Difficile Ribotype Research Articles

Impact of the introduction of nucleic acid amplification testing on Clostridioides difficile detection and ribotype distribution in Wales

ObjectivesTo determine the impact of the 2018 introduction of nucleic acid amplification tests (NAATs) for C. difficile detection on the laboratory diagnosis of C. difficile infection (CDI), and the distribution of C. difficile ribotypes. MethodsA retrospective analysis of five years (2015–2019) of C. difficile diagnostic laboratory and PCR ribotyping test results. ResultsA total of 255,104 diagnostic results, from 136,353 patients were analysed: 199,794 samples where glutamate dehydrogenase (GDH) was used as the primary screen; and 55,310 where NAATs were employed. An overall decrease in frontline positivity from 2015 to 2019 (10.3% [n = 5017] to 6% [n = 3190] - p < 0.0001) was observed, despite an increase in the number of samples tested (48,778 to 52,839). NAAT positivity was lower than GDH (p < 0.0001) for the two years where it was implemented. The variance was accounted for by increased overall C. difficile isolation and reduced toxin negative strain culture from NAAT positive samples (p < 0.0001). Ribotype distribution (6546 samples) remained stable with decreasing RT27 isolation in each year except 2017 (p < 0.0001). RT78 was associated with toxin A/B EIA positivity (p < 0.0001). ConclusionsUse of NAAT for the detection of C. difficile, as part of a 2-step algorithm, has not led to an increase in CDI laboratory diagnostic test positivity. In spite of ribotype distribution being comparable for screening in toxin A/B positive samples, there is a significantly greater correlation between NAAT positivity and culture of toxigenic strains compared to GDH.

Phylogenomic analysis of Clostridioides difficile ribotype 106 strains reveals novel genetic islands and emergent phenotypes

Clostridioides difficile infection (CDI) is a major healthcare-associated diarrheal disease. Consistent with trends across the United States, C. difficile RT106 was the second-most prevalent molecular type in our surveillance in Arizona from 2015 to 2018. A representative RT106 strain displayed robust virulence and 100% lethality in the hamster model of acute CDI. We identified a unique 46 KB genomic island (GI1) in all RT106 strains sequenced to date, including those in public databases. GI1 was not found in its entirety in any other C. difficile clade, or indeed, in any other microbial genome; however, smaller segments were detected in Enterococcus faecium strains. Molecular clock analyses suggested that GI1 was horizontally acquired and sequentially assembled over time. GI1 encodes homologs of VanZ and a SrtB-anchored collagen-binding adhesin, and correspondingly, all tested RT106 strains had increased teicoplanin resistance, and a majority displayed collagen-dependent biofilm formation. Two additional genomic islands (GI2 and GI3) were also present in a subset of RT106 strains. All three islands are predicted to encode mobile genetic elements as well as virulence factors. Emergent phenotypes associated with these genetic islands may have contributed to the relatively rapid expansion of RT106 in US healthcare and community settings.

Degree of concordance of Clostridioides difficile strains in adults with community-associated C. difficile infection and infants with C. difficile colonization.

Infants asymptomatically excrete Clostridioides difficile during their first year of life, suggesting that they may represent a source of infection for adults who acquire community-associated C. difficile infection (CA-CDI). The genetic relationship of C. difficile strains from asymptomatic infants and adults with CA-CDI is not well defined. In this study, 50 infants were recruited at birth, and stool samples were collected at routine well-child visits. Adult stool samples collected during the same period and geographical area from patients who were diagnosed with CA-CDI were selected for comparison. C. difficile was cultivated and probed by PCR for toxin genes and were typed by PCR fluorescent ribotyping. Isolates from adults and infants with shared ribotypes were subjected to whole-genome sequencing (WGS). Of these 50 infants, 36 were positive for C. difficile at least once in their first year of life, with a peak incidence at 6 months. Among 180 infant stool samples, 48 were positive. Of 48 isolates from positive stools, 29 were toxigenic by polymerase chain reaction (PCR) and 8 of 48 stool samples were positive for toxin by enzyme immunoassays (EIAs). Ribotypes F106 and F014-020 were present in both colonized infants and adults with CA-CDI. WGS identified 1 adult-infant pair that differed by 5 single-nucleotide polymorphisms (SNPs). Also, 4 additional adult-infant clusters differed by ≤16 SNPs. Infants that are colonized with C. difficile share ribotypes with adults from the same geographical region with CA-CDI. Selected isolates in the 2 populations show a genetic relationship by WGS.

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.

Optimization of an Assay To Determine Colonization Resistance to Clostridioides difficile in Fecal Samples from Healthy Subjects and Those Treated with Antibiotics.

A healthy, intact gut microbiota is often resistant to colonization by gastrointestinal pathogens. During periods of dysbiosis, however, organisms such as Clostridioides difficile can thrive. We describe an optimized in vitro colonization resistance assay for C. difficile in stool (CRACS) and demonstrate the utility of this assay by assessing changes in colonization resistance following antibiotic exposure. Fecal samples were obtained from healthy volunteers (n = 6) and from healthy subjects receiving 5 days of moxifloxacin (n = 11) or no antibiotics (n = 10). Samples were separated and either not manipulated (raw) or sterilized (autoclaved or filtered) prior to inoculation with C. difficile ribotype 027 spores and anaerobic incubation for 72 h. Different methods of storing fecal samples were also investigated in order to optimize the CRACS. In healthy, raw fecal samples, incubation with spores did not lead to increased C. difficile total viable counts (TVCs) or cytotoxin detection. In contrast, increased C. difficile TVCs and cytotoxin detection occurred in sterilized healthy fecal samples or those from antibiotic-treated individuals. The CRACS was functional with fecal samples stored at either 4°C or -80°C but not with those stored with glycerol (12% or 30% [vol/vol]). Our data show that the CRACS successfully models in vitro the loss of colonization resistance and subsequent C. difficile proliferation and toxin production. The CRACS could be used as a proxy for C. difficile infection in clinical studies or to determine if an individual is at risk of developing C. difficile infection or other potential infections occurring due to a loss of colonization resistance.

Clostridium difficile in soil conditioners, mulches and garden mixes with evidence of a clonal relationship with historical food and clinical isolates.

With rates of community-associated Clostridium difficile infection (CA-CDI) increasing worldwide, potential reservoirs/sources of C. difficile in the community are being sought. Since C. difficile is found in animal manure and human biosolids, which are composted for agricultural purposes, composted products could be a source. In this study, the presence of C. difficile in composted products, and their genetic relatedness to other previously isolated strains from humans, root vegetables and the environment in Western Australia, was investigated. Overall, C. difficile was found in 22.5% (16/71) of composted products [29.7% (11/37) of soil conditioners, 16.7% (2/12) of mulches and 13.6% (3/22) of garden mixes]. Fifteen C. difficile PCR ribotypes (RTs) were identified, the most common toxigenic strains being RTs 020 and 056. Clostridium difficile RT 056 is commonly associated with CDI in humans and has also been isolated from cattle, root vegetables and the environment (veterinary clinics and lawn) in Australia. High-resolution core-genome analysis of 29 C. difficile RT 056 strains revealed clonal relationships between isolates derived from humans, vegetables, composted products and the environment. These findings provide support for an intricate transmission network between human, food and the environment, further highlighting the importance of a 'One Health' approach for managing CDI.

Burden of Clostridium difficile Infection (CDI) Across Whole Healthcare Economies and European Borders; COMBACTE-CDI Results

Background: The burden of C. difficile infection (CDI) on healthcare facilities is well recognized. However, studies focusing on inpatient settings, in addition to ascertainment bias in general, have led to a paucity of data on the true burden of CDI across whole healthcare economies. Methods: Sites testing both inpatient and community samples were recruited from 12 European countries (1 site per 3 million population). On 2 selected days, all diarrheal fecal samples (regardless of tests requested) were sent to the European Coordinating Laboratory (ECL) for C. difficile toxin testing and culture. The CDI results and tests not requested at each submitting site were compared with the ECL results to determine the number of missed CDIs. Contemporaneous C. difficile isolates from food and animal sources were collected. All isolates underwent PCR ribotyping and toxinotyping; prevalences of ribotypes among regions of Europe and reservoir settings were compared. Results: Overall, 3,163 diarrheal fecal samples were received from 119 sites. The burden of CDI varied by country (positivity rates, 0–15.8%) and by European region; the highest positivity rate in Eastern Europe was 13.1%. The testing and positivity rates in community samples were 29.6% and 1.4% vs 74.9% and 5.0% in hospital samples; 16% and 55% of samples positive for CDI at ECL were not diagnosed in hospitals and the community. The most common C. difficile ribotypes from hospital samples were 027 (11%), 181 (12%), and 014 (8%), although prevalence varied by country. The highest prevalence of toxinotype IIIb (ribotypes 027, 181, and 176) was seen in Eastern Europe (55% of all isolates), which also had the lowest testing rate. For hospital samples, the proportion of toxinotype IIIb was inversely related to the testing rate (r = −0.79) (Fig. 1). The most common ribotypes from food sources were 078 (23%) and 126 (13%) (toxinotype V), and most common ribotypes from community samples were 078 (9%) and 039 (9%). Overall, 106 different ribotypes were identified: 25 in both the hospital and community and 16 in the hospital, community, and food chain. Conclusions: The diagnosed burden of CDI varies markedly among countries in both hospital and community settings. Reduced sampling/testing in Eastern Europe is inversely related to the proportion of toxinotype IIIb strains identified, suggesting that lack of suspicion leads to underdiagnosis and outbreaks of infection. The proportion of missed CDIs in the community was ~3.5× higher than in hospitals, indicating major underrecognition in the former setting. There were marked differences in ribotypes in different reservoir settings, emphasizing the complex epidemiology of C. difficile.Funding: Proprietary organization: COMBACTE-CDI is an EU funded (Horizon2020) consortium of academic and EFPIA partners (bioMerieux, GSK, Sanofi Pasteur, Astra Zeneca, Pfizer, Da Volterra) with additional Funding: from the EFPIA partners.Disclosures: Submitter: Kerrie Davies; the work presented is funded via the EU and EFPIA (commercial) partners in a consortium.

Characterisation and Antibiotic Susceptibility Profile of Clostridioides (Clostridium) Difficile Isolated from Chicken Carcasses.

IntroductionClostridioides (Clostridium) difficile is a Gram+, anaerobic, spore-forming, rod-shaped bacterium that can produce toxins, and it is mainly because its virulence is attributed. The objective of this study was to evaluate the presence of C. difficile and hyper virulent ribotypes in chicken carcasses and the antibiotic susceptibility of isolated strains.Material and MethodsC. difficile was isolated from chicken carcasses by microbiological methods, its ribotypes were identified by means of PCR, the toxin production ability was defined by ELISA, and the susceptibility of the isolates to selected antibiotics was determined by minimum inhibitory concentration evaluator strips.ResultsThe bacterium was isolated from 69 out of 185 (37.3%) examined chicken carcass samples, and six out of the 69 (8.7%) isolates were identified as ribotype 027. All isolates were susceptible to amoxicillin-clavulanic acid (100.0%), vancomycin (97.1%), metronidazole (88.4%), and tetracycline (95.7%), whereas they were resistant to cefotaxime (97.1%) and imipenem (89.9%).ConclusionThe results of this study demonstrate the presence of toxigenic C. difficile isolates such as ribotype 027 (one of the most common causes of C. difficile infection in humans) in chicken carcasses. Although there is no case for stating that C. difficile is a food-borne pathogen, the presence of C. difficile in chicken may be considered to be a potential risk to consumers.

An outbreak of Clostridioides difficile infections due to a 027-like PCR ribotype 181 in a rehabilitation centre: Epidemiological and microbiological characteristics

Clostridioides difficile is one of the most important healthcare-associated pathogens. Recently, several new 027-like types have been found that all belong to the multilocus sequence typing (MLST) Clade 2. We report a rapidly spreading outbreak of C. difficile infections (CDI) due to a newly identified PCR ribotype (RT) 181 in a Rehabilitation Centre (RC). Genomic analysis revealed the outbreak strain, not previously identified in Greece, belonged to clade 2, sequence type (ST) 1 and had a 18bp deletion in tcdC at position 311 together with a single nucleotide deletion at position 117, similarly to RT 027. The presence of a clonal outbreak was confirmed by whole genome sequencing, yet the source of this ribotype remained unclear. The emergence and rapid spread of new C. difficile ribotypes highlights the need for ongoing C. difficile surveillance and better understanding of overall Clade 2 phylogeny.

Successful management of a Clostridioides difficile ribotype 027 outbreak with a lean intervention bundle

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.

Clostridioides difficile ribotype distribution in a large teaching hospital in Serbia

BackgroundThe global epidemic of nosocomial diarrhea caused by Clostridioides (Clostridium) difficile started in 2000, with high mortality rates and emergence of a new hypervirulent strain NAP1/BI/027. The aim of this study was to assess the presence of ribotype 027 and other C. difficile ribotypes in a Serbian University Hospital, compare the temporal variability of ribotypes 3 years apart, as well as to compare clinical, demographic and laboratory characteristics and disease outcome among patients infected with 027 and non-027 ribotype. This was a prospective observational cohort study addressing 4-month intervals during 2014/2015 and 2017/2018.ResultsRibotyping was performed in 64 non-duplicate C. difficile strains. Ribotype 027 was the most prevalent, and was detected in 53 (82.8%) patients (43/45 and 10/19 patients in 2014–2015 and 2017/2018, respectively). Other detected ribotypes were 001/072 in 4 (6.3%), 002 in 4 (6.3%), 014/020 in 2 (3.1%) and 176 in 1 (1.5%) patient. The percentage of the patients infected with ribotype 027 significantly decreased during the 3-year period, from 95.6 to 52.6% (p < 0.001). Ribotype 027 infection was associated with fluoroquinolone treatment more frequently than infection with other ribotypes [33 (62.3%) vs. 2 (18.2%), p = 0.010)]. A severe C. difficile infection was diagnosed more often in patients with the detected ribotype 027 compared to those infected with non-027 ribotypes (p = 0.006). No significant difference in the mortality and recurrence rates was found between the patients infected with ribotype 027 and those infected with other ribotypes [10/53 (18.8%) vs. 2/11 (18.2%), p = 0.708, and 10/35 (28.6%) vs. 0/2 (0%), p = 1.000, respectively].ConclusionClostridium difficile ribotype 027 was the most prevalent ribotype among patients in a large Serbian hospital, but there is a clear decreasing trend.

Microbiota in vitro modulated with polyphenols shows decreased colonization resistance against Clostridioides difficile but can neutralize cytotoxicity

While the knowledge on gut microbiota - C. difficile interactions has improved over the years, the understanding of the underlying mechanisms providing colonization resistance as well as preventative measures against the infection remain incomplete. In this study the antibiotic clindamycin and polyphenol extracts from pomegranate and blueberries were used individually and in combination to modulate fecal microbial communities in minibioreactor arrays (MBRA). Modulated communities were inoculated with C. difficile (ribotype 027). Subsequent 7-day periodical monitoring included evaluation of C. difficile growth and activity of toxins TcdA and TcdB as well as analysis of MBRA bacterial community structure (V3V4 16 S metagenomics). Polyphenols affected multiple commensal bacterial groups and showed different synergistic and antagonistic effects in combination with clindamycin. Exposure to either clindamycin or polyphenols led to the loss of colonization resistance against C. difficile. The successful growth of C. difficile was most significantly correlated with the decrease in Collinsella and Lachnospiraceae. Additionally, we demonstrated that Clostridium sporogenes decreased the activity of both C. difficile toxins TcdA and TcdB. The feature was shown to be common among distinct C. sporogenes strains and could potentially be applicable as a non-antibiotic agent for the alleviation of C. difficile infection.

Low prevalence of Clostridium difficile colonization in patients in long-term care facilities in Graz, Austria: A point-prevalence study

We aimed to determine the prevalence of asymptomatic colonization by C. difficile in stool of residents in four long-term care facilities (LTCFs) in Graz, Austria and to identify factors associated with colonization. We conducted a point-prevalence study in March 2018. Stool samples were examined by GDH enzyme immunoassay and when positive a toxin A/B-enzyme immunoassay was carried out. Additionally, all samples were tested by toxin A and B PCR and were plated manually as well as in automated fashion onto selective C. difficile agar. In 4/144 (2.8%) residents the GDH assay was positive. Each resident was colonized by a different C. difficile ribotype. C. difficile was not detected in any of the environmental samples. Significantly more colonized residents (60%) had stayed at a hospital in the 3 months previous to the study compared to 10% of non-colonized patients (p=0.01). The prevalence of colonization by toxigenic C. difficile was 2.8% in patients in LTCFs in Graz, Austria.

Biological characteristics associated with virulence in Clostridioides difficile ribotype 002 in Hong Kong

ABSTRACT Clostridioides difficile infection (CDI) is a common cause of nosocomial diarrhea and can sometimes lead to pseudo-membranous colitis and toxic megacolon. We previously reported that the PCR ribotype 002 was a common C. difficile ribotype in Hong Kong that was associated with increased mortality. In this study, we assessed in vitro bacteriological characteristics and in vivo virulence of ribotype 002 compared to other common ribotypes, including ribotypes 012, 014 and 046. We observed significantly higher toxin A (p < 0.05) and toxin B (p < 0.05) production, sporulation (p < 0.001) and germination rates (p < 0.0001) in ribotype 002 than other common ribotypes. In a murine model of C. difficile infection, ribotype 002 caused significantly more weight loss (p < 0.001) and histological damage (p < 0.001) than other common ribotypes. These findings may have contributed to the higher prevalence and mortality observed, and provided mechanistic insights that can help public surveillance and develop novel therapeutics to combat against this infection.

Ribotypes in isolates and testing algorithm of C. difficile infections in the studied sample.

Clostridium (Clostridioides) difficile is the most common pathogen of nosocomial and antibiotic-related diarrhea in health-care facilities. The aim of the analysis was to show the testing algorithm and to identify hypervirulent strains (suspected RT 027). The retrospective analysis of patient samples suspected on CDI was carried out by a two-step algorithm. Biological specimens were analysed by GDH or culture, immunoenzymatic assay on toxins A/B and selected samples also by a real-time PCR. In 1006 specimen suspected on CDI, 202 specimens were evaluated as positive in the two-step algorithm. Conflicting results (64 C. difficile isolates) were tested in a three-step algorithm by a real-time PCR and revealed 59 toxigenic and non RT 027 ribotypes. Statistically significant dependence among the independent variables, such as: diagnostic parameters and length of hospitalization (p = 0.175) and C. difficile (suspected RT027) ribotypes was not found. The results of PCR ribotyping showed ahigh prevalence of hypervirulent and toxigenic ribotypes in the studied sample. A resistance to vancomycin was found in one isolate. The PCR method contributed to the rapid laboratory diagnosis and thus treatment of high risk patients or was used as athird step in in the case of unclear results of standard diagnostic methods(Tab. 1, Fig. 4, Ref. 18). Text in PDF www.elis.sk.

PCR ribotypes of Clostridioides difficile across Texas from 2011 to 2018 including emergence of ribotype 255

ABSTRACT Clostridioides difficile infection (CDI) is the most prevalent healthcare-associated infection in the United States and carries a significant healthcare system burden. As part of an ongoing, active surveillance system of C. difficile throughout Texas, the objective of this study was to assess changes in C. difficile ribotypes of clinical isolates obtained from hospitalized patients in Texas over the past seven years. Fifty hospitals located in Texas, USA sent C. difficile positive stool specimens to a centralized laboratory for PCR ribotyping and toxin characterization between 2011 and 2018. Data collected included specimen collection date, patient age, and sex. Strain genotypes were compiled, and changes in ribotype distribution over time were assessed. Overall, 7796 samples were ribotyped from predominately female patients (58.4%) aged 62 ± 19 years. Samples were obtained from all geographic regions of Texas including Houston/Southwest region (n = 5129; 85%), Dallas/North Texas (n = 579, 9.6%), Central Texas (n = 164; 2.7%), and South Texas (n = 162; 2.6%). The 10 most common ribotypes comprised 73% of all isolates tested during the study period. The most common ribotypes were 027 (17.5%), followed by 014–020 (16.1%), 106 (11.6%), and 002 (9.1%). The prevalence of ribotypes 027, 001, and 078–126 declined significantly over time, while ribotypes 106 and 054 increased in prevalence (P < 0.001). Furthermore, the emergence of a novel ribotype 255 strain was observed. Differences in ribotype distribution were also noted based on age and geographic distribution (P < 0.001, each). This seven-year study demonstrated changing molecular epidemiology of C. difficile in Texas, including the emergence of a novel ribotype 255.

In vitro and in vivo anti-clostridial activity of newly isolated Pediococcus acidilactici SPM138 against Clostridium difficile

Clostridium difficile infection (CDI) has become a growing health concern, as evident from the increase in the mortality rate among elderly or hospitalized patients. Treatment of CDI is usually based on antibiotics (metronidazole and vancomycin), but it has some limitations, including cost and antibiotic resistance. Probiotics could offer an effective remedy to prevent CDI and could be an auxiliary agent in treatment CDI. Here, the anti-clostridial activity of a newly isolated probiotic strain, Pediococcus acidilactici SPM138 (SPM138) was evaluated. The cultivation of C. difficile (CD) with SPM138, inhibited the growth of CD and significantly reduced CD toxins level. The result of MTT assay showed that, incubation with 25% CD culture supernatant decreased the survival rate of HT-29 cells to less than 20%. However, the survival rate of these cells increased to 98% in the presence of the 25% CD + SPM138 supernatant. The mRNA expression of IL-6, IL-8 and PTGS2 in HT-29 cells decreased by more than 60% upon incubation with CD + SPM138 co-cultures as compared to the levels observed after treatment with CD supernatant only. The concentration of IL-8 also decreased by more than 60% upon treatment with CD + SPM138 co-culture supernatant. In a C. difficile PCR ribotype 027-infected mouse model, the concentration of CD toxin in stool samples of SPM138-fed mice was only 37% of that reported in C. difficile 027-infected group. These findings show that P. acidilactici SPM138 may be a promising probiotic in CDI.

Constitutive expression of the cryptic vanGCd operon promotes vancomycin resistance in Clostridioides difficile clinical isolates.

To describe, for the first time (to the best of our knowledge), the genetic mechanisms of vancomycin resistance in clinical isolates of Clostridioides difficile ribotype 027. Clinical isolates and laboratory mutants were analysed: genomically to identify resistance mutations; by transcriptional analysis of vanGCd, the vancomycin resistance operon encoding lipid II d-alanine-d-serine that is less bound by vancomycin than native lipid II d-alanine-d-alanine; by imaging of vancomycin binding to cell walls; and for changes in vancomycin bactericidal activity and autolysis. Vancomycin-resistant laboratory mutants and clinical isolates acquired mutations to the vanSR two-component system that regulates vanGCd. The substitutions impaired VanSR's function, resulting in constitutive transcription of vanGCd. Resistance was reversed by silencing vanG, encoding d-alanine-d-serine ligase in the vanGCd operon. In resistant cells, vancomycin was less bound to the cell wall septum, the site where vancomycin interacts with lipid II. Vancomycin's bactericidal activity was reduced against clinical isolates and laboratory mutants (64 and ≥1024 mg/L, respectively) compared with WT strains (4 mg/L). Truncation of the potassium transporter TrkA occurred in laboratory mutants, which were refractory to autolysis, accounting for their survival in high drug concentrations. Ribotype 027 evolved first-step resistance to vancomycin by constitutively expressing vanGCd, which is otherwise silent. Experimental evolutions and bactericidal assays show that ribotype 027 can acquire mutations to drastically enhance its tolerance to vancomycin. Thus, further epidemiological studies are warranted to examine the extent to which vancomycin resistance impacts clinical outcomes and the potential for these strains to evolve higher-level resistance, which would be devastating.

Lactotrehalose, an Analog of Trehalose, Increases Energy Metabolism Without Promoting Clostridioides difficile Infection in Mice

Trehalose is a disaccharide that might be used in the treatment of cardiometabolic diseases. However, trehalose consumption promotes the expansion of Clostridioides difficile ribotypes that metabolize trehalose via trehalose-6-phosphate hydrolase. Furthermore, brush border and renal trehalases can reduce the efficacy of trehalose by cleaving it into monosaccharides. We investigated whether a trehalase-resistant analogue of trehalose (lactotrehalose) has the same metabolic effects of trehalose without expanding C difficile. We performed studies with HEK293 and Caco2 cells, primary hepatocytes from mice, and human intestinal organoids. Glucose transporters were overexpressed in HEK293 cells, and glucose tra2nsport was quantified. Primary hepatocytes were cultured with or without trehalose or lactotrehalose, and gene expression patterns were analyzed. C57B6/J mice were given oral antibiotics and trehalose or lactotrehalose in drinking water, or only water (control), followed by gavage with the virulent C difficile ribotype 027 (CD027); fecal samples were analyzed for toxins A (ToxA) or B (ToxB) by enzyme-linked immunosorbent assay. Other mice were given trehalose or lactotrehalose in drinking water for 2 days before placement on a chow or 60% fructose diet for 10 days. Liver tissues were collected and analyzed by histologic, serum biochemical, RNA sequencing, autophagic flux, and thermogenesis analyses. We quantified portal trehalose and lactotrehalose bioavailability by gas chromatography mass spectrometry. Fecal microbiomes were analyzed by 16S ribosomal RNA sequencing and principal component analyses. Lactotrehalose and trehalose each blocked glucose transport in HEK293 cells and induced a gene expression pattern associated with fasting in primary hepatocytes. Compared with mice on the chow diet, mice on the high-fructose diet had increased circulating cholesterol, higher ratios of liver weight-to-body weight, hepatic lipid accumulation (steatosis), and liver gene expression patterns of carbohydrate-responsive de novo lipogenesis. Mice given lactotrehalose while on the high-fructose diet did not develop any of these features and had increased whole-body caloric expenditure compared with mice given trehalose or water and fed a high-fructose diet. Livers from mice given lactotrehalose had increased transcription of genes that regulate mitochondrial energy metabolism compared with liver from mice given trehalose or controls. Lactotrehalose was bioavailable in venous and portal circulation and fecal samples. Lactotrehalose reduced fecal markers of microbial branched-chain amino acid biosynthesis and increased expression of microbial genes that regulate insulin signaling. In mice given antibiotics followed by CD027, neither lactotrehalose nor trehalose increased levels of the bacteria or its toxin in stool-in fact, trehalose reduced the abundance of CD027 in stool. Lactotrehalose and trehalose reduced markers of inflammation in rectal tissue after CD027 infection. Lactotrehalose is a trehalase-resistant analogue that increases metabolic parameters, compared with trehalose, without increasing the abundance or virulence of C difficile strain CD027. Trehalase-resistant trehalose analogues might be developed as next-generation fasting-mimetics for the treatment of diabetes and nonalcoholic fatty liver disease.

Clostridioides (Clostridium) Difficile in Food-Producing Animals, Horses and Household Pets: A Comprehensive Review.

Clostridioides (Clostridium) difficile is ubiquitous in the environment and is also considered as a bacterium of great importance in diarrhea-associated disease for humans and different animal species. Food animals and household pets are frequently found positive for toxigenic C. difficile without exposing clinical signs of infection. Humans and animals share common C. difficile ribotypes (RTs) suggesting potential zoonotic transmission. However, the role of animals for the development of human infection due to C. difficile remains unclear. One major public health issue is the existence of asymptomatic animals that carry and shed the bacterium to the environment, and infect individuals or populations, directly or through the food chain. C. difficile ribotype 078 is frequently isolated from food animals and household pets as well as from their environment. Nevertheless, direct evidence for the transmission of this particular ribotype from animals to humans has never been established. This review will summarize the current available data on epidemiology, clinical presentations, risk factors and laboratory diagnosis of C. difficile infection in food animals and household pets, outline potential prevention and control strategies, and also describe the current evidence towards a zoonotic potential of C. difficile infection.