Clostridium difficile toxin-positive diarrheal stool specimens submitted to eight Canadian hospital laboratories from 2013 to 2015 were cultured. Polymerase chain reaction ribotyping of isolates was ...performed using an internationally standardized, high-resolution capillary gel-based electrophoresis protocol and antimicrobial susceptibility testing conducted by CLSI-defined agar dilution (M11-A8, 2012). Among the 1310 isolates of C. difficile cultured, 141 different ribotypes were identified; the most common ribotypes were 027 (24.5% of isolates), 014 (7.7%), 020 (6.6%), 106 (6.1%), and 002 (4.6%). Ribotype 027 was the commonest ribotype in all geographic regions of Canada and was more frequently isolated from patients aged ≥80 years (40.6%) than younger patients (P<0.00001). Ribotype 027 isolates were frequently moxifloxacin-resistant (92.2% of isolates) and multidrug-resistant (49.5%). Fidaxomicin demonstrated the greatest in vitro potency (lowest MIC90, 0.5 μg/mL; lowest maximum MIC, 2 μg/mL) of eight antimicrobial agents tested and was the most active agent against each of the five commonest ribotypes (MIC90, 0.25–1 μg/mL).
•1310 isolates of Clostridium difficile were cultured from toxin-positive stools.•141 ribotypes were identified among the 1310 isolates.•The commonest ribotypes were 027 (24.5% of isolates), 014 (7.7%), and 020 (6.6%).•49.5% of ribotype 027 isolates were multidrug-resistant; 92.2% were moxifloxacin-resistant.•Fidaxomicin had the lowest MIC90 (0.5 μg/mL) of eight agents tested.
Abstract
Objectives
To investigate the relationship between
Clostridium (Clostridioides) difficile
strain characteristics and
C. difficile
infection (CDI) outcome.
Methods
Between October and ...December 2017, 16 hospitals collected epidemiological data according to the European Centre for Disease Prevention and Control (ECDC) surveillance protocol for CDI.
C. difficile
isolates were characterized by ribotyping, toxin genes detection and antibiotic susceptibility testing to metronidazole, vancomycin and moxifloxacin.
Results
The overall mean CDI incidence density was 4.5 95% CI 3.6–5.3 cases per 10,000 patient-days. From the 433 CDI cases, 330 (76.2%) were healthcare-associated, 52 (12.0%) cases were community-associated or of unknown origin and 51 (11.8%) CDI cases recurrent; a complicated course of CDI was reported in 65 cases (15.0%). Eighty-eight (20.3%) of patients died and 59 of them within 30 days after the CDI diagnosis.
From the 379
C. difficile
isolates, the most prevalent PCR ribotypes were 001 (
n
= 127, 33.5%) and 176 (
n
= 44, 11.6%). A total of 186 (49.1%) isolates showed a reduced susceptibility to moxifloxacin (> 4 mg/L) and 96.4% of them had Thr82Ile in the GyrA. Nineteen isolates revealed reduced susceptibility to metronidazole and two isolates to vancomycin (> 2 mg/L).
A fatal outcome was associated with a reduced susceptibility to moxifloxacin, the advanced age of the patients and a complicated course of CDI (
p
<0.05). No association between ribotype, binary toxin and a reduced susceptibility to moxifloxacin and complicated course or recurrent CDI was found.
Conclusions
A reduced susceptibility to moxifloxacin, in causative
C. difficile
strains was associated with fatal outcome of the patients, therefore it is an important marker in surveillance of CDI.
We report a patient case of pseudomembranous colitis associated with a monotoxin-producing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR) ribotype ...(RT) 151. To understand why this isolate was not identified using a routine commercial test, we performed a genomic analysis of RT151.
Illumina short-read sequencing was performed on n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. Subsequently, we used PacBio circular consensus sequencing to determine the complete genome sequence of isolates belonging to cryptic clades C–I and C-II, which includes the patient isolate.
We found that 1) RT151s are polyphyletic with isolates falling into clades 1 and cryptic clades C–I and C-II; 2) RT151 contains both nontoxigenic and toxigenic isolates and 3) RT151 C-II isolates contained monotoxin pathogenicity loci. The isolate from our patient case report contains a novel-pathogenicity loci insertion site, lacked tcdA and had a divergent tcdB sequence that might explain the failure of the diagnostic test.
This study shows that RT151 encompasses both typical and cryptic clades and provides conclusive evidence for C. difficile infection due to clade C-II isolates that was hitherto lacking. Vigilance towards C. difficile infection as a result of cryptic clade isolates is warranted.
Clostridium difficile is an anaerobic, spore forming, rod shaped bacterium frequently isolated from butchery animals in recent years. The aim of this study is to evaluate the presence of C. difficile ...(especially ribotype 027 and 078) in cattle and sheep carcasses and to investigate the antibiotic susceptibility of isolates. The bacterium was isolated in 83 out of 247 (33.6%) cattle and 78 out of 308 (25.3%) sheep carcass samples. 15/83 (18.1%) cattle and 6/78 (7.7%) sheep isolates were identified as ribotype 027, whereas the other hypervirulent isolate ribotype 078 could not be detected among the analysed samples. Almost all isolates were susceptible to amoxicillin-clavulanic acid (98.8%), vancomycin (96.9%) and tetracycline (97.5%), whereas resistant to cefotaxim (97.5%) and imipenem (87.0%). In conclusion, the results demonstrate the presence of toxigenic C. difficile isolates in cattle and sheep carcasses on the slaughter line. As a result, the results of this study demonstrate the presence of toxigenic C. difficile isolates in cattle and sheep carcasses on the slaughter line.
Background. Clostridium difficile infection (CDI) can cause severe disease and death, especially in older adults. A better understanding of risk factors for adverse outcomes is needed. This study ...tests the hypotheses that infection with specific ribotypes and presence of stool toxins independently associate with severity and constructs predictive models of adverse outcomes. Methods. Cases of non-recurrent CDI were prospectively included after positive stool tests for toxins A and/or B by enzyme immunoassay (EIA) or tcdB by polymerase chain reaction. Outcomes included severe CDI (intensive care unit admission, colectomy, or death attributable to CDI within 30 days of diagnosis) and 30-day all-cause mortality. Adjusted models were developed to test hypotheses and predict outcomes. Results. In total, 1144 cases were included. The toxin EIA was positive in 37.2% and 35.6% of patients were of age >65 years. One of the 137 unique ribotypes was ribotype 027 (16.2%). Detectable stool toxin did not associate with outcomes. Adjusting for covariates, including age, Ribotype 027 was a significant predictor of severe CDI (90 cases; odds ratio OR, 1.73; 95% confidence interval CI, 1.03–2.89; P = .037) and mortality (89 cases; OR, 2.02; 95% CI, 1.19–3.43; P = .009). Concurrent antibiotic use associated with both outcomes. Both multivariable predictive models had excellent performance (area under the curve >0.8). Conclusions. Detection of stool toxin A and/or B by EIA does not predict severe CDI or mortality. Infection with ribotype 027 independently predicts severe CDI and mortality. Use of concurrent antibiotics is a potentially modifiable risk factor for severe CDI.
The unarmored dinoflagellate Margalefidinium polykrikoides is a well-known cosmopolitan harmful alga that forms intensive blooms causing massive fish kills and mortality of other marine animals. ...Globally distributed populations of M. polykrikoides have been categorized into four ribotypes: East Asian, American/Malaysian, Philippines, and Mediterranean, according to their characteristic sequences in LSU rRNA gene and primary geographic distributions, while no discernible morphological feature has been observed to distinguish. Here we examined the toxicity of four strains of M. polykrikoides including MPJZB-C3 and MPJZB-D6 (East Asian ribotype, from China), CP1 (American/Malaysian ribotype, from USA), and MPCoKK23 (American/Malaysian ribotype, from Malaysia), to three aquatic animals including the marine medaka (Oryzias melastigma), rotifer (Brachionus plicatilis, L-type), and brine shrimp (Artemia salina). With equivalent initial cell densities ranged from 1000 to 7000 cells mL−1, CP1 and MPCoKK23 displayed acute toxicity in a dose-response manner to the three target animals (100% mortality at higher cell densities) within 24 h, MPJZB-C3 also displayed acute toxicity to the fish and rotifer in 24 h (up to 50% mortality) when the cell densities were above 3000 cells mL−1. The strain MPJZB-D6 did not exhibit significant toxicity to all three animals in 24 h, but, in 120 h, caused 50–100% mortalities in fish when the cell density was >3000 cells mL−1 and 17% mortality in brine shrimp when the cell density was >5000 cells mL−1. Statistical analysis of the data indicated that the strains CP1 and MPCoKK23 exhibited significantly higher toxicity (50–100% mortalities) to all three animals than the strains MPJZB-C3 and MPJZB-D6 (0–100% mortality) did within 24 h exposure, while, however, the difference in toxicity between the American and Malaysian strains and that between the two Chinese strains were both not significant in terms of the mortality of test animals. While the cell-free culture media (by filtration) and sonicated cultures were less toxic to test animals than the live cultures for all M. polykrikoides strains, sonicated cultures were even less toxic than the cell-free culture media. Among the three target animals, the marine medaka exhibited most sensitive to the toxicity, followed by rotifer and then brine shrimp. Collectively, while our results demonstrated that all strains, or both ribotypes, of M. polykrikoides are toxic, the results may also suggest the differences in toxicity to three test animals among the four cultures of M. polykrikoides reflect the inherent difference in toxicity between East Asian and American/Malaysian ribotypes, which stimulated us to propose a further investigation on whether or not the four different ribotypes also differ inherently in their toxicity in the future, via comparing more strains of all four ribotypes and relevant genetic characterizations.
•Two M. polykrikoides strains from China could be toxic to marine animals.•Two M. polykrikoides strains from USA and Malaysia are significantly more toxic.•Sonicated cultures are less toxic than cell-free culture media and live cultures.•Different ribotypes of M. polykrikoides may differ in toxicity.
infections (CDI) are considered worldwide as emerging health threat. Uptake of
spores may result in asymptomatic carrier status or lead to CDI that could range from mild diarrhea, eventually ...developing into pseudomembranous colitis up to a toxic megacolon that often results in high mortality. Most epidemiological studies to date have been performed in middle- and high income countries. Beside others, the use of antibiotics and the composition of the microbiome have been identified as major risk factors for the development of CDI. We therefore postulate that prevalence rates of CDI and the distribution of
strains differ between geographical regions depending on the regional use of antibiotics and food habits. A total of 593 healthy control individuals and 608 patients suffering from diarrhea in communities in Germany, Ghana, Tanzania and Indonesia were selected for a comparative multi-center cross-sectional study. The study populations were screened for the presence of
in stool samples. Cultured
strains (
= 84) were further subtyped and characterized using PCR-ribotyping, determination of toxin production, and antibiotic susceptibility testing. Prevalence rates of
varied widely between the countries. Whereas high prevalence rates were observed in symptomatic patients living in Germany and Indonesia (24.0 and 14.7%), patients from Ghana and Tanzania showed low detection rates (4.5 and 6.4%). Differences were also obvious for ribotype distribution and toxin repertoires. Toxin A
/B
ribotypes 001/072 and 078 predominated in Germany, whereas most strains isolated from Indonesian patients belonged to toxin A
/B
ribotype SLO160 and toxin A
/B
ribotype 017. With 42.9-73.3%, non-toxigenic strains were most abundant in Africa, but were also found in Indonesia at a rate of 18.2%. All isolates were susceptible to vancomycin and metronidazole. Mirroring the antibiotic use, however, moxifloxacin resistance was absent in African
isolates but present in Indonesian (24.2%) and German ones (65.5%). This study showed that CDI is a global health threat with geographically different prevalence rates which might reflect distinct use of antibiotics. Significant differences for distributions of ribotypes, toxin production, and antibiotic susceptibilities were observed.
The epidemiology of
infection (CDI) has drastically changed since the emergence of the epidemic strain BI/NAP1/027, also known as ribotype 027 (R027). However, the relationship between the infecting
...strain and clinical outcomes is still debated. We hypothesized that certain subpopulations of R027 isolates could be associated with unfavorable outcomes. We applied high-resolution multilocus variable-number tandem-repeat analysis (MLVA) to characterize
R027 isolates collected from confirmed CDI patients recruited across 10 Canadian hospitals from 2005 to 2008. PCR ribotyping was performed first to select R027 isolates that were then analyzed by MLVA (
= 450). Complicated CDI (cCDI) was defined by the occurrence of any of admission to an intensive care unit, colonic perforation, toxic megacolon, colectomy, and if CDI was the cause or contributed to death within 30 days after enrollment. Three major MLVA clusters were identified, MC-1, MC-3, and MC-10. MC-1 and MC-3 were exclusive to Quebec centers, while MC-10 was found only in Ontario. Fewer cases infected with MC-1 developed cCDI (4%) than those infected with MC-3 and MC-10 (15% and 16%, respectively), but a statistically significant difference was not reached. Our data did not identify a clear association between subpopulations of R027 and different clinical outcomes; however, the data confirmed the utility of MLVA's higher discrimination potential to better characterize CDI populations in an epidemiological analysis. For a patient with CDI, the progression toward an unfavorable outcome is a complex process that probably includes several interrelated strain and host characteristics.
Trichomonas gallinae, a single-celled protozoan parasite, is a causative agent of the disease trichomonosis, which is distributed worldwide and has recently been highlighted as a pandemic threat to ...several wild bird species. The aim of this study was to determine the prevalence and genotypic diversity of T. gallinae in Riyadh, Saudi Arabia. For this purpose, 273 oral swab samples from different bird species (feral pigeon Columba livia, common mynah Acridotheres tristis, chicken Gallus gallus domesticus, turkey Meleagris gallopavo, and ducks Anatidae) were collected and tested for T. gallinae infection with InPouch™ TV culture kits. The results showed that the overall prevalence of T. gallinae in these samples was 26.4% (n = 72). The PCRs were used to detect the internal transcribed spacer (ITS) region of T. gallinae, and the results of the sequence analysis indicated genetic variation. Among 48 sequences, we found 15 different ribotypes, of which 12 were novel. Three had been previously described as ribotypes A, C, and II. To our knowledge, this study demonstrated the presence of T. gallinae strain diversity in Saudi Arabian birds for the first time and revealed that ribotypes A and C are predominant among Riyadh birds.
In a 2015 point-prevalence study, Clostridioides difficile 027, a hypervirulent ribotype, was absent from healthcare institutions in Switzerland. In late 2016, we detected an outbreak of C. difficile ...infection (CDI) with ribotype 027 occurring across several hospitals in the same hospital network.
The first cases of CDI due to ribotype 027 triggered an outbreak investigation, including whole genome sequencing (WGS) to identify outbreak strains.
Twenty-eight patients with CDI caused by ribotype 027 between December 2016 and December 2017 were identified, out of which 20 were caused by a single clone. Commonalities among these patients were hospitalization in the same room or on the same ward, receiving care from the same healthcare workers, and shared toilet areas. In addition to the epidemiological links suggesting possible transmission pathways between cases, WGS confirmed the clonality of this C. difficile 027 outbreak. The outbreak was contained by isolation precautions, raising awareness among healthcare workers, harmonizing diagnostic algorithms, and switching to a sporicidal agent for environmental disinfection. Of note, neither default gowning and gloving nor hand washing with water and soap were implemented.
This C. difficile 027 outbreak was recognized belatedly due to lack of screening for this ribotype in some hospitals, and was contained by a swift response with simple infection prevention measures and adapting the laboratory approach. In order to have a better understanding of C. difficile epidemiology, diagnostic approaches should be standardized, CDI declared notifiable, and longitudinal data on prevalent ribotypes collected in countries where this is not established.
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