To survive in the host, pathogenic bacteria need to be able to react to the unfavorable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more.... Show moreTo survive in the host, pathogenic bacteria need to be able to react to the unfavorable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions. Show less
Aim: The bacterial microbiota is well-recognized for its role in Clostridioides difficile colonization and infection, while fungi and yeasts remain understudied. The aim of this study was to... Show moreAim: The bacterial microbiota is well-recognized for its role in Clostridioides difficile colonization and infection, while fungi and yeasts remain understudied. The aim of this study was to analyze the predictive value of the mycobiota and its interactions with the bacterial microbiota in light of C. difficile colonization and infection. Methods: The mycobiota was profiled by ITS2 sequencing of fecal DNA from C. difficile infection (CDI) patients (n = 29), asymptomatically C. difficile colonization (CDC) patients (n = 38), and hospitalized controls with C. difficile negative stool culture (controls; n = 38). Previously published 16S rRNA gene sequencing data of the same cohort were used additionally for machine learning and fungal-bacterial network analysis. Results: CDI patients were characterized by a significantly higher abundance of Candida spp. (MD 0.270 ± 0.089, P = 0.002) and Candida albicans (MD 0.165 ± 0.082, P = 0.023) compared to controls. Additionally, they were deprived of Aspergillus spp. (MD -0.067 ± 0.026, P = 0.000) and Penicillium spp. (MD -0.118 ± 0.043, P = 0.000) compared to CDC patients. Network analysis revealed a positive association between several fungi and bacteria in CDI and CDC, although the analysis did not reveal a direct association between Clostridioides spp. and fungi. Furthermore, the microbiota machine learning model outperformed the models based on the mycobiota and the joint microbiota-mycobiota model. The microbiota classifier successfully distinguished CDI from CDC [Area Under the Receiver Operating Characteristic (AUROC) = 0.884] and CDI from controls (AUROC = 0.905). Blautia and Bifidobacterium were marker genera associated with CDC patients and controls. Conclusion: The gut mycobiota differs between CDI, CDC, and controls and may affect Clostridioides spp. through indirect interactions. The mycobiota data alone could not successfully discriminate CDC from controls or CDI patients and did not have additional predictive value to the bacterial microbiota data. The identification of bacterial marker genera associated with CDC and controls warrants further investigation. Show less
This thesis is divided into three parts. The first part of this thesis describes the epidemiology of infections with Clostridioides difficile, meticillin-resistant Staphylococcus aureus and... Show moreThis thesis is divided into three parts. The first part of this thesis describes the epidemiology of infections with Clostridioides difficile, meticillin-resistant Staphylococcus aureus and colistin-resistant Enterobacterales. An important treatment strategy for recurrent C. difficile infections is restoring the disturbed gut microbiota by faecal microbiota transplantation (FMT). The second part discusses the risk of transmission of pathogenic and/or multidrug-resistant bacteria via FMT and procedures to prevent this. Apart from recurrent C. difficile infections, several new potential indications of FMT are being explored. In the third part of this thesis, FMT is explored as a potential new treatment strategy for several neurological disorders, with a main focus on Parkinson’s disease. Show less
Clostridioides difficile infections (CDI) have a high morbidity and mortality rate and have always been considered a nosocomial disease. Nonetheless, the number of cases of community-acquired CDI... Show moreClostridioides difficile infections (CDI) have a high morbidity and mortality rate and have always been considered a nosocomial disease. Nonetheless, the number of cases of community-acquired CDI is increasing, and new evidence suggests additional C. difficile reservoirs exist. Pathogenic C. difficile strains have been found in livestock, domestic animals, and meat, so a zoonotic transmission has been proposed. Objective: The goal of this study was to isolate C. difficile strains in dogs at a veterinary clinic in Rio de Janeiro, Brazil, and characterize clinical and pathological findings associated with lower gastrointestinal tract disorders. Methods: Fifty stool samples and biopsy fragments from dogs were obtained and cultured in the CDBA selective medium. All suggestive C. difficile colonies were confirmed by MALDI-TOF MS and PCR (tpi gene). Vancomycin, metronidazole, moxifloxacin, erythromycin, and rifampicin were tested for antibiotic susceptibility. Biofilm, motility assays, and a PCR for the toxins (tcdA, tcdB, and cdtB), as well as ribotyping, were also performed. Results: Blood samples and colonic biopsy fragments were examined in C. difficile positive dogs. Ten animals (20%) tested positive for C. difficile by using stool samples, but not from biopsy fragments. Most C. difficile strains were toxigenic: six were A+B+ belonging to RT106; two were A+B+ belonging to RT014/020; and two were AB- belonging to RT010. All strains were biofilm producers. In the motility test, 40% of strains were as motile as the positive control, CD630 (RT012). In the disc diffusion test, two strains (RT010) were resistant to erythromycin and metronidazole; and another to metronidazole (RT014/020). In terms of C. difficile clinicopathological correlations, no statistically significant morphological changes, such as pseudomembranous and "volcano" lesions, were observed. Regarding hematological data, dogs positive for C. difficile had leucopenia (p = 0.02) and lymphopenia (p = 0.03). There was a significant correlation between senility and the presence of C. difficile in the dogs studied (p = 0,02). Conclusions: Although C. difficile has not been linked to canine diarrheal disorders, it appears to be more common in dogs with intestinal dysfunctions. The isolation of ribotypes frequently involved in human CDI outbreaks around the world supports the theory of C. difficile zoonotic transmission. Show less
Clostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut... Show moreClostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut microbiota and increases the risk of C. difficile infection (CDI), antibiotics are also the primary treatment for CDI. However, only a few antibiotics, including vancomycin, fidaxomicin, and rifaximin, are effective against CDI, and resistance to these antibiotics has emerged recently. In this study, we report the identification of two RT027 C. difficile clinical isolates (TGH35 and TGH64) obtained from symptomatic CDI-diagnosed patients in Tampa, Florida in 2016. These two strains showed an elevated minimum inhibitory concentration (MIC) of vancomycin (MIC = 4 mu g/mL, compared to the EUCAST breakpoint of 2 mu g/mL) and contained a vanR(Cd) 343A>G mutation resulting in a Thr115Ala substitution in the VanR(Cd) response regulator. This mutation was absent in the vancomycin-sensitive control epidemic strain RT027/R20291. TGH64 was also resistant to rifaximin (MIC >= 128 mu g/mL) and carried the previously reported Arg505Lys and Ile548Met mutations in RpoB. Furthermore, we report on the antimicrobial resistance (AMR) and genomic characterization of additional C. difficile isolates, including RT106/TGH120, RT017/TGH33, and RT017/TGH51, obtained from the same patient sample cohort representing the highly prevalent and regionally distributed C. difficile ribotypes worldwide. Considering that the VanR(Cd) Thr115Ala mutation was also independently reported in seven C. difficile clinical isolates from Texas and Israel in 2019, we recommend epidemiological surveillance to better understand the impact of this mutation on vancomycin resistance.IMPORTANCE The perpetually evolving antimicrobial resistance (AMR) of C. difficile is an important contributor to its epidemiology and is a grave concern to global public health. This exacerbates the challenge of treating the infections caused by this multidrug-resistant causative organism of potentially life-threatening diarrhea. Further, the novel resistance-determining factors can be transferred between different strains and species of bacteria and cause the spread of AMR in clinical, environmental, and community settings. In this study, we have identified a mutation (vanR(Cd) 343A>G) that causes a Thr115Ala substitution and is linked to an increased MIC of vancomycin in clinical isolates of C. difficile obtained from Florida in 2016. Understanding the mechanisms of AMR, especially those of newly evolving strains, is essential to effectively guide antibiotic stewardship policies to combat antibiotic resistance as well as to discover novel therapeutic targets. Show less
Clostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut... Show moreClostridioides difficile, the primary cause of nosocomial antibiotic-associated diarrhea, has a complex relationship with antibiotics. While the use of broad-spectrum antibiotics disrupts the gut microbiota and increases the risk of C. difficile infection (CDI), antibiotics are also the primary treatment for CDI. However, only a few antibiotics, including vancomycin, fidaxomicin, and rifaximin, are effective against CDI, and resistance to these antibiotics has emerged recently. In this study, we report the identification of two RT027 C. difficile clinical isolates (TGH35 and TGH64) obtained from symptomatic CDI-diagnosed patients in Tampa, Florida in 2016. These two strains showed an elevated minimum inhibitory concentration (MIC) of vancomycin (MIC = 4 μg/mL, compared to the EUCAST breakpoint of 2 μg/mL) and contained a vanRCd 343A>G mutation resulting in a Thr115Ala substitution in the VanRCd response regulator. This mutation was absent in the vancomycin-sensitive control epidemic strain RT027/R20291. TGH64 was also resistant to rifaximin (MIC ≥ 128 μg/mL) and carried the previously reported Arg505Lys and Ile548Met mutations in RpoB. Furthermore, we report on the antimicrobial resistance (AMR) and genomic characterization of additional C. difficile isolates, including RT106/TGH120, RT017/TGH33, and RT017/TGH51, obtained from the same patient sample cohort representing the highly prevalent and regionally distributed C. difficile ribotypes worldwide. Considering that the VanRCd Thr115Ala mutation was also independently reported in seven C. difficile clinical isolates from Texas and Israel in 2019, we recommend epidemiological surveillance to better understand the impact of this mutation on vancomycin resistance. Show less
Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human... Show moreClostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 10(7)) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log(10)-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI. Show less
Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenicC. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human... Show moreClostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenicC. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrentCDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventingCDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (ata dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain(210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas;bacterial populations and cytotoxin production were determined using viable counting and Vero cellcytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculatedsingly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. Inexperiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescentand failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous toCD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked othermetronidazole resistance determinants. This study showed that RT027 was unable to elicit simulatedinfection in the presence of NTCD-E4 following stimulation by four different antimicrobials. Thesedata complement animal and clinical studies in suggesting NTCD offer prophylactic potential in themanagement of human CDI. Show less
Clostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human... Show moreClostridioides difficile infection (CDI) remains a significant healthcare burden. Non-toxigenic C. difficile (NTCD) strains have shown a benefit in preventing porcine enteritis and in human recurrent CDI. In this study, we evaluated the efficacy of metronidazole-resistant NTCD-E4 in preventing CDI facilitated by a range of antimicrobials in an in vitro human gut model. NTCD-E4 spores (at a dose of 107) were instilled 7 days before a clinical ribotype (RT) 027 (at the same dose) strain (210). In separate experiments, four different antimicrobials were used to perturb gut microbiotas; bacterial populations and cytotoxin production were determined using viable counting and Vero cell cytotoxicity, respectively. RT027 and NTCD-E4 proliferated in the in vitro model when inoculated singly, with RT027 demonstrating high-level cytotoxin (3-5-log10-relative units) production. In experiments where the gut model was pre-inoculated with NTCD-E4, RT027 was remained quiescent and failed to produce cytotoxins. NTCD-E4 showed mutations in hsmA and a gene homologous to CD196-1331, previously linked to medium-dependent metronidazole resistance, but lacked other metronidazole resistance determinants. This study showed that RT027 was unable to elicit simulated infection in the presence of NTCD-E4 following stimulation by four different antimicrobials. These data complement animal and clinical studies in suggesting NTCD offer prophylactic potential in the management of human CDI. Show less
Background: Awareness and compliance with international guidelines for diagnosis and clinical management of Clostridioides difficile infection (CDI) are unknown.Aim: To compare the awareness and... Show moreBackground: Awareness and compliance with international guidelines for diagnosis and clinical management of Clostridioides difficile infection (CDI) are unknown.Aim: To compare the awareness and compliance with the recommended strategies for diagnosis and clinical management of CDI across Europe in 2018-2019.Methods: Hospital sites and their associated community practices across 12 European countries completed an online survey in 2018-2019, to report on their practices in terms of surveillance, prevention, diagnosis, and treatment of CDI. Responses were collected from 105 hospitals and 39 community general practitioners (GPs).Findings: Hospital sites of 11 countries reported participation in national surveillance schemes compared with six countries for international schemes. The European Society of Clinical Microbiology and Infectious Diseases (ESCMID)-recommended CDI testing meth-odologies were used by 82% (86/105) of hospitals, however countries reporting the highest incidence of CDI used non-recommended tests. Over 75% (80/105) of hospitals were aware of the most recent European CDI treatment guidelines at the time of this survey compared with only 26% (10/39) of surveyed GPs. However, up to 15% (16/105) of hospitals reported using the non-recommended metronidazole for recurrent CDI cases, sites in countries with lower awareness of CDI treatment guidelines. Only 37% (39/105) of hospitals adopted contact isolation precautions in case of suspected CDI.Conclusion: Good awareness of guidelines for the management of CDI was observed across the surveyed European hospital sites. However, low compliance with diagnostic testing guidelines, infection control measures for suspected CDI, and insufficient awareness of treatment guidelines continued to be reported in some countries. 2022 The Author(s). Published by Elsevier Ltd on behalf of The Healthcare Infection Society. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Show less
Background: During the COVID-19 pandemic, several factors, such as improved hand hygiene, social distancing, and restricted hospital referral, may have had an influence on the epidemiology of... Show moreBackground: During the COVID-19 pandemic, several factors, such as improved hand hygiene, social distancing, and restricted hospital referral, may have had an influence on the epidemiology of Clostridioides difficile infections (CDI). Methods: The annual CDI incidence rate of nine hospitals participating in the Dutch sentinel CI surveillance with complete data was compared between 2020 and the previous five surveillance years. Trends in characteristics of hos-pitalised CDI patients in 21-24 participating hospitals were compared between the first (March 13-May 12, 2020) or second Dutch COVID-19 wave (September 17, 2020-January 1, 2021) and the same calendar periods in 2015 through 2019. All analyses were adjusted for trend changes over time. Findings: The annual CDI incidence rate in 2020 was lower compared to previous years. During the second wave, the percentage of CDI patients with severe CDI was higher compared to earlier (25.8% in 2020 vs 17.9% in 2015-2019 (RR 1.6; 95%CI 1.1-2.3)). After adjustment for delayed C. difficile diagnostics (>= 8 days from start symptoms), the increase disappeared. Delayed C. difficile diagnostics was indeed more common during the second wave (RR 1.7; 95%CI 1.1-2.6), but only for community-onset CDI (CO-CDI). Interpretation: This study shows that a higher percentage of severe CDI cases was observed during the second COVID-19 wave. This may partially be caused by delayed diagnostics, potentially due to decreased visits to a physi-cian or restricted hospital referral for CO-CDI patients. Funding Dutch ministry of Health. Copyright (C) 2022 The Authors. Published by Elsevier Ltd. Show less
Aptekorz, M.; Sacha, K.; Gofron, Z.; Kabala, M.; Harmanus, C.; Kuijper, E.; Martirosian, G. 2022
Clostridioides difficile is an important health care-associated pathogen. The aim of this study was to analyze the antibiotic susceptibility of C. difficile isolates from feces of patients from 13... Show moreClostridioides difficile is an important health care-associated pathogen. The aim of this study was to analyze the antibiotic susceptibility of C. difficile isolates from feces of patients from 13 hospitals in Silesia, Poland. The incidence of CDI per 100.000 people in Silesia in 2018-2019 was higher than the average in Poland (39.3-38.7 vs. 30.2-29.5, respectively). The incidence doubled from 26.4 in 2020 to 55.1 in 2021. Two hundred and thirty stool samples tested positive for GDH (glutamate dehydrogenase) and toxins were cultured anaerobically for C. difficile. The isolates were characterized, typed, and tested for susceptibility to 11 antibiotics by E-test (EUCAST, 2021). The genes of toxins A/B and binary were detected by mPCR. Of 215 isolates, 166 (77.2%) were classified as RT 027 and 6 (2.8%) as related RT 176. Resistance to ciprofloxacin (96.7%), moxifloxacin (79.1%), imipenem (78.1%), penicillin (67%), and rifampicin (40.5%) was found. The ermB gene was detected in 79 (36.7%) strains. Multidrug resistance (MDR) was confirmed in 50 (23.3%) strains of RT 027 (94%). We concluded that a high prevalence of MDR among hypervirulent RT 027/176 C. difficile was found in the Silesian region of Poland, emphasizing the need to enhance regional infection control on CDI and antibiotic stewardships. Show less
Kachrimanidou, M.; Metallidis, S.; Tsachouridou, O.; Harmanus, C.; Lola, V.; Protonotariou, E.; ... ; Kuijper, E. 2022
Objectives: The epidemiology of Clostridioides difficile infection (CDI) has undergone many changes since the beginning of this century and continues to evolve based on recent studies. Here, we... Show moreObjectives: The epidemiology of Clostridioides difficile infection (CDI) has undergone many changes since the beginning of this century and continues to evolve based on recent studies. Here, we performed a molecular analysis of C. difficile isolates in northern Greece across 10 health-care facilities, spanning from 2016 to 2019. Methods: 221 C. difficile isolates were cultured from stool samples of hospitalized patients with diarrhea and screened by PCR for the presence of the toxin A (tcdA), toxin B (tcdB), the binary toxin (cdtA and cdtB) genes and the regulating gene of tcdC. PCR ribotyping of the cultured isolates was performed by a standardized protocol for capillary gel-based PCR ribotyping and an international database with well documented reference strains. Results: Thirty-five different PCR ribotypes were identified. The most common RTs identified were: 181 (36%, 80/221), 017 (10%, 21/221), 126 (9%, 19/221), 078 (4%, 9/221) and 012 (4%, 8/221). Notably, the predominant RT181, with toxin profile tcdA thorn tcdB thorn cdtA thorn cdtB thorn , was identified in seven out of ten participating hospitals. Conclusions: Multiple C. difficile ribotypes have been circulating in the northern Greece region with RTs 181 (closely related to 027), 017, 126 and 078 being predominant. (C) 2022 Elsevier Ltd. All rights reserved. Show less
Rossen, T.M. van; Ooijevaar, R.E.; Vandenbroucke-Grauls, C.M.J.E.; Dekkers, O.M.; Kuijper, E.J.; Keller, J.J.; Prehn, J. van 2022
Objectives: Clostridioides difficile infection (CDI), its subsequent recurrences (rCDIs), and severe CDI (sCDI) provide a significant burden for both patients and the healthcare system. Identifying... Show moreObjectives: Clostridioides difficile infection (CDI), its subsequent recurrences (rCDIs), and severe CDI (sCDI) provide a significant burden for both patients and the healthcare system. Identifying patients diagnosed with initial CDI who are at increased risk of developing sCDI/rCDI could lead to more cost-effective therapeutic choices. In this systematic review we aimed to identify clinical prognostic factors associated with an increased risk of developing sCDI or rCDI.Methods: PubMed, Embase, Emcare, Web of Science and COCHRANE Library databases were searched from database inception through March, 2021. The study eligibility criteria were cohort and caseecontrol studies. Participants were patients >= 18 years old diagnosed with CDI, in which clinical or laboratory factors were analysed to predict sCDI/rCDI. Risk of bias was assessed by using the Quality in Prognostic Research (QUIPS) tool and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) tool modified for prognostic studies. Study selection was performed by two independent reviewers. Overview tables of prognostic factors were constructed to assess the number of studies and the respective effect direction and statistical significance of an association.Results: 136 studies were included for final analysis. Greater age and the presence of multiple comorbidities were prognostic factors for sCDI. Identified risk factors for rCDI were greater age, healthcareassociated CDI, prior hospitalization, proton pump inhibitors (PPIs) started during or after CDI diagnosis, and previous rCDI.Conclusions: Prognostic factors for sCDI and rCDI could aid clinicians to make treatment decisions based on risk stratification. We suggest that future studies use standardized definitions for sCDI/rCDI and systematically collect and report the risk factors assessed in this review, to allow for meaningful metaanalysis of risk factors using data of high-quality trials. (C) 2021 The Author(s). Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases. Show less
Meijs, A.P.; Gijsbers, E.F.; Hengeveld, P.D.; Kuijper, E.J.; Dierikx, C.M.; Greeff, S.C. de; Duijkeren, E. van 2022
Veterinary healthcare workers are in close contact with many different animals and might be at an increased risk of acquiring Clostridioides difficile. In this cross-sectional study, we assessed... Show moreVeterinary healthcare workers are in close contact with many different animals and might be at an increased risk of acquiring Clostridioides difficile. In this cross-sectional study, we assessed the prevalence and risk factors of C. difficile carriage in Dutch veterinary healthcare workers. Participants provided a faecal sample and filled out a questionnaire covering potential risk factors for C. difficile carriage. C. difficile culture positive isolates were polymerase chain reaction (PCR) ribotyped and the presence of toxin genes tcdA, tcdB and cdtA/cdtB was determined. Eleven of 482 [2.3%; 95% confidence interval (CI) 1.3-4.0] veterinary healthcare workers were carriers of C. difficile. Three persons carried C. difficile ribotype 078 (0.6%; 95% CI 0.2-1.8). Risk factors for carriage were health/medication and hygiene related, including poor hand hygiene after patient (animal) contact, and did not include occupational contact with certain animal species. In conclusion, the prevalence of C. difficile carriage in veterinary healthcare workers was low and no indications were found that working in veterinary care is a risk for C. difficile carriage. Show less
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... Show moreClostridioides 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. Show less
Kabala, M.; Gofron, Z.; Aptekorz, M.; Burdynowski, K.; Harmanus, C.; Kuijper, E.; Martirosian, G. 2022
116 environmental samples from a 504 bed clinical hospital obtained in 2017/19 were inoculated into C diff Banana Broth (TM). Six C. difficile and 12 C. pefringens strains were isolated. Antibiotic... Show more116 environmental samples from a 504 bed clinical hospital obtained in 2017/19 were inoculated into C diff Banana Broth (TM). Six C. difficile and 12 C. pefringens strains were isolated. Antibiotic-resistant Clostridium spp. dominated in hospital environment. To determine Clostridium spp. in hospital environment suitable medium like C diff Banana Broth (TM) should be used. (C) 2021 Published by Elsevier Ltd. Show less
Clostridioides difficile is often resistant to the actions of antibiotics to treat other bacterial infections and the resulting C. difficile infection (CDI) is among the leading causes of... Show moreClostridioides difficile is often resistant to the actions of antibiotics to treat other bacterial infections and the resulting C. difficile infection (CDI) is among the leading causes of nosocomial infectious diarrhea worldwide. The primary virulence mechanism contributing to CDI is the production of toxins. Treatment failures and recurrence of CDI have urged the medical community to search for novel treatment options. Strains that do not produce toxins, so called non-toxigenic C. difficile, have been known to colonize the colon and protect the host against CDI. In this review, a comprehensive description and comparison of the immune responses to toxigenic C. difficile and non-toxigenic adherence, and colonization factors, here called non-toxin proteins, is provided. This revealed a number of similarities between the host immune responses to toxigenic C. difficile and non-toxin proteins, such as the influx of granulocytes and the type of T-cell response. Differences may reflect genuine variation between the responses to toxigenic or non-toxigenic C. difficile or gaps in the current knowledge with respect to the immune response toward non-toxigenic C. difficile. Toxin-based and non-toxin-based immunization studies have been evaluated to further explore the role of B cells and reveal that plasma cells are important in protection against CDI. Since the success of toxin-based interventions in humans to date is limited, it is vital that future research will focus on the immune responses to non-toxin proteins and in particular non-toxigenic strains. Show less
Current assays for Clostridioides difficile in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a... Show moreCurrent assays for Clostridioides difficile in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a more rapid point-of-care assay featuring a "turn-on " bioluminescent readout of a C. difficile-specific protease, PPEP-1. NanoLuc, a bright and stable luciferase, was "caged " with a PPEP-1-responsive peptide tail that inhibited luminescence. Upon proteolytic cleavage, the peptide was released and NanoLuc activity was restored, providing a visible readout. The bioluminescent sensor detected PPEP-1 concentrations as low as 10 nM. Sensor uncaging was achieved within minutes, and signal was captured using a digital camera. Importantly, the sensor was also functional at ambient temperature and compatible with fecal material, suggesting that it can be readily deployed in a variety of settings. Show less
