to erythromycin, clindamycin, and moxifloxacin; resistance to imipenem demonstrated 97%, to rifampicin - 45% isolates. C. difficile strains could be grouped by MLVA into 5 distinct clusters, and the largest cluster II contains 16 strains. The comparison of rifampicin GM MIC of cluster II (n=16 strains) with all others (n=13) showed that strains from clusters I, III, IV and V possessed significantly (p <0.005) higher GM MIC and were more resistant to rifampicin. CONCLUSION: MLVA analysis proved transmission and recognized outbreak due to multidrug-resistant RT 027 C. difficile among patients of tertiary care hospital in Southern Poland. The reason for this is probably the widespread occurrence of spores in the hospital environment, which includes, among others, neglect of hygienic procedures and epidemic supervision. High resistance to imipenem (97%) and to rifampicin (45%) among C. difficile RT 027 Silesian isolates is threatening and requires further studies to elucidate this phenomenon.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

In vivo animal models confirm an increased virulence potential and pathogenicity of the NAP1/RT027/ST01 genotype within the Clostridium difficile MLST Clade 2.

BACKGROUND: Based on MLST analyses the global population of C. difficile is distributed in eight clades, of which Clade 2 includes the "hypervirulent" NAP1/RT027/ST01 strain along with various unexplored sequence types (STs). METHODS: To clarify whether this clinically relevant phenotype is a widespread feature of C. difficile Clade 2, we used the murine ileal loop model to compare the in vivo pro-inflammatory (TNF-α, IL-1ß, IL-6) and oxidative stress activities (MPO) of five Clade 2 clinical C. difficile isolates from sequence types (STs) 01, 41, 67, and 252. Besides, we infected Golden Syrian hamsters with spores from these strains to determine their lethality, and obtain a histological evaluation of tissue damage, WBC counts, and serum injury biomarkers (LDH, ALT, AST, albumin, BUN, creatinine, Na+, and Cl-). Genomic distances were calculated using Mash and FastANI to explore whether the responses were dictated by phylogeny. RESULTS: The ST01 isolate tested ranked first in all assays, as it induced the highest overall levels of pro-inflammatory cytokines, MPO activity, epithelial damage, biochemical markers, and mortality measured in both animal models. Statistically indistinguishable or rather similar outputs were obtained for a ST67 isolate in tests such as tissue damage, neutrophils count, and lethal activity. The results recorded for the two ST41 isolates tested were of intermediate magnitude and the ST252 isolate displayed the lowest pathogenic potential in all animal experiments. This ordering matched the genomic distance of the ST01 isolate to the non-ST01 isolates. CONCLUSIONS: Despite their close phylogenic relatedness, our results demonstrate differences in pathogenicity and virulence levels in Clade 2 C. difficile strains, confirm the high severity of infections caused by the NAP1/RT027/ST01 strain, and highlight the importance of C. difficile typing.

Origin, genomic diversity and microevolution of the Clostridium difficile B1/NAP1/RT027/ST01 strain in Costa Rica, Chile, Honduras and Mexico.

Clostridium difficile B1/NAP1/RT027/ST01 has been responsible for outbreaks of antibiotic-associated diarrhoea in clinical settings worldwide and is associated with severe disease presentations and increased mortality rates. Two fluoroquinolone-resistant (FQR) lineages of the epidemic B1/NAP1/RT027/ST01 strain emerged in the USA in the early 1990s and disseminated trans continentally (FQR1 and FQR2). However, it is unclear when and from where they entered Latin America (LA) and whether isolates from LA exhibit unique genomic features when compared to B1/NAP1/RT027/ST01 isolates from other regions of the world. To answer the first issue we compared whole-genome sequences (WGS) of 25 clinical isolates typed as NAP1, RT027 or ST01 in Costa Rica (n=16), Chile (n=5), Honduras (n=3) and Mexico (n=1) to WGS of 129 global isolates from the same genotype using Bayesian phylogenomics. The second question was addressed through a detailed analysis of the number and type of mutations of the LA isolates and their mobile resistome. All but two B1/NAP1/RT027/ST01 isolates from LA belong to the FQR2 lineage (n=23, 92 %), confirming its widespread distribution. As indicated by analysis of a dataset composed of 154 WGS, the B1/NAP1/RT027/ST01 strain was introduced into the four LA countries analysed between 1998 and 2005 from North America (twice) and Europe (at least four times). These events occurred soon after the emergence of the FQR lineages and more than one decade before the first report of the detection of the B1/NAP1/RT027/ST01 in LA. A total of 552 SNPs were identified across all genomes examined (3.8-4.3 Mb) in pairwise comparisons to the R20291 reference genome. Moreover, pairwise SNP distances were among the smallest distances determined in this species so far (0 to 55). Despite this high level of genomic conservation, 39 unique SNPs (7 %) in genes that play roles in the infection process (i.e. slpA) or antibiotic resistance (i.e. rpoB, fusA) distinguished the LA isolates. In addition, isolates from Chile, Honduras and Mexico had twice as many antibiotic resistance genes (ARGs, n=4) than related isolates from other regions. Their unique set of ARGs includes a cfr-like gene and tetM, which were found as part of putative mobile genetic elements whose sequences resemble undescribed integrative and conjugative elements. These results show multiple, independent introductions of B1/NAP1/RT027/ST01 isolates from the FQR1 and FQR2 lineages from different geographical sources into LA and a rather rapid accumulation of distinct mutations and acquired ARG by the LA isolates.

Unique Clindamycin-Resistant Clostridioides difficile Strain Related to Fluoroquinolone-Resistant Epidemic BI/RT027 Strain.

During a surveillance study of patients in a long-term care facility and the affiliated acute care hospital in the United States, we identified a Clostridioides difficile strain related to the epidemic PCR ribotype (RT) 027 strain associated with hospital outbreaks of severe disease. Fifteen patients were infected with this strain, characterized as restriction endonuclease analysis group DQ and RT591. Like RT027, DQ/RT591 contained genes for toxin B and binary toxin CDT and a tcdC gene of identical sequence. Whole-genome sequencing and multilocus sequence typing showed that DQ/RT591 is a member of the same multilocus sequence typing clade 2 as RT027 but in a separate cluster. DQ/RT591 produced a similar cytopathic effect as RT027 but showed delayed toxin production in vitro. DQ/RT591 was susceptible to moxifloxacin but highly resistant to clindamycin. Continued surveillance is warranted for this clindamycin-resistant strain that is related to the fluoroquinolone-resistant epidemic RT027 strain.

PCR based detection of tcdCΔ117 in Clostridium difficile infection identifies patients at risk for recurrence - A hospital-based prospective observational study.

OBJECTIVES: Increasing incidence and severity of Clostridium difficile infection (CDI) in the last decades has been attributed to the emergence of hypervirulent C. difficile strain PCR-ribotype 027 (RT027). Commercial multiplex real-time PCR tests allow the presumptive identification of RT027 by detecting a single-base deletion at nt117 in the tcdC gene (tcdCΔ117). The clinical usefulness of the detection of tcdCΔ117 is unclear. Therefore, we evaluated test performance and clinical association of the detection of tcdCΔ117 in patients with CDI in a prospective observational study conducted in a tertiary care hospital in Germany. METHODS: From June to October 2015, stool from all patients with suspected CDI was tested for C. difficile according to ESCMID guidelines. C. difficile was cultured from positive samples and ribotyping was performed. Clinical data were collected prospectively from all C. difficile positive patients. RESULTS: From 1121 tested stool samples 107 patients with CDI were included in the study. TcdCΔ117 was detected in 18 (16.8%) of these patients. Multivariable logistic regression analysis revealed an independent association of detection of tcdCΔ117 with a further episode of CDI (OR 14.6; 95% CI 3.6-58.3; p < 0.001) and death within 30 days of the positive test (OR 5.1; 95% CI 1.0-25.7; p = 0.046). As follow up data are limited, it remains unclear, whether the further episode of CDI was due to tcdCΔ117 (recurrence) or another type. CONCLUSION: In our setting, PCR-based detection of tcdCΔ117 identified patients at risk for recurrent CDI and increased mortality and thus may guide therapeutic choices in CDI patients at the time of diagnosis.

Occurrence of Clostridium difficile ribotype 027 in hospitals of Silesia, Poland.

Clostridium difficile is an important healthcare-associated pathogen, responsible for a broad spectrum of diarrheal diseases. The aim of this prospective study was to determine the occurrence of C. difficile infection (CDI), to characterize cultured C. difficile strains and to investigate the association of fecal lactoferrin with CDI. Between January 2013 and June 2014, 148 stool samples were obtained from adult diarrheal patients (C. difficile as a suspected pathogen) hospitalized in different healthcare facilities of 15 Silesian hospitals. Out of 134 isolated C. difficile strains, 108 were ribotyped: 82.4% belonged to Type 027, 2.8% to Type 176, 2.8% to Type 014, 1.9% to Type 010 and 0.9% to Types 001, 018, 020 and 046 each. In total, 6.5% non-typable strains were identified. All Type 027 isolates contained both toxin genes tcdA & tcdB, and binary toxin genes (cdtA &cdtB). Susceptibility testing revealed that all Type 027 isolates were sensitive to metronidazole and vancomycin and resistant to moxifloxacin, ciprofloxacin, imipenem and erythromycin. Of 89 Type 027 strains, 16 had a ermB (688 bp) gene coinciding with high levels of erythromycin resistance (MIC >256 µg/mL). Of 16 ermB positive strains, 14 demonstrated also high level of resistance to clindamycin (>256 µg/mL). A significant difference (p = 0.004) in lactoferrin level was found between C. difficile toxin-positive (n = 123; median 185.9 µg/mL; IQR 238.8) and toxin-negative (n = 25; median 22.4 µg/mL; IQR 141.7) fecal samples. Stool samples from n = 89 patients with CDI caused by Type 027 demonstrated significantly higher (p = 0.03) lactoferrin level (median 173.0 µg/mL; IQR 237.3) than from patients with CDI caused by other ribotypes and non-typable C. difficile strains (median 189.4 µg/mL; IQR 190.8).

CRISPR Diversity and Microevolution in Clostridium difficile.

Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes.

The Clostridium difficile PCR ribotype 027 lineage: a pathogen on the move.

Clostridium difficile is a Gram-positive, spore-forming, human and animal pathogen that is the major cause of antibiotic-associated diarrhoea worldwide. The past decade has seen the rapid emergence of the hypervirulent PCR ribotype (RT) 027 complex, which has been associated with increases in the incidence and severity of disease and mortality. In this review, we describe the potential virulence factors that have been reported in strains from the RT 027 complex. We review the emergence, population structure, dissemination and evolution of this lineage.