Comparison of Whole-Genome Sequence-Based Methods and PCR Ribotyping for Subtyping of Clostridioides difficile.

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks the discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better and provide standardized and interlaboratory exchangeable data. Using a well-characterized collection of diverse strains (N = 630; 100 unique ribotypes [RTs]), we compared the discriminatory power of core genome multilocus sequence typing (cgMLST) (SeqSphere and EnteroBase), whole-genome MLST (wgMLST) (EnteroBase), and single-nucleotide polymorphism (SNP) analysis. A unique cgMLST profile (more than six allele differences) was observed in 82 of 100 RTs, indicating that cgMLST could distinguish most, but not all, RTs. Application of cgMLST in two outbreak settings with RT078 and RT181 (known to have low intra-RT allele differences) showed no distinction between outbreak and nonoutbreak strains in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize, and offers higher discrimination. However, adjusted cutoff thresholds and epidemiological data are necessary to recognize outbreaks of some specific RTs. We propose to use an allelic threshold of three alleles to identify outbreaks.

Community-Onset Clostridioides Difficile Infection in Hospitalized Patients in The Netherlands.

BACKGROUND: Clostridioides difficile infection (CDI) is increasingly reported in the community. The aim of this study was to analyze characteristics of hospitalized patients with community-onset CDI (CO-CDI). METHODS: In the Netherlands, 24 hospitals (university-affiliated and general hospitals) participate in the sentinel CDI surveillance program. Clinical characteristics and 30-day outcomes of hospitalized patients >2 years old diagnosed with CDI are registered. Samples of these patients are sent to the national reference laboratory for polymerase chain reaction ribotyping. Data obtained for this surveillance from May 2012 to May 2018 were used to compare CO-CDI with hospital-onset (HO)-CDI episodes. RESULTS: Of 5405 registered cases, 2834 (52.4%) were reported as HO-CDI, 2174 (40.2%) were CO-CDI, and 339 (6.3%) had onset of symptoms in another healthcare facility (eg, nursing home). The proportion of CO-CDI increased over the years and was lower during winter months. Hospitalized patients with CO-CDI were younger (63.8 vs 68.0 years, P < .001) and more often females (53.0% vs 49.6%, P = .02) than patients with HO-CDI. Median time between onset of symptoms and CDI testing was longer in CO-CDI (4 vs 1 day, P < .001). Similar ribotypes were found in CO-CDI and HO-CDI, but ribotype 001 was more frequent among HO-CDI, whereas ribotype 023 was more frequent in CO-CDI. Six of 7 (85.7%) surgeries due to CDI, 27 of 50 (54%) ICU admissions due to CDI, and 48 of 107 (44.9%) of CDI-associated deaths were attributable to CO-CDI. CONCLUSIONS: Our study demonstrates that patients hospitalized with CO-CDI contribute substantially to the total number of CDI episodes and CDI-associated complications in hospitals, stressing the need for awareness and early testing for CDI in community and outpatient settings and also in patients admitted from community with diarrhoea. Surveillance programs that also target nonhospitalized CDI patients are needed to understand the true burden and dynamics of CDI.

Identification and validation of two peptide markers for the recognition of Clostridioides difficile MLST-1 and MLST-11 by MALDI-MS.

OBJECTIVES: Clostridioides difficile infection (CDI) has become the main cause of nosocomial infective diarrhoea. To survey and control the spread of different C. difficile strains, there is a need for suitable rapid tests. The aim of this study was to identify peptide/protein markers for the rapid recognition of C. difficile strains by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). METHODS: We analysed 44 well-characterized strains, belonging to eight different multi-locus sequence types (MLST), using ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS. The amino acid sequence of two peptide markers specific for MLST-1 and MLST-11 strains was elucidated by MALDI-TOF-MS/MS. The investigation of 2689 C. difficile genomes allowed the determination of the sensitivity and specificity of these markers. C18-solid-phased extraction was used to enrich the MLST-1 marker. RESULTS: Two peptide markers (m/z 4927.81 and m/z 5001.84) were identified and characterized for MLST-1 and MLST-11 strains, respectively. The MLST-1 marker was found in 786 genomes of which three did not belong to MLST-1. The MLST-11 marker was found in 319 genomes, of which 14 did not belong to MLST-11. Importantly, all MLST-1 and MLST-11 genomes were positive for their respective marker. Furthermore, a peptide marker (m/z 5015.86) specific for MLST-15 was found in 59 genomes. We translated our findings into a fast and simple method that allowed the unambiguous identification of the MLST-1 marker on a MALDI-TOF-MS platform. CONCLUSIONS: MALDI-FTICR MS-based peptide profiling resulted in the identification of peptide markers for C. difficile MLST-1 and MLST-11.

Clostridium difficile in wild rodents and insectivores in the Netherlands.

With wild rodents and insectivores being present around humans and their living, working and food production environments, it is important to gain knowledge of the zoonotic pathogens present in these animals. The enteropathogen Clostridium difficile, an opportunistic anaerobic bacteria, can be carried by both animals and humans, and is distributed globally. It is known that there is genetic overlap between human and animal sources of C. difficile. In this study, the aim was to assess the presence of C. difficile in rodents and insectivores trapped on and around pig and cattle farms in the Netherlands. In total 347 rodents and insectivores (10 different species) were trapped and 39·2% tested positive for presence of C. difficile. For all positive samples the ribotype (RT) was determined, and in total there were 13 different RTs found (in descending order of frequency: 057, 010, 029, 005, 073, 078, 015, 035, 454, 014, 058, 062, 087). Six of the RTs isolated from rodents and insectivores are known to be associated with human C. difficile infection; RT005, RT010, RT014, RT015, RT078 and RT087. The presence of rodents and insectivores in and around food production buildings (e.g. farms) could contribute to the spread of C. difficile in the human environment. In order to enable on-farm management for pathogen control, it is essential to comprehend the role of wild rodents and insectivores that could potentially affect the ecology of disease agents on farms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that rodents and insectivores in and around food production buildings (e.g. farms) can carry Clostridium difficile ribotypes associated with human C. difficile infection (CDI). C. difficile spores in rodent and insectivore droppings are able to survive in the environment for prolonged periods, leading to host-to-host exposure and transmission. Therefore we can state that rodent and insectivore presence on farms is a risk for zoonotic pathogen transmission of C. difficile.

Wild mice in and around the city of Utrecht, the Netherlands, are carriers of Clostridium difficile but not ESBL-producing Enterobacteriaceae, Salmonella spp. or MRSA.

Mice in buildings are a hygiene hazard because they harbour several zoonoses and animal diseases. The aim of this study was to gather information on specific bacteria in house mice caught in the urban environment. Mice caught in snap traps during pest control activities were collected in and around the city of Utrecht, the Netherlands, during May-June 2014, October-November 2015 and September-November 2016. The gut contents were analysed for ESBL/AmpC-producing Enterobacteriaceae, Salmonella spp., and Clostridium difficile and the buccal cavities were swabbed for methicillin-resistant S. aureus (MRSA). In total, 109 house mice (Mus musculus) and 22 wood mice (Apodemus sylvaticus) were examined. One mouse was found positive for Enterobacter spp. Salmonella spp. and MRSA were not found. Of n = 80 mice, 35·0% carried C. difficile (ribotypes in descending order of frequency: 014/020, 258, 002, 005, 013, 056, 081 and two unknown ribotypes). In conclusion, mouse droppings are a hazard for transmission of C. difficile to humans and their environment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that mice in buildings can carry Clostridium difficile ribotypes that are associated with clinical disease in humans. Whether the mice are the source or whether they picked up these bacteria from the human environment has not been investigated. Either way, mouse droppings in the indoor environment are a hazard for transmission of C. difficile to humans.

An outbreak of Clostridium difficile infections due to new PCR ribotype 826: epidemiologic and microbiologic analyses.

OBJECTIVES: To investigate an unusual outbreak of five patients with a total of eight episodes of a Clostridium difficile infection on a gastrointestinal surgical ward of a Dutch tertiary-care, university-affiliated hospital. METHODS: Clinical case investigations and laboratory analyses were performed. Laboratory analyses included PCR ribotyping, multiple-locus variable-number tandem repeat analysis typing, toxin typing, antimicrobial susceptibility testing and whole genome sequencing. RESULTS: The outbreak was associated with recurrent and severe disease in two of five patients. All episodes were due to a unique ribotype that was not recognized in the collection of an international network of reference laboratories and was assigned PCR ribotype 826. PCR ribotype 826 is a toxin A-, toxin B- and binary toxin-positive ribotype which according to molecular typing belongs to clade 5 and resembles the so-called hypervirulent ribotype 078. The presence of a clonal outbreak was confirmed by whole genome sequencing, yet the source of this newly identified ribotype remained unclear. CONCLUSIONS: This newly identified C. difficile PCR ribotype 826 is part of clade 5 and might also have increased virulence. The recognition of this outbreak highlights the need for ongoing C. difficile infection surveillance to monitor new circulating ribotypes with assumed increased virulence.

Zoonotic Transfer of Clostridium difficile Harboring Antimicrobial Resistance between Farm Animals and Humans.

The emergence of Clostridium difficile as a significant human diarrheal pathogen is associated with the production of highly transmissible spores and the acquisition of antimicrobial resistance genes (ARGs) and virulence factors. Unlike the hospital-associated C. difficile RT027 lineage, the community-associated C. difficile RT078 lineage is isolated from both humans and farm animals; however, the geographical population structure and transmission networks remain unknown. Here, we applied whole-genome phylogenetic analysis of 248 C. difficile RT078 strains from 22 countries. Our results demonstrate limited geographical clustering for C. difficile RT078 and extensive coclustering of human and animal strains, thereby revealing a highly linked intercontinental transmission network between humans and animals. Comparative whole-genome analysis reveals indistinguishable accessory genomes between human and animal strains and a variety of antimicrobial resistance genes in the pangenome of C. difficile RT078. Thus, bidirectional spread of C. difficile RT078 between farm animals and humans may represent an unappreciated route disseminating antimicrobial resistance genes between humans and animals. These results highlight the importance of the "One Health" concept to monitor infectious disease emergence and the dissemination of antimicrobial resistance genes.

Molecular typing and antimicrobial susceptibility testing to six antimicrobials of Clostridium difficile isolates from three Czech hospitals in Eastern Bohemia in 2011-2012.

In 2011-2012, a survey was performed in three regional hospitals in the Czech Republic to determine the incidence of Clostridium difficile infections (CDIs) and to characterize bacterial isolates. C. difficile isolates were characterized by PCR ribotyping, toxin genes detection, multiple-locus variable-number tandem-repeat analysis (MLVA), and antimicrobial susceptibility testing to fidaxomicin, vancomycin, metronidazole, clindamycin, LFF571, and moxifloxacin using agar dilution method. The incidence of CDI in three studied hospitals was 145, 146, and 24 cases per 100,000 inhabitants in 2011 and 177, 258, and 67 cases per 100,000 inhabitants in 2012. A total of 64 isolates of C. difficile was available for molecular typing and antimicrobial susceptibility testing. 60.9% of the isolates were classified as ribotype 176. All 41 isolates of ribotypes 176 and 078 were positive for the presence of binary toxin genes. Ribotype 176 also carried 18-bp deletion in the regulatory gene tcdC. Tested isolates of C. difficile were fully susceptible to vancomycin and metronidazole, whereas 65.1% of the isolates were resistant to moxifloxacin. MLVA results indicated that isolates from three different hospitals were genetically related, suggesting transmission between healthcare facilities.

Faecal shedding of antimicrobial-resistant Clostridium difficile strains by dogs.

OBJECTIVES: To longitudinally assess the shedding of antimicrobial resistant Clostridium difficile strains by clinically healthy dogs raised at breeding facilities. METHODS: 18 puppies from three different litters (#1, 2 and 3) were sampled weekly from parturition to day 20-55 postpartum. Faecal samples from the mothers of litters #2 and 3 were also available for analysis. Bacterial isolates were ribotyped, tested for in vitro antimicrobial susceptibility and further characterised. RESULTS: C. difficile was recovered from all sampled animals of litters #1 and 2, and a third of puppies from litter #3, but marked differences in C. difficile recovery were detected in different age groups (0-100%). Recovered PCR ribotypes included 056 (22 isolates), 010 (6 isolates), 078 and 213 (2 isolates each), and 009 and 020 (1 isolate each). Different ribotypes were shed by four individual animals. Regardless of their origin and ribotype, all isolates demonstrated full resistance to levofloxacin. Additionally, all but one isolate (belonging to ribotype 078) were resistant to ertapenem, and all ribotype 010 isolates displayed high-level resistance to clindamycin, clarithromycin and erythromycin. A single ribotype 078 isolate showed metronidazole heteroresistance. CLINICAL SIGNIFICANCE: Healthy dogs can shed antimicrobial-resistant C. difficile strains.

Increased incidence of Clostridium difficile PCR ribotype 027 in Hesse, Germany, 2011 to 2013.

After the first outbreak of Clostridium difficile PCR ribotype (RT) 027 in Germany in 2007, no further outbreaks were reported until the recent re-emergence of RT 027 in Hesse, a federal state with 6 million inhabitants located in south-west Germany. We undertook a survey to determine the prevalence of RT 027 and other strains in a prospective study. From January 2011 to July 2013, we analysed 291 specimens from patients diagnosed with C. difficile infection (CDI) in 40 healthcare facilities in Hesse. The mean incidence of CDI in hospitals including at least 10 patients in the survey was 9.9 per 10,000 patient days (range 4.8-22.8) in November 2012. We obtained 214 toxigenic C. difficile isolates. RT 001 was the most prevalent (31.8%). RT 027, the second most common type (26.6%), was prevalent in all hospitals (n=14) from which at least seven isolates were available for typing, but its frequency varied considerably (range: 9.1­70%). The annual frequency of RT 027 increased from 21.4% in 2011 to 30.0% in 2013 (p=0.04). Our study indicates that infections with C. difficile RT 027 are now prevalent in Hesse. It underscores the need for surveillance programmes to analyse the molecular epidemiology of C. difficile.

Emergence of Clostridium difficile infection in tuberculosis patients due to a highly rifampicin-resistant PCR ribotype 046 clone in Poland.

Clostridium difficile infection (CDI) is a major cause of nosocomial diarrhea. CDI is known to develop after antibiotic administration, but anti-tuberculosis agents have rarely been implicated. We documented an outbreak caused by a highly rifampicin-resistant C. difficile strain of polymerase chain reaction (PCR) ribotype 046 in patients with active tuberculosis.

Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates.

The presence and characteristics of Clostridium difficile were investigated in 839 faecal samples from seven different animal species in the Netherlands. The number of positive samples ranged from 3.4% (cattle) to 25.0% (dogs). Twenty-two different PCR ribotypes were identified. Among 96 isolates, 53% harboured toxin genes. All C. difficile isolates from pigs, cattle and poultry were toxinogenic, whereas the majority of isolates from pet animals consisted of non-toxinogenic PCR ribotypes 010 and 039. Ribotype 012 was most prevalent in cattle and ribotype 078 in pigs. No predominant ribotypes were present in horse and poultry samples. Overall, PCR ribotypes 012, 014 and 078 were the most frequently recovered toxinogenic ribotypes from animal samples. Comparison with human isolates from the Dutch Reference Laboratory for C. difficile at Leiden University Medical Centre (LUMC) showed that these types were also recovered from human hospitalized patients in 2009/2010, encompassing 0.8%, 11.4% and 9.8% of all isolates, respectively. Application of multiple-locus variable-number tandem-repeat analysis indicated a genotypic relation of animal and human ribotype 078 strains, but a clear genotypic distinction for ribotypes 012 and 014. We conclude that toxinogenic C. difficile PCR ribotypes found in animals correspond to PCR ribotypes associated with human disease in hospitalized patients in the Netherlands. Contrary to PCR ribotype 078, significant genetic differences were observed between animal and human PCR ribotype 012 and 014 isolates.

Type-specific risk factors and outcome in an outbreak with 2 different Clostridium difficile types simultaneously in 1 hospital.

BACKGROUND: Clostridium difficile infection (CDI) due to polymerase chain reaction (PCR) ribotype 027 (type 027) has been described worldwide. In some countries, an increase was reported of toxin A-negative PCR ribotype 017 (type 017). We encountered an outbreak due to these 2 types occurring simultaneously in a 980-bed teaching hospital in the Netherlands. METHODS: In a case-control study from May 2005 through January 2007, we investigated general and type-specific risk factors as well as outcome parameters for CDI due to type 027 or 017. Clonal dissemination was investigated by multilocus variable number of tandem repeat analysis (MLVA). RESULTS: We identified 168 CDI patients: 57 (34%) with type 017, 46 (27%) with type 027, and 65 (39%) with 1 of 36 different other types. As controls, we included 77 non-CDI diarrheal patients and 162 patients without diarrhea. Risk factors for CDI were nasogastric intubation, recent hospitalization, and use of cephalosporins and clindamycin. Type-specific risk factors were older age for both types 017 and 027, use of clindamycin and immunosuppressive agents for type 017, and use of fluoroquinolones for type 027. At day 30 of follow-up, the overall mortality among patients with types 017, 027, other types, non-CDI diarrheal patients, and nondiarrheal patients was 23%, 26%, 3%, 2%, and 6%, respectively. MLVA showed persistent clonal dissemination of types 017 and 027, despite appropriate infection control measures. CONCLUSIONS: Patients with CDI have type-specific risk factors and mortality rates, with prolonged clonal spread of type 027 or 017.

Isolation of the first three cases of Clostridium difficile polymerase chain reaction ribotype 027 in Singapore.

INTRODUCTION: The incidence of Clostridium (C.) difficile infection (CDI) was on the rise from 2001 to 2006 in Singapore. Recent unpublished data suggests that its incidence had remained stable or decreased in most local public hospitals between 2006 and 2010. It is, however, not known if the polymerase chain reaction (PCR) ribotype 027 strains have been circulating, although reports suggest that this strain is emerging in Asia, with the first cases reported from Japan in 2007, as well as in Hong Kong and Australia in 2009. We initiated a culture-based surveillance to detect this epidemic strain in Singapore. METHODS: From September 2008 to December 2009, all non-duplicate toxin-positive stool samples from the three largest public hospitals in Singapore were collected for culture and further analysis. RESULTS: Out of the 366 samples collected, 272 viable isolates were cultured. Of these, 240 tested toxin-positive and ten tested positive for the binary toxin gene; 35 different PCR ribotypes were found. Three isolates that tested positive for binary toxin contained the same PCR ribotyping pattern as the C. difficile 027 control strain. All three had the 18-bp deletion and single nucleotide tcdC deletion at position 117. Susceptibility testing was performed, demonstrating susceptibility to erythromycin and moxifloxacin. CONCLUSION: We report the first three isolates of C. difficile 027 from Singapore. However, their susceptibility patterns are more consistent with the historical 027 strains. Rising CDI incidence may not be associated with the emergence of the epidemic 027 strain at this time.

Clostridium difficile infection in an endemic setting in the Netherlands.

The purpose of this investigation was to study risk factors for Clostridium difficile infection (CDI) in an endemic setting. In a 34-month prospective case-control study, we compared the risk factors and clinical characteristics of all consecutively diagnosed hospitalised CDI patients (n = 93) with those of patients without diarrhoea (n = 76) and patients with non-CDI diarrhoea (n = 64). The incidence of CDI was 17.5 per 10,000 hospital admissions. C. difficile polymerase chain reaction (PCR) ribotype 014 was the most frequently found type (15.9%), followed by types 078 (12.7%) and 015 (7.9%). Independent risk factors for endemic CDI were the use of second-generation cephalosporins, previous hospital admission and previous stay at the intensive care unit (ICU). The use of third-generation cephalosporins was a risk factor for diarrhoea in general. We found no association of CDI with the use of fluoroquinolones or proton pump inhibitors (PPIs). The overall 30-day mortality among CDI patients, patients without diarrhoea and patients with non-CDI diarrhoea was 7.5%, 0% and 1.6%, respectively. In this endemic setting, risk factors for CDI differed from those in outbreak situations. Some risk factors that have been ascribed to CDI earlier were, in this study, not specific for CDI, but for diarrhoea in general. The 30-day mortality among CDI patients was relatively high.

Acquisition of Clostridium difficile by piglets.

Clostridium difficile is recognized as an important cause of nosocomial diarrhoea in humans especially in association with administration of antibiotics. In pigs, C. difficile can cause neonatal enteritis and can be isolated from faeces from both diseased and healthy animals. The presented prospective study describes how soon C. difficile can be isolated from newborn piglets after normal parturition and how C. difficile spreads within a pig farm. Six sows, their farrowing crates and their litters at one farm were sampled until C. difficile was found in all piglets. Within 48 h after birth, all 71 piglets became positive for C. difficile (two piglets were already positive within 1h post partum), all sows became positive within 113 h after parturition and the farrowing crates were found intermittently positive. C. difficile could also be detected in air samples and in samples of teats of the sows. All isolates belonged to PCR ribotype 078. Twenty-one C. difficile ribotype 078 isolates, found at the farm, were further analyzed by MLVA (multiple-locus variable-number tandem repeat analysis) and belonged to one clonal complex, except one isolate. To be sure that piglets were not born already infected with C. difficile ribotype 078, 38 caesarean derived piglets were sampled immediately after surgery. All piglets tested negative at delivery and stayed negative for C. difficile ribotype 078 during the 21 days in which they were kept in sterile incubators. This study shows that C. difficile ribotype 078 spreads easily between sows, piglets and the environment. Vertical transmission of C. difficile ribotype 078 was not found and is very unlikely to occur.

Relatedness of human and animal Clostridium difficile PCR ribotype 078 isolates determined on the basis of multilocus variable-number tandem-repeat analysis and tetracycline resistance.

Totals of 102 and 56 Clostridium difficile type 078 strains of human and porcine origins, respectively, from four European countries were investigated by an optimized multilocus variable-number tandem-repeat analysis (MLVA) and for tetracycline susceptibility. Eighty-five percent of all isolates were genetically related, irrespective of human or porcine origin. Human strains were significantly more resistant to tetracycline than porcine strains. All tetracycline-resistant strains contained the Tn916-like transposon harboring the tet(M) gene. We conclude that strains from human and porcine origins are genetically related, irrespective of the country of origin. This may reflect a lack of diversity and/or common source.

Application of multiple-locus variable-number tandem-repeat analysis to determine clonal spread of toxin A-negative Clostridium difficile in a general hospital in Buenos Aires, Argentina.

Isolates from patients with Clostridium difficile infection (CDI) usually produce both toxin A (TcdA) and toxin B (TcdB), but an increasing number of reports from Europe and Asia mention infections with TcdA-negative, TcdB-positive (A-/B+) strains, usually characterized as PCR ribotype 017 (type 017). Incidence rates of CDI per 10 000 admissions in a 200-bed Argentinean general hospital were 37, 84, 67, 43, 48 and 42 for the years 2000 to 2005, respectively. The annual percentages of type 017 CDI were 7.7%, 64.6%, 91.4%, 92.0%, 75.0% and 86.4%, respectively. Comparison of 112 017-CDI patients with 41 non-017-CDI patients revealed that 017-CDI patients were more often male (68.8% vs. 46.3%; odds ratio 2.55, 95% confidence interval 1.23-5.50). All type 017 strains tested belonged to toxinotype VIII and had a 1.8-kb deletion in tcdA. In addition, 90% of tested type 017 isolates had high-level resistance to clindamycin and erythromycin, determined by the presence of the ermB gene. Multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to 56 Argentinean isolates and 15 isolates from seven other countries. Country-specific clonal complexes were found in each country. Among 56 Argentinean isolates, four clonal complexes were recognized, accounting for 61% of all isolates. These clonal complexes did not show correlation over time, but seemed to be restricted to specific wards, mainly internal medicine and pulmonology wards. A total of 56% of recurrent infections were caused by a different isolate, despite identification of an identical PCR-ribotype. We conclude that C. difficile type 017 gradually replaced other circulating PCR ribotypes and that MLVA provides detailed insight into nosocomial spread.