Rapid Isothermal Detection of Pathogenic Clostridioides difficile Using Recombinase Polymerase Amplification.

Nosocomial-associated diarrhea due to Clostridioides difficile infection (CDI) is diagnosed after sample precultivation by the detection of the toxins in enzyme immunoassays or via toxin gene nucleic acid amplification. Rapid and direct diagnosis is important for targeted treatment to prevent severe cases and recurrence. We developed two singleplex and a one-pot duplex fluorescent 15 min isothermal recombinase polymerase amplification (RPA) assays targeting the toxin genes A and B (tcdA and tcdB). Furthermore, we adapted the singleplex RPA to a 3D-printed microreactor device. Analytical sensitivity was determined using a DNA standard and DNA extracts of 20 C. difficile strains with different toxinotypes. Nineteen clostridial and gastrointestinal bacteria strains were used to determine analytical specificity. Adaptation of singleplex assays to duplex assays in a 50 µL volume required optimized primer and probe concentrations. A volume reduction by one-fourth (12.4 µL) was established for the 3D-printed microreactor. Mixing of RPA was confirmed as essential for optimal analytical sensitivity. Detection limits (LOD) ranging from 119 to 1411 DNA molecules detected were similar in the duplex tube format and in the singleplex 3D-printed microreactor format. The duplex RPA allows the simultaneous detection of both toxins important for the timely and reliable diagnosis of CDI. The 3D-printed reaction chamber can be developed into a microfluidic lab-on-a-chip system use at the point of care.

Non-human Clostridioides difficile Reservoirs and Sources: Animals, Food, Environment.

Clostridioides difficile is ubiquitous and is found in humans, animals and in variety of environments. The substantial overlap of ribotypes between all three main reservoirs suggests the extensive transmissions. Here we give the overview of European studies investigating farm, companion and wild animals, food and environments including water, soil, sediment, wastewater treatment plants, biogas plants, air, and households. Studies in Europe are more numerous especially in last couple of years, but are still fragmented in terms of countries, animal species, or type of environment covered. Soil seem to be the habitat of divergent unusual lineages of C. difficile. But the most important aspect of animals and environment is their role in C. difficile transmissions and their potential as a source for human infection is discussed.

SARS-CoV-2 molecular epidemiology in Slovenia, January to September 2021.

BackgroundSequencing of SARS-CoV-2 PCR-positive samples was introduced in Slovenia in January 2021. Our surveillance programme comprised three complementary schemes: (A) non-targeted sequencing of at least 10% of samples, (B) sequencing of samples positive after PCR screening for variants of concern (VOC) and (C) sequencing as per epidemiological indication.AimWe present the analysis of cumulative data of the non-targeted surveillance of SARS-CoV-2 and variant-dependent growth kinetics for the five most common variants in Slovenia for the first 9 months of 2021.MethodsSARS-CoV-2 PCR-positive samples, from January to September 2021, were selected for sequencing according to the national surveillance plan. Growth kinetics studies were done on Vero E6 cells.ResultsAltogether 15,175 genomes were sequenced and 64 variants were detected, of which three successively prevailed. Variant B.1.258.17 was detected in ca 80% of samples in January and was replaced, within 9 weeks, by the Alpha variant. The number of cases decreased substantially during the summer of 2021. However, the introduction of the Delta variant caused a fourth wave and completely outcompeted other variants. Other VOC were only detected in small numbers. Infection of Vero E6 cells showed higher replication rates for the variants Alpha and Delta, compared with B.1.258.17, B.1.258, and B.1.1.70, which dominated in Slovenia before the introduction of the Alpha and Delta variants.ConclusionInformation on SARS-CoV-2 variant diversity provided context to the epidemiological data of PCR-positive cases, contributed to control of the initial spread of known VOC and influenced epidemiological measures.

Clostridioides difficile positivity rate and PCR ribotype distribution on retail potatoes in 12 European countries, January to June 2018.

BackgroundWhile human-to-human transmission of Clostridioides difficile occurs often, other infection sources, including food, animals and environment, are under investigation.AimWe present a large study on C. difficile in a food item in Europe, encompassing 12 European countries (Austria, France, Greece, Ireland, Italy, the Netherlands, Poland, Slovakia, Spain, Sweden, Romania and the United Kingdom).MethodsPotato was selected because of availability, ease of sampling and high C. difficile positivity rates. Identical protocols for sampling and isolation were used, enabling a direct comparison of the C. difficile positivity rate.ResultsFrom C. difficile-positive potato samples (33/147; 22.4%), we obtained 504 isolates, grouped into 38 PCR ribotypes. Positivity rates per country varied (0-100%) and were at least 10% in 9/12 countries. No geographical clustering of samples with high positivity rates or in PCR ribotype distribution was observed. The most frequently detected PCR ribotypes (014/020, 078/126, 010 and 023) are also commonly reported in Europe among human clinically relevant isolates, in animal isolates and in the environment. Whole genome sequencing revealed several genetically related strain pairs (Spain/RT126, France/RT010, Austria and Sweden/RT276) and a cluster of very similar strains in RT078/126.ConclusionOur results suggest, the high potato contamination rates could have public health relevance. They indicate potatoes can serve as a vector for introducing C. difficile spores in the household environment, where the bacterium can then multiply in sensitive hosts with disrupted or unmature microbiota. Potato contamination with PCR ribotypes shared between humans, animals and soil is supportive of this view.

A point-prevalence study on community and inpatient Clostridioides difficile infections (CDI): results from Combatting Bacterial Resistance in Europe CDI (COMBACTE-CDI), July to November 2018.

BackgroundThere is a paucity of data on community-based Clostridioides difficile infection (CDI) and how these compare with inpatient CDI.AimTo compare data on the populations with CDI in hospitals vs the community across 12 European countries.MethodsFor this point-prevalence study (July-November 2018), testing sites sent residual diagnostic material on sampling days to a coordinating laboratory for CDI testing and PCR ribotyping (n = 3,163). Information on whether CDI testing was requested at the original site was used to identify undiagnosed CDI. We used medical records to identify differences between healthcare settings in patient demographics and risk factors for detection of C. difficile with or without free toxin.ResultsThe CDI positivity rate was 4.4% (country range: 0-16.2) in hospital samples, and 1.3% (country range: 0-2.2%) in community samples. The highest prevalence of toxinotype IIIb (027, 181 and 176) was seen in eastern European countries (56%; 43/77), the region with the lowest testing rate (58%; 164/281). Different predisposing risk factors were observed (use of broad-spectrum penicillins in the community (OR: 8.09 (1.9-35.6), p = 0.01); fluoroquinolones/cephalosporins in hospitals (OR: 2.2 (1.2-4.3), p = 0.01; OR: 2.0 (1.1-3.7), p = 0.02)). Half of community CDI cases were undetected because of absence of clinical suspicion, accounting for three times more undiagnosed adults in the community compared with hospitals (ca 111,000 vs 37,000 cases/year in Europe).ConclusionThese findings support recommendations for improving diagnosis in patients presenting with diarrhoea in the community, to guide good practice to limit the spread of CDI.

Low Clostridioides difficile positivity rate in wild animal shelter in Slovenia.

Here we review literature on Clostridioides difficile in captive wild animals and describe results from a single wild animal shelter in Slovenia. C. difficile was found in four out of 22 samples from animals of 15 different species (mammals n = 3; birds n = 12). Isolates were cultured only from bird samples and typed as RT 078, 002, 014 and additional unknown type. All three known ribotypes are commonly shared between humans and/or animals and environment.

Clostridioides difficile in national food surveillance, Slovenia, 2015 to 2017.

BackgroundClostridioides difficile is an important human and animal intestinal pathogen. Because of increasing indications of an association between C. difficile and food, in 2015, the Administration of the Republic of Slovenia for Food Safety, Veterinary Sector and Plant Protection (UVHVVR) included C. difficile in its national food surveillance.AimWe aim to report the results and experience with a nationwide and long-term testing of food for C. difficile as a part of a regular national food surveillance programme.MethodsRetail minced meat and meat preparations (beef, pork and poultry) were sampled within a three-year period, 2015 to 2017. Selected raw retail vegetables, leaf salads and root vegetables, and ready-to-eat salads were only sampled during 2016 and 2017. Seafood was only sampled in 2017.ResultsAltogether, 434 samples were tested, with 12 of 336 (3.6%) meat samples and 6 of 98 (6.1%) raw vegetables contaminated with C. difficile. Twelve of 18 recovered food isolates were toxigenic (toxinotypes 0, III, V, XII). The isolates belonged to 13 different PCR ribotypes, 001 being most common (5 isolates). Several food types with an increased potential of being contaminated with C. difficile were detected by surveillance.ConclusionThe three-year C. difficile testing within the national food surveillance revealed a low proportion of C. difficile-contaminated food and high genotype variability. Because the risk of C. difficile infection associated with C. difficile-contaminated food is unknown, no measures were recommended in the case of positive results.

Isolation of Clostridioides difficile from different outdoor sites in the domestic environment.

Clostridioides difficile was isolated from 12 of 25 samples collected from the gardens of five individual houses and 132 isolates belonged to 12 PCR ribotypes. Compost material had the highest positivity rate and the highest PCR ribotype diversity in comparison to soil from vegetable and flower gardens. Isolated PCR ribotypes overlap with common human and animal types but also with divergent C. difficile lineages common for the soil environment.

High Clostridium difficile contamination rates of domestic and imported potatoes compared to some other vegetables in Slovenia.

Clostridium difficile, recently reclassified to Clostridioides difficile, is among most important causes of intestinal infections in humans. Zoonotic potential and foodborne transmissions are considered to be partially involved in C. difficile spread. Here we report prevalence of C. difficile in 142 retail and 12 homegrown vegetables in Slovenia between years 2014 and 2017. The overall prevalence of C. difficile on vegetables was 18,2% (28/154). A total of 115 isolates were obtained which belonged to 25 PCR ribotypes. Ten of those were toxigenic and PCR ribotype 014/020 was the most prevalent. Most of 25 determined PCR ribotypes were previously reported in humans, animals, soil or water in Slovenia. Among tested vegetables, potatoes had the highest positivity rate (28,0% vs. 6,7% and 9,4% for ginger and leaf vegetables). Altogether 66,7% of C. difficile positive potato samples were imported from 12 different countries of three different continents. The origin of contamination could be any point between production and retail store, however, our results suggest a possibility that potatoes represent a transnational and transcontinental way of C. difficile transmissions.

MRSA diversity and the emergence of LA-MRSA in a large teaching hospital in Slovenia.

The methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of a variety of infections in hospitals and the community. One of the most prominent changes in the MRSA epidemiology is the emergence of livestock-associated MRSA (LA-MRSA) strains in the human population. The aim of this study was to follow the MRSA epidemiology in a large teaching hospital during an 8-year time period (2006-2013). Altogether 519 MRSA, cultured from screening or clinical samples, were distributed into 77 spa types, of which three (t003 and t001, associated with CC5; and t015; associated with CC45) were the most common. LA-MRSA-associated spa types (t011, t034, t108, t899; associated with CC398) started to emerge in the year 2009 and continued to be found annually at a frequency from 3.9% to 12.7% of all MRSA strains examined. Only 6 of 27 LA-MRSA strains were associated with infections.

Clostridium (Clostridioides) difficile shedding by polar bears (Ursus maritimus) in the Canadian Arctic.

Clostridium (Clostridioides) difficile has been identified in humans and a wide range of animal species, but there has been little study of remote animal populations with limited human contact. The objective of this study was to determine the prevalence and types of C. difficile in wild and captive polar bears (Ursus maritimus). Fecal samples were collected from two populations of wild polar bears in Nunavut Canada; M'Clintock Channel and Hudson Strait (Davis Strait or Foxe Basin), as well as from a facility (PBJ) in Churchill, Manitoba that temporarily houses nuisance polar bears and from captive bears in a zoological park. Enrichment culture was performed and isolates were characterized by ribotyping and toxinotyping. Clostridium difficile was isolated from 24/143 (16.8%) of samples; 18/120 (15%) wild bear samples, 4/7 (57%) from the PBJ and 2/16 (13%) samples from three zoo bears. The prevalence of C. difficile was significantly higher in bears that were housed at the PBJ vs wild bears (P = 0.0042), but there was no difference between wild bears from M'Clintock Channel (14/100, 14%) and those from Hudson Strait (4/20, 20%) (P = 0.50). Fourteen of the 24 (58%) isolates were toxigenic; 13/18 (72%) wild bear isolates, 0/4 PBJ isolate and 1/2 zoo isolates. Four toxigenic ribotypes were identified, with one that possessed tcdB and cdtA predominating. None of the toxigenic isolates were ribotypes that have been identified previously by the authors. There was no overlap in toxigenic ribotypes between the different populations. Clostridium difficile was not uncommonly identified in polar bears, with differences in type distribution amongst the different regions. The presence of strains that have not been identified in humans or domestic animals suggests that polar bears may be a natural reservoir of unique strains of this important bacterium.

Molecular epidemiology of Clostridioides (previously Clostridium) difficile isolates from a university hospital in Minas Gerais, Brazil.

The molecular epidemiology of 38 non-duplicate toxigenic Clostridioides (previously Clostridium) difficile isolates from inpatients from a hospital in Brazil during a 6-year period (2012-2017) were investigated by multilocus sequence typing (MLST) and ribotyping. These isolates were classified into 20 sequence types (ST), six (30%) of which were novel, revealing a high diversity in a single hospital. Classic hypervirulent strains ST1/RT027 and ST11/RT078 were not identified, while ST42 (almost all RT106) was the most common type, being detected in 11 (28.9%) strains. Noteworthy, six (15.8%) isolates were classified into five STs from clade 2, four of which were new ST and RT. Our study suggests that possible hypervirulent strains other than ST1/RT027 might be inadvertently circulating in Brazilian hospitals and highlights the importance of permanent surveillance on circulating strains in a national scale.

Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Genome-Wide Typing of Clostridium difficile.

Clostridium difficile, recently renamed Clostridioides difficile, is the most common cause of antibiotic-associated nosocomial gastrointestinal infections worldwide. To differentiate endogenous infections and transmission events, highly discriminatory subtyping is necessary. Today, methods based on whole-genome sequencing data are increasingly used to subtype bacterial pathogens; however, frequently a standardized methodology and typing nomenclature are missing. Here we report a core genome multilocus sequence typing (cgMLST) approach developed for C. difficile Initially, we determined the breadth of the C. difficile population based on all available MLST sequence types with Bayesian inference (BAPS). The resulting BAPS partitions were used in combination with C. difficile clade information to select representative isolates that were subsequently used to define cgMLST target genes. Finally, we evaluated the novel cgMLST scheme with genomes from 3,025 isolates. BAPS grouping (n = 6 groups) together with the clade information led to a total of 11 representative isolates that were included for cgMLST definition and resulted in 2,270 cgMLST genes that were present in all isolates. Overall, 2,184 to 2,268 cgMLST targets were detected in the genome sequences of 70 outbreak-associated and reference strains, and on average 99.3% cgMLST targets (1,116 to 2,270 targets) were present in 2,954 genomes downloaded from the NCBI database, underlining the representativeness of the cgMLST scheme. Moreover, reanalyzing different cluster scenarios with cgMLST were concordant to published single nucleotide variant analyses. In conclusion, the novel cgMLST is representative for the whole C. difficile population, is highly discriminatory in outbreak situations, and provides a unique nomenclature facilitating interlaboratory exchange.

Non-human C. difficile Reservoirs and Sources: Animals, Food, Environment.

Clostridium difficile is ubiquitous and is found in humans, animals and in variety of environments. The substantial overlap of ribotypes between all three main reservoirs suggests the extensive transmissions. Here we give the overview of European studies investigating farm, companion and wild animals, food and environments including water, soil, sediment, waste water treatment plants, biogas plants, air and households. Studies in Europe are more numerous especially in last couple of years, but are still fragmented in terms of countries, animal species or type of environment covered. Soil seem to be the habitat of divergent unusual lineages of C. difficile. But the most important aspect of animals and environment is their role in C. difficile transmissions and their potential as a source for human infection is discussed.

Clostridium difficile and Clostridioides difficile: Two validly published and correct names.

The current status of the names Clostridium difficile and Clostridioides difficile is explained in view of the current confusion about the correct name of this well-known pathogen. Both names have been validly published under the provisions of the Prokaryotic Code and both names can be used.

The incidence of Clostridioides difficile and Clostridium perfringens netF-positive strains in diarrheic dogs.

The aim of this study was to examine the incidence of Clostridioides (previously Clostridium) difficile and Clostridium perfringens in the feces of diarrheic and non-diarrheic dogs. Also, the presence of other common canine enteropathogens was examined. Toxigenic C. difficile and C. perfringens positive for the NetF-encoding gene (netF) were detected in 11 (11.9%) and seven (7.6%) diarrheic dogs, respectively. Three dogs were diagnosed simultaneously with toxigenic C. difficile and netF-positive C. perfringens. Among other enteropathogens, Giardia sp. was the most common agent detected in dogs positive for toxigenic C. difficile or netF-positive C. perfringens. The results suggest that C. difficile and C. perfringens occur more frequently as a primary cause of diarrhea.

Clinical epidemiology of Clostridium difficile infection among hospitalized patients with antibiotic-associated diarrhea in a university hospital of Brazil.

A cohort of 110 adult individuals was analyzed to compare clinical characteristics of hospitalized patients who received antibiotics and developed Clostridium difficile infection (CDI) with those who received antibiotics and did not develop the disease in a university Hospital in Brazil. CDI was diagnosed by toxigenic culture and C. difficile isolates were characterized by PCR ribotyping. Stool samples were also screened for Clostridium perfringens, methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella oxytoca. The prevalence of CDI among patients with AAD was 31.8%. C. difficile diarrhea was significantly associated with the severity of underlying comorbidities at admission (OR = 1.21; 95% CI, 1.04-1.40) and with the number of antibiotics used during hospitalization (OR = 1.43; 95% CI, 1.07-1.92). Diabetes mellitus was markedly associated with a higher risk of death in patients with AAD (OR = 6.38; 95% CI, 1.33-30.7). PCR ribotypes 014/020 and 106 (20.6% each) were the most common among the isolates. Binary toxin-encoding gene (cdtB) was detected in six samples, but previously described hypervirulent ribotypes 027 and 078 were not found. K. oxytoca and enterotoxigenic C. perfringens were not detected, while only one patient (0.9%) was positive for MRSA. Our results indicate that comorbidity severity and the number of antibiotics used during hospitalization are strong independent predictors of nosocomial C. difficile diarrhea. Diabetes was associated with a higher mortality among patients with AAD. A huge diversity of C. difficile ribotypes was observed in our study, although classical hypervirulent strains were not observed.

An Update on Clostridium difficile Toxinotyping.

Toxinotyping is a PCR-restriction fragment length polymorphism (RFLP)-based method for differentiation of Clostridium difficile strains according to the changes in the pathogenicity locus (PaLoc), a region coding for toxins A and B. Toxinotypes are a heterogenous group of strains that are important in the development of molecular diagnostic tests and vaccines and are a good basis for C. difficile phylogenetic studies. Here we describe an overview of the 34 currently known toxinotypes (I to XXXIV) and some changes in nomenclature.