Epidemiology of Primary and Recurrent Healthcare-Associated and Community-Associated Pediatric Clostridioides difficile Infection in Canada, 2015-2020.

Clostridioides difficile infection (CDI) among children remains a concerning cause of morbidity in hospital settings. We present epidemiological and molecular trends in healthcare- and community-associated CDI among children in Canadian inpatient and outpatient settings, including those who experienced recurrent infections.

Trends in Clostridioides difficile infection rates in Canadian hospitals during the coronavirus disease 2019 (COVID-19) pandemic.

The coronavirus disease 2019 (COVID-19) pandemic has placed significant burden on healthcare systems. We compared Clostridioides difficile infection (CDI) epidemiology before and during the pandemic across 71 hospitals participating in the Canadian Nosocomial Infection Surveillance Program. Using an interrupted time series analysis, we showed that CDI rates significantly increased during the COVID-19 pandemic.

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.

Direct Clostridioides difficile ribotyping from stool using capillary electrophoresis.

Clostridioides difficile(C. difficile) genotyping is essential for surveillance of emerging strains, transmissions, and outbreak investigations, but culture is lengthy and may not be routinely performed, which necessitates culture-independent genotyping methods. We aimed to develop a direct from stool C. difficile PCR ribotyping algorithm using capillary electrophoresis. Ribotypes were generated directly from 66.8% of stools with 33.2% requiring broth enrichment. 16S and tcdB cycle thresholds (Ct) were significantly lower (P< 0.001) in directly ribotyped stools compared to enriched stools, and Ct correlated with direct ribotyping (area under the curve: 0.97 and 0.96, respectively). Direct and isolate ribotypes were 94.7% concordant. Mixed C. difficile ribotypes were presumptively identified in 14 (7.5%) samples with 12 (6.4%) mixtures confirmed. We have developed a rapid PCR ribotyping algorithm allowing for direct C. difficile genotyping from stool using capillary electrophoresis with occasional detection of mixed C. difficile populations in stool, which is a limitation of conventional isolate genotyping.

Emergence of pstS-Null Vancomycin-Resistant Enterococcus faecium Clone ST1478, Canada, 2013-2018.

Rates of vancomycin-resistant enterococci bloodstream infections have remained relatively low in Canada. We recently observed an increase of 113% in these infections rates, which coincided with emergence of Enterococcus faecium pstS-null sequence type 1478. The proportion of this sequence type increased from 2.7% to 38.7% for all tested isolates from 2013-2018.

The evolving epidemiology of Clostridium difficile infection in Canadian hospitals during a postepidemic period (2009-2015).

BACKGROUND: The clinical and molecular epidemiology of health care-associated Clostridium difficile infection in nonepidemic settings across Canada has evolved since the first report of the virulent North American pulsed-field gel electrophoresis type 1 (NAP1) strain more than 15 years ago. The objective of this national, multicentre study was to describe the evolving epidemiology and molecular characteristics of health care-associated C. difficile infection in Canada during a post-NAP1-epidemic period, particularly patient outcomes associated with the NAP1 strain. METHODS: Adult inpatients with C. difficile infection were prospectively identified, using a standard definition, between 2009 and 2015 through the Canadian Nosocomial Infection Surveillance Program (CNISP), a network of 64 acute care hospitals. Patient demographic characteristics, severity of infection and outcomes were reviewed. Molecular testing was performed on isolates, and strain types were analyzed against outcomes and epidemiologic trends. RESULTS: Over a 7-year period, 20 623 adult patients admitted to hospital with health care-associated C. difficile infection were reported to CNISP, and microbiological data were available for 2690 patients. From 2009 to 2015, the national rate of health care-associated C. difficile infection decreased from 5.9 to 4.3 per 10 000 patient-days. NAP1 remained the dominant strain type, but infection with this strain has significantly decreased over time, followed by an increasing trend of infection with NAP4 and NAP11 strains. The NAP1 strain was significantly associated with a higher rate of death attributable to C. difficile infection compared with non-NAP1 strains (odds ratio 1.91, 95% confidence interval [CI] 1.29-2.82). Isolates were universally susceptible to metronidazole; one was nonsusceptible to vancomycin. The proportion of NAP1 strains within individual centres predicted their rates of health care-associated C. difficile infection; for every 10% increase in the proportion of NAP1 strains, the rate of health care-associated C. difficile infection increased by 3.3% (95% CI 1.7%-4.9%). INTERPRETATION: Rates of health care-associated C. difficile infection have decreased across Canada. In nonepidemic settings, NAP4 has emerged as a common strain type, but NAP1, although decreasing, continues to be the predominant circulating strain and remains significantly associated with higher attributable mortality.

Emergence of an outbreak-associated Clostridium difficile variant with increased virulence.

The prevalence of Clostridium difficile infections has increased due to the emergence of epidemic variants from diverse genetic lineages. Here we describe the emergence of a novel variant during an outbreak in a Costa Rican hospital that was associated with severe clinical presentations. This C. difficile variant elicited higher white blood cell counts and caused disease in younger patients than did other strains isolated during the outbreak. Furthermore, it had a recurrence rate, a 30-day attributable disease rate, and disease severity as great as those of the epidemic strain NAP1. Pulsed-field gel electrophoresis genotyping indicated that the outbreak strains belong to a previously undescribed variant, designated NAPCR1. Whole-genome sequencing and ribotyping indicated that the NAPCR1 variant belongs to C. difficile ribotype 012 and sequence type 54, as does the reference strain 630. NAPCR1 strains are resistant to fluoroquinolones due to a mutation in gyrA, and they possess an 18-bp deletion in tcdC that is characteristic of the epidemic, evolutionarily distinct, C. difficile NAP1 variant. NAPCR1 genomes contain 10% more predicted genes than strain 630, most of which are of hypothetical function and are present on phages and other mobile genetic elements. The increased virulence of NAPCR1 was confirmed by mortality rates in the hamster model and strong inflammatory responses induced by bacteria-free supernatants in the murine ligated loop model. However, NAPCR1 strains do not synthesize toxin A and toxin B at levels comparable to those in NAP1 strains. Our results suggest that the pathogenic potential of this emerging C. difficile variant is due to the acquisition of hypothetical functions associated with laterally acquired DNA.

Development and validation of an internationally-standardized, high-resolution capillary gel-based electrophoresis PCR-ribotyping protocol for Clostridium difficile.

PCR-ribotyping has been adopted in many laboratories as the method of choice for C. difficile typing and surveillance. However, issues with the conventional agarose gel-based technique, including inter-laboratory variation and interpretation of banding patterns have impeded progress. The method has recently been adapted to incorporate high-resolution capillary gel-based electrophoresis (CE-ribotyping), so improving discrimination, accuracy and reproducibility. However, reports to date have all represented single-centre studies and inter-laboratory variability has not been formally measured or assessed. Here, we achieved in a multi-centre setting a high level of reproducibility, accuracy and portability associated with a consensus CE-ribotyping protocol. Local databases were built at four participating laboratories using a distributed set of 70 known PCR-ribotypes. A panel of 50 isolates and 60 electronic profiles (blinded and randomized) were distributed to each testing centre for PCR-ribotype identification based on local databases generated using the standard set of 70 PCR-ribotypes, and the performance of the consensus protocol assessed. A maximum standard deviation of only ±3.8bp was recorded in individual fragment sizes, and PCR-ribotypes from 98.2% of anonymised strains were successfully discriminated across four ribotyping centres spanning Europe and North America (98.8% after analysing discrepancies). Consensus CE-ribotyping increases comparability of typing data between centres and thereby facilitates the rapid and accurate transfer of standardized typing data to support future national and international C. difficile surveillance programs.

Characterization of a stable, metronidazole-resistant Clostridium difficile clinical isolate.

BACKGROUND: Clostridium difficile are gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15-35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking. METHODOLOGY/PRINCIPAL FINDINGS: Genotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production. CONCLUSIONS/SIGNIFICANCE: This is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.

Multidrug-resistant North American pulsotype 2 Clostridium difficile was the predominant toxigenic hospital-acquired strain in the province of Manitoba, Canada, in 2006-2007.

The objective of the current study was to determine whether the antimicrobial susceptibility profile or genotype of hospital-acquired isolates of Clostridium difficile differed from isolates causing community-acquired disease. Five hundred diarrhoeal stool samples (one >2 ml sample per patient) from patients across Manitoba, Canada, in 2006-2007 that were reported as C. difficile toxin positive were cultured, resulting in 432 isolates of toxin-positive C. difficile for analysis. Of these 432 isolates, acquisition status could be determined for 235 (54.4%); 182 (77.4%) isolates were hospital acquired and 53 (22.6%) were community acquired. North American pulsotype (NAP) designations based on SmaI PFGE could be defined for 52.3% of the 432 isolates, with NAP2 (n=122) being the most common. Ninety-one per cent (71/78) of NAP2 isolates were recovered from patients with hospital-acquired C. difficile disease. Other NAP types and isolates with non-NAP-type PFGE patterns were less frequently associated with hospital-acquired disease. Community-acquired disease (35.3% of isolates) was associated with a wide variety of NAP types. NAP2 isolates were homogeneous (85.5% had SmaI PFGE pattern 0003) and demonstrated low susceptibility to moxifloxacin (6.6%) and clindamycin (1.6%) compared with non-NAP2 isolates (64.1-93.2% moxifloxacin susceptible; 14.1-28.2% clindamycin susceptible). All isolates of C. difficile in Manitoba were susceptible to metronidazole, piperacillin-tazobactam, amoxicillin-clavulanate and meropenem. NAP2 isolates of toxigenic C. difficile were approximately three times more common than NAP1 isolates (28.2 vs 9.1%) in Manitoba in 2006-2007, and these isolates demonstrated high levels of clonality and multidrug resistance, and were associated with hospital acquisition.

Detection of Clostridium difficile in retail ground meat products in Manitoba.

The aim of the present study was to determine whether Clostridium difficile was present in uncooked retail ground beef and ground pork products sold in Winnipeg, Manitoba. Using an alcohol treatment protocol and inoculation of cultures on C difficile Moxalactam Norfloxacin (CDMN), toxigenic C difficile was found in 6.3% of 48 meat samples. The C difficile isolates belonged to different pulsotypes, all of which had been previously isolated from the stool of Manitoba patients with C difficile disease. Because cooking of meat will not eradicate C difficile spores, this raises a concern regarding potential foodborne transmissibility of this organism.The aim of the present study was to determine whether Clostridium difficile was present in uncooked retail ground beef and ground pork products sold in Winnipeg, Manitoba. Using an alcohol treatment protocol and inoculation of cultures on C difficile Moxalactam Norfloxacin (CDMN), toxigenic C difficile was found in 6.3% of 48 meat samples. The C difficile isolates belonged to different pulsotypes, all of which had been previously isolated from the stool of Manitoba patients with C difficile disease. Because cooking of meat will not eradicate C difficile spores, this raises a concern regarding potential foodborne transmissibility of this organism.

La présente étude visait à déterminer si on décelait du Clostridium difficile dans les produits de viande hachée et de porc haché non cuits vendus au détail à Winnipeg, au Manitoba. Au moyen d'un protocole de traitement par l'alcool et d'inoculation de cultures sur la C difficile Moxalactam Norfloxacin (CDMN), les chercheurs ont trouvé du C difficile toxicogénique dans 6,3 % des 48 échantillons de viande. Les isolats de C difficile appartenaient à divers pulsotypes, tous déjà isolés dans les selles de patients manitobains atteints d'une maladie à C difficile. Puisque la cuisson de la viande n'éradique pas les spores C difficile, ce phénomène soulève de l'inquiétude au sujet de la transmissibilité potentielle de cet organisme par voie alimentaire.

VanG-type vancomycin-resistant Enterococcus faecalis strains isolated in Canada.

Enterococcus faecalis G1-0247 (vancomycin MIC, 16 microg/ml) was found to harbor a vanG operon 99% identical to the vanG operon in E. faecalis BM4518. E. faecalis N03-0233 (vancomycin MIC, 16 microg/ml) was found to harbor a novel vanG operon, vanG2, on an element in a different chromosomal location than the vanG-harboring elements in G1-0247 and BM4518.

Translocation of Clostridium difficile toxin B across polarized Caco-2 cell monolayers is enhanced by toxin A.

Clostridium difficile is the etiological agent of antibiotic-associated diarrhea; the most common form of nosocomial infectious diarrhea. The basis for the shock-like systemic symptoms observed in severe cases of this infection are not known. It is hypothesized that the invasion of C difficile toxins A and/or B from the gut mucosa may contribute to these symptoms.A polarized tissue culture model employing Caco-2 cells grown on transwell inserts was established to study the translocation of purified C difficile toxins A and B. C difficile toxins were (125)I labelled and inoculated onto confluent polarized Caco-2 cell monolayers to study translocation dynamics. Electrical resistance measurements were used to monitor monolayer confluence and tight junction integrity. Samples were taken from the apical and basal sides of the insert, as well as the insert itself, and tested using the human foreskin fibroblasts cell cytotoxicity assay to monitor partitioning of the radiolabelled toxins. Toxin A produced a 50% reduction in electrical resistance in 3 h whereas the same concentration of toxin B required at least 7 h to achieve the same effect. Both toxins A and B were able to translocate across confluent monolayers of Caco-2 cells. The combination of toxin A and B together was synergistic with respect to promoting the translocation of toxin B. Although the addition of toxin A resulted in a 100% increase in the amount of toxin B able to translocate, no increases in toxin A translocation were observed. These findings suggest a model of pathogenesis in which C difficile toxin A facilitates the translocation of toxin B from the gut into submucosal areas where it may play a role in inflammatory damage.