Search

Clostridioides difficile is a Gram-positive, anaerobic, and spore-forming bacterial member of the human gut microbiome. The primary virulence factors of C. difficile are toxin A and toxin B. These toxins damage the cell cytoskeleton and cause various diseases, from diarrhea to severe pseudomembranous colitis. Evidence suggests that bacteriophages can regulate the expression of the pathogenicity locus (PaLoc) genes of C. difficile. We previously demonstrated that the genome of the C. difficile RT027 strain NCKUH-21 contains a prophage-like DNA sequence, which was found to be markedly similar to that of the φCD38-2 phage. In the present study, we investigated the mechanisms underlying the φNCKUH-21-mediated regulation of the pathogenicity and the PaLoc genes expression in the lysogenized C. difficile strain R20291. The carriage of φNCKUH-21 in R20291 cells substantially enhanced toxin production, bacterial motility, biofilm formation, and spore germination in vitro. Subsequent mouse studies revealed that the lysogenized R20291 strain caused a more severe infection than the wild-type strain. We screened three φNCKUH-21 genes encoding DNA-binding proteins to check their effects on PaLoc genes expression. The overexpression of NCKUH-21_03890, annotated as a transcriptional regulator (phage transcriptional regulator X, PtrX), considerably enhanced toxin production, biofilm formation, and bacterial motility of R20291. Transcriptome analysis further confirmed that the overexpression of ptrX led to the upregulation of the expression of toxin genes, flagellar genes, and csrA. In the ptrX-overexpressing R20291 strain, PtrX influenced the expression of flagellar genes and the sigma factor gene sigD, possibly through an increased flagellar phase ON configuration ratio., Competing Interests: Declaration of Interest Statement The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Background/purpose: Although metronidazole is not recommended to treat Clostridioides difficile infection (CDI) in Western countries, it was still to be recommended for the treatment of non-severe CDI among Taiwanese adults in 2020. This controversy in the clinical role of metronidazole therapy for CDI was examined in a prospective clinical study., Methods: The study was conducted from January 2015 to December 2016 in three hospitals in Taiwan. Metronidazole treatment failure (MTF) was defined as the persistence of diarrhea after six days of treatment, medication modification (shifting to oral vancomycin), or death after five days of therapy., Results: Overall, 325 patients receiving metronidazole for CDI were included. The overall MTF rate was 48.6% (158 patients). Leukocyte counts of >15,000 cells/mL in peripheral blood (odd ratio [OR] 1.81; P = 0.04) and congestive heart failure (OR 3.26; P = 0.02) were independently associated with MTF. The MTF rate for patients with leukocyte counts of ≤15,000 cells/mL and no congestive heart failure, leukocyte counts of >15,000 cells/mL and no congestive heart failure, leukocyte counts of ≤15,000 cells/mL and congestive heart failure, and leukocyte counts of >15,000 cells/mL and congestive heart failure were 44.2%, 51.8%, 73.3%, and 66.7%, respectively. Of note, patients who experienced MTF had a higher recurrence rate of CDI than those with metronidazole treatment success (13.9% vs. 6.0%, P = 0.02)., Conclusion: For Taiwanese adults with CDI, the failure rate of metronidazole therapy approached 50%, which suggests the reappraisal of the therapeutic role of metronidazole therapy, especially for patients with leukocytosis or underlying congestive heart failure., Competing Interests: Declaration of competing interest The authors have no conflicts of interest relevant to this article., (Copyright © 2022 Formosan Medical Association. Published by Elsevier B.V. All rights reserved.)

This is the first report to discover Clostridiodes difficile (C. difficile) ribotype RT126 and RT598 (both ribotypes belong to RT078-lineage) in a river water system in southern Taiwan. Fluoroquinolone resistance was also found. The connection between clinical isolates and those from the environment needs further investigation., (Copyright © 2022. Published by Elsevier B.V.)

Background: Several studies have highlighted the incidence of Clostridioides difficile infections (CDIs) in Taiwan and certain ribotypes have been related to severe clinical diseases. A study was conducted to investigate the polymerase chain reaction (PCR) ribotypes and genetic relatedness of clinical C. difficile strains collected from January 2009 to December 2015 at a hospital in northeastern Taiwan., Material and Methods: A modified two-step typing algorithm for C. difficile was used by combining a modified 8-plex and 3'-truncated tcdA screening PCR. In addition, MLVA typing was adopted for investigation of bacterial clonality and transmission., Results: Among a total of 86 strains, 24 (28%) were nontoxigenic and 62 (72%) had both tcdA and tcdB (A + B+). No tcdA-negative and tcdB-positive (A - B + ) strains were identified. Binary toxin (CDT)-producing (cdtA+/cdtB+) strains were started to be identified in 2013. The 21 (34%) A + B + clinical strains with binary toxin and tcdC deletion were identified as RT127 strains, which contained both RT078-lineage markers and fluoroquinolone (FQ)-resistant mutations (Thr82Ile in gyrA). Multiple loci variable-number tandem repeat analysis (MLVA) for phylogenetic relatedness of RT127 strains indicated that 20 of 21 strains belonged to a clonal complex that was identical to a clinical strain collected from southern Taiwan in 2011, suggestive of a clonal expansion in Taiwan., Conclusion: A two-step typing method could rapidly confirm species identification and define the toxin gene profile of C. difficile isolates. The clonal expansion of RT127 strains in Taiwan indicates monitoring and surveillance of toxigenic C. difficile isolates from human, animal, and environment are critical to develop One Health prevention strategies., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest in this work., (Copyright © 2022. Published by Elsevier B.V.)

Clostridioides difficile is one of the most common nosocomial gastrointestinal pathogens, and recurrence is a problematic issue because approximately 20-30% of patients experience at least one episode of recurrence, even after treatment with a therapeutic drug of choice for C. difficile infection (CDI), such as vancomycin. CDI recurrence has a multifactorial complex mechanism, in which gut microbiota disruption coincident with viable C. difficile spores, is considered the most important factor. The effectiveness of an anti-C. difficile antimicrobial agent against CDI cannot guarantee its inhibitory effect on C. difficile spores and vice versa. However, an antimicrobial agent, such as fidaxomicin, which has a good inhibitory effect on both C. difficile vegetative cells and spores is assumed to not only treat CDI but also prevent its recurrence. Prolonged adherence to the exosporium has been proposed as a possible mechanism of inhibiting spores, and as a result, redesigning anti-C. difficile antimicrobial agents with the ability to adhere to the exosporium may provide another pathway for the development of anti-C. difficile spore agents. For example, vancomycin lacks an inhibitory effect against C. difficile spores, but a vancomycin-loaded spore-targeting iron oxide nanoparticle that selectively binds to C. difficile spores has been developed to successfully delay spore germination. Some new antimicrobial agents in phase II clinical trials, including cadazolid and ridinilazole, have shown exceptional anti-C. difficile and spore-inhibiting effects that can be expected to not only treat CDI but also prevent its recurrence in the future., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2021. Published by Elsevier B.V.)

Background: Clostridium difficile infections (CDIs) cause significant mortality and morbidity. Critically ill patients are susceptible to CDIs and tend to have severe CDIs, and their clinical presentations are not merely diarrhea., Materials and Methods: From September 2017 to March 2018, the adults with CDIs in the ICUs were included. Fecal specimens with positive results of glutamate dehydrogenase assay were cultured for C. difficile, and toxinotyping and ribotyping for available C. difficile isolates were done. The CDI cases were categorized into the diarrheal group and ileus group. Difficult-to-treat cases with the presentations of life-threatening complications (bowel perforation or bacteremia), toxic megacolon, and refractory diarrhea, were analyzed., Results: Totally 23 cases, including 6 cases of ileus and 17 of diarrhea, were included. Overall, the incidence of CDI in the ICUs was 10.7 cases per 10,000 patient-days. The ileus group tended to have more severe presentation, shorter ICU stay, higher ICU mortality, and receive initial intravenous metronidazole therapy. Severe and fulminant CDIs accounted for 65.2% (15 cases). The ICU mortality rate was 39.1%, but only one death was directly related to CDI (4.3%). Of nine (39.1%) difficult-to-treat cases, there was only one isolate of RT611 with tcdC deletion and cdtA/cdtB from a case with toxic megacolon. No hypervirulent isolates of RT027 or 078 were detected., Conclusion: Severe CDIs in the ICU were not rare. Clinicians should be aware of abdominal symptoms and signs other than diarrhea, such as ileus, to make timely diagnosis and management of CDI., Competing Interests: Declaration of competing interest This work was supported by the grants from Ministry of Science and Technology (MOST 108-2321-B-006-004) and Ministry of Health and Welfare (MOHW 108-TDU-B-211-13303), Taiwan., (Copyright © 2020. Published by Elsevier B.V.)

Background: Among the individuals with hematological malignancy (HM) complicated with Clostridioides difficile infection (CDI), the variables associated with in-hospital mortality and recurrence of CDI were investigated., Material and Methods: Including adults with HM and those without malignancy suffering from CDI from January 2015 to December 2016 in three hospitals in Taiwan., Results: Totally 314 patients including 77 with HM and 237 patients without malignancy were included. HM patients more often had low leukocyte counts (<500 cells/mL: 28.6% vs. 2.1%) than those without malignancy and more patients without malignancy had severe CDI than patients with HM (31.6% vs. 14.3%, P = .003), according to the severity score of IDSA/SHEA. Patients with HM had a higher recurrence rate of CDI (14.3%, 11/77 vs. 7.2%, 17/237; P = .07) and longer hospital stay (47.2 ± 40.8 days vs. 33.3 ± 37.3 days; P = .006) than those without malignancy. In the multivariate analyses for those with HM and CDI, the in-hospital mortality was associated with vancomycin-resistant Enterococcus (VRE) colonization or infection (odds ratio [OR] 7.72; P = .01), and C. difficile ribotype 078 complex infection (OR 9.22; P = .03). Moreover underlying hematological malignancy (OR 2.74; P = .04) and VRE colonization/infection (OR 2.71; P = .02) were independently associated with CDI recurrence., Conclusion: Patients with HM complicated with CDI were often regarded as non-severe infection, but had a similar in-hospital mortality rate as those without malignancy. CDI due to ribotype 078 complex isolates heralded a poor prognosis among HM patients., Competing Interests: Declaration of competing interest All authors report no conflicts of interest relevant to this article., (Copyright © 2021. Published by Elsevier B.V.)

The importance of the detection of relevant toxins or toxin genes to diagnose Clostridioides difficile infection (CDI) or the prediction of clinical outcomes of CDI has been emphasized in recent years. Although stool culture of C. difficile is not routinely recommended in the era of nonculture methods as the preferred tools for CDI diagnosis, the clinical significance of toxigenic C. difficile growth (tCdG) in stool cultures was analyzed. A clinical study was conducted in medical wards of Tainan Hospital, Ministry of Health and Welfare, in southern Taiwan. Diarrheal adults with fecal glutamate dehydrogenase and C. difficile toxin between January 2013 and April 2020 were included. Of the 209 patients with CDI, 158 (75.6%) had tCdG found in stool cultures, and the rest (51, 24.4%) had no tCdG in stool. Only prior ceftazidime or ceftriaxone therapy was independently associated with no tCdG in stool (odds ratio [OR] 2.17, P =  0.02). Compared to the patients with tCDG in stool, those without tCdG in stool experienced treatment success more often (97.1% versus 67.0%, P <  0.001) if treated with metronidazole or vancomycin but had a similar in-hospital mortality or recurrence rate. In the multivariate analysis among 114 patients with CDI treated with metronidazole or vancomycin, treatment success was independently associated with no tCdG in stool (OR 12.7, P =  0.02). Despite the limited utility of stool cultures in CDI diagnoses, no tCdG in stool culture heralds a favorable therapeutic outcome among adults with CDI treated with metronidazole or vancomycin. IMPORTANCE The importance of detecting toxins or toxin genes when diagnosing Clostridioides difficile infections (CDIs) or predicting the severity and outcomes of CDI has been emphasized in recent years. Although the yielding of C. difficile from stool cultures might implicate higher bacterial loads in fecal samples, in an era of nonculture methods for the standard diagnosis of CDIs, clinical significance of positive stool cultures of toxigenic C. difficile was analyzed in this study. Despite the limited ability of stool cultures in CDI diagnoses, no yielding of C. difficile growth might predict the successful CDI therapy.

Clostridioides difficile is a gram-positive, spore-forming anaerobic bacterium, and the leading cause of antibiotic-associated diarrhea worldwide. During C. difficile infection, spores germinate in the presence of bile acids into vegetative cells that subsequently colonize the large intestine and produce toxins. In this study, we demonstrated that C. difficile spores can universally adhere to, and be phagocytosed by, murine macrophages. Only spores from toxigenic strains were able to significantly stimulate the production of inflammatory cytokines by macrophages and subsequently induce significant cytotoxicity. Spores from the isogenic TcdA and TcdB double mutant induced significantly lower inflammatory cytokines and cytotoxicity in macrophages, and these activities were restored by pre-exposure of the spores to either toxins. These findings suggest that during sporulation, spores might be coated with C. difficile toxins from the environment, which could affect C. difficile pathogenesis in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Clostridium difficile is the leading cause of nosocomial infectious diarrhea. Hospitalized patients were at risk of C. difficile-associated diarrhea (CDAD). However the risk factors of CDAD in patients with different hospitalization period are not clear., Material and Methods: A prospective investigation was conducted in medical wards of a district hospital in southern Taiwan, from January 2011 to January 2013. We arbitrary divided patients into two groups: hospitalized for at most 14 days and 15-30 days, and analyzed their risk factors for CDAD., Results: Overall 451 patients were enrolled. The multivariable analysis of 19 (8.0%) patients developing CDAD within 14 days' hospital stay and 216 patients hospitalized for ≤ 14 days without CDAD showed malignancy (odds ratio [OR] 7.15, 95% confidence interval [CI] 1.82-28.09; P = 0.005), prior cephalosporin (OR 10.8, 95% CI 1.3-93.9; P = 0.03) and proton pump inhibitor (PPI; OR 7.1, 95% CI 2.1-24.7; P = 0.002) therapy were independently related to CDAD (Table 3), but hypertension (OR 0.2, 95% CI 0.1-0.7; P = 0.01) was reversely related to CDAD. However, of 9 (4.2%) patients developing CDAD later (15-30 days' hospital stay) and 207 patients with longer hospitalization (15-30 days) but free of CDAD, malignancy (OR 14.0, 95% CI 1.6-124.9; P = 0.02) and underlying diabetes mellitus (OR 20.5, 95% CI 2.9-144.9; P = 0.002) were independent risk factors of CDAD., Conclusion: Risk factors for CDAD among hospitalized patients varied by the duration of hospital stay. Intervention strategies to prevent CDAD may be different in terms of hospital stay duration., (Copyright © 2019. Published by Elsevier B.V.)

Clostridium difficile is a Gram-positive, spore-forming bacterium, and major cause of nosocomial diarrhea. Related studies have identified numerous factors that influence virulence traits such as the production of the two primary toxins, toxin A (TcdA) and toxin B (TcdB), as well as sporulation, motility, and biofilm formation. However, multiple putative transcriptional regulators are reportedly encoded in the genome, and additional factors are likely involved in virulence regulation. Although the leucine-responsive regulatory protein (Lrp) has been studied extensively in Gram-negative bacteria, little is known about its function in Gram-positive bacteria, although homologs have been identified in the genome. This study revealed that disruption of the lone lrp homolog in C. difficile decelerated growth under nutrient-limiting conditions, increased TcdA and TcdB production. Lrp was also found to negatively regulate sporulation while positively regulate swimming motility in strain R20291, but not in strain 630. The C. difficile Lrp appeared to function through transcriptional repression or activation. In addition, the lrp mutant was relatively virulent in a mouse model of infection. The results of this study collectively demonstrated that Lrp has broad regulatory function in C. difficile toxin expression, sporulation, motility, and pathogenesis., (Copyright © 2019 Chen, Rathod, Chiu, Chen, Tsai and Huang.)

Clostridioides difficile ( Clostridium difficile ) sequence type 11 (ST11) is well established in production animal populations worldwide and contributes considerably to the global burden of C. difficile infection (CDI) in humans. Increasing evidence of shared ancestry and genetic overlap of PCR ribotype 078 (RT078), the most common ST11 sublineage, between human and animal populations suggests that CDI may be a zoonosis. We performed whole-genome sequencing (WGS) on a collection of 207 ST11 and closely related ST258 isolates of human and veterinary/environmental origin, comprising 16 RTs collected from Australia, Asia, Europe, and North America. Core genome single nucleotide variant (SNV) analysis identified multiple intraspecies and interspecies clonal groups (isolates separated by ≤2 core genome SNVs) in all the major RT sublineages: 078, 126, 127, 033, and 288. Clonal groups comprised isolates spread across different states, countries, and continents, indicative of reciprocal long-range dissemination and possible zoonotic/anthroponotic transmission. Antimicrobial resistance genotypes and phenotypes varied across host species, geographic regions, and RTs and included macrolide/lincosamide resistance (Tn 6194 [ ermB ]), tetracycline resistance (Tn 6190 [ tetM ] and Tn 6164 [ tet44 ]), and fluoroquinolone resistance ( gyrA / B mutations), as well as numerous aminoglycoside resistance cassettes. The population was defined by a large "open" pan-genome (10,378 genes), a remarkably small core genome of 2,058 genes (only 19.8% of the gene pool), and an accessory genome containing a large and diverse collection of important prophages of the Siphoviridae and Myoviridae This study provides novel insights into strain relatedness and genetic variability of C. difficile ST11, a lineage of global One Health importance. IMPORTANCE Historically, Clostridioides difficile ( Clostridium difficile ) has been associated with life-threatening diarrhea in hospitalized patients. Increasing rates of C. difficile infection (CDI) in the community suggest exposure to C. difficile reservoirs outside the hospital, including animals, the environment, or food. C. difficile sequence type 11 (ST11) is known to infect/colonize livestock worldwide and comprises multiple ribotypes, many of which cause disease in humans, suggesting CDI may be a zoonosis. Using high-resolution genomics, we investigated the evolution and zoonotic potential of ST11 and a new closely related ST258 lineage sourced from diverse origins. We found multiple intra- and interspecies clonal transmission events in all ribotype sublineages. Clones were spread across multiple continents, often without any health care association, indicative of zoonotic/anthroponotic long-range dissemination in the community. ST11 possesses a massive pan-genome and numerous clinically important antimicrobial resistance elements and prophages, which likely contribute to the success of this globally disseminated lineage of One Health importance., (© Crown copyright 2019.)

Clostridium difficile infection (CDI) is the leading cause of nosocomial infection in hospitalized patients receiving long-term antibiotic treatment. An excessive host inflammatory response is believed to be the major mechanism underlying the pathogenesis of C. difficile infection, and various proinflammatory cytokines such as IL-1β are detected in patients with C. difficile infection. IL-1β is known to be processed by caspase-1, a cysteine protease that is regulated by a protein complex called the inflammasome, which leads to a specialized form of cell death called pyroptosis. The function of inflammasome activation-induced pyroptosis is to clear or limit the spread of invading pathogens via infiltrated neutrophils. Here, we focused on inflammasome activation induced by intact C. difficile to re-evaluate the nature of inflammasome activation in CDI pathogenesis, which could provide information that leads to an alternative therapeutic strategy for the treatment of this condition in humans. First, we found that caspase-1-dependent IL-1β production was induced by C. difficile pathogens in macrophages and increased in a time-dependent manner. Moreover, intracellular toxigenic C. difficile was essential for ATP-P2X 7 pathway of inflammasome activation and subsequent caspase-1-dependent pyroptotic cell death, leading to the loss of membrane integrity and release of intracellular contents such as LDH. Notably, we also observed that bacterial components such as surface layer proteins (SLPs) were released from pyroptotic cells. In addition, pro-IL-1β production was completely MyD88 and partially TLR2 dependent. Finally, to investigate the role of the caspase-1-dependent inflammasome in host defense, we found that colonic inflammasome activation was also induced by CDI and that caspase-1 inhibition by Ac-YVAD-CMK led to increased disease progression and C. difficile load. Taken together, the present results suggest that MyD88 and TLR2 are critical component in pro-IL-1β production and intracellular C. difficile following the ATP-P2X 7 pathway of inflammasome activation and pyroptosis, which play important roles in host defense through the utilization of inflammation-mediated bacterial clearance mechanisms during C. difficile infection.

Clinical management of Clostridium difficile infection is still far from satisfactory as bacterial spores are resistant to many chemical agents and physical treatments. Certain types of nanoparticles have been demonstrated to exhibit anti-microbial efficacy even in multi-drug resistance bacteria. However, most of these studies failed to show biocompatibility to the mammalian host cells and no study has revealed in vivo efficacy in C. difficile infection animal models. The spores treated with 500 µg/mL Fe 3-δ O 4 nanoparticles for 20 minutes, 64% of the spores were inhibited from transforming into vegetative cells, which was close to the results of the sodium hypochlorite-treated positive control. By cryo-electron micro-tomography, we demonstrated that Fe 3-δ O 4 nanoparticles bind on spore surfaces and reduce the dipicolinic acid (DPA) released by the spores. In a C. difficile infection animal model, the inflammatory level triple decreased in mice with colonic C. difficile spores treated with Fe 3-δ O 4 nanoparticles. Histopathological analysis showed a decreased intense neutrophil accumulation in the colon tissue of the Fe 3-δ O 4 nanoparticle-treated mice. Fe 3-δ O 4 nanoparticles, which had no influence on gut microbiota and apparent side effects in vivo, were efficacious inhibitors of C. difficile spore germination by attacking its surface and might become clinically feasible for prophylaxis and therapy.

The diarrheal pathogen Clostridium difficile consists of at least six distinct evolutionary lineages. The RT017 lineage is anomalous, as strains only express toxin B, compared to strains from other lineages that produce toxins A and B and, occasionally, binary toxin. Historically, RT017 initially was reported in Asia but now has been reported worldwide. We used whole-genome sequencing and phylogenetic analysis to investigate the patterns of global spread and population structure of 277 RT017 isolates from animal and human origins from six continents, isolated between 1990 and 2013. We reveal two distinct evenly split sublineages (SL1 and SL2) of C. difficile RT017 that contain multiple independent clonal expansions. All 24 animal isolates were contained within SL1 along with human isolates, suggesting potential transmission between animals and humans. Genetic analyses revealed an overrepresentation of antibiotic resistance genes. Phylogeographic analyses show a North American origin for RT017, as has been found for the recently emerged epidemic RT027 lineage. Despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents., (Copyright © 2017 Cairns et al.)

Ribotypes and toxin genotypes of clinical C. difficile isolates in Taiwan are rarely reported. A prospective surveillance study from January 2011 to January 2013 was conducted at the medical wards of a district hospital in southern Taiwan. Of the first toxigenic isolates from 120 patients, 68 (56.7%) of 120 isolates possessed both tcdA and tcdB. Of 52 (43.3%) with tcdB and truncated tcdA (tcdA-/tcdB+), all were ribotype 017 and none had binary toxin or tcdC deletion. Eighteen (15%) toxigenic isolates harbored binary toxins (cdtA and cdtB) and all had tcdC deletion, including Δ39 (C184T) deletion (14 isolates), Δ18 in-frame deletion (3 isolates), and Δ18 (Δ117A) deletion (1 isolate). Eleven of 14 isolates with Δ39 (C184T) deletion belonged to the ribotype 078 family, including ribotype 127 (6 isolates), ribotype 126 (4 isolates), and ribotype 078 (1 isolate). Among 8 patients with consecutive C. difficile isolates, these isolates from 6 (75%) patients were identical, irrespective of the presence or absence of diarrhea, suggestive of persistent fecal carriage or colonization. In conclusion in southern Taiwan, ribotype 017 isolates with a tcdA-/tcdB+ genotype were not uncommon and of C. difficile isolates with binary toxin, the ribotype 078 family was predominant., Competing Interests: The authors have declared that no competing interests exist

Clostridium difficile is the major cause of nosocomial diarrhea. We have previously demonstrated that in southern Taiwan, severe C. difficile-associated diarrhea (CDAD) cases were due to the C. difficile RT 126 strain infection, indicating the arrival of an epidemic C. difficile clone in southern Taiwan. RT126 has a close genetic relationship with RT078. However, the RT078 family is the predominant strain of C. difficile in animals worldwide, particularly in swine. In this study, we surveyed C. difficile strains isolated from swine at several farms in Taiwan from August 2011 to March 2015. We found that all swine strains, namely RT078 (32.5%, 37 of 114), RT126 (28.9%, 33 of 114) and RT127 (37.7%, 43 of 114), belonged to the toxigenic RT078 family. All strains had high gyrA mutation rate (57.9%, 66/114), which was linked to quinolone resistance. Notably, Rep-PCR revealed that 3 RT078 animal strains had the same fingerprint as human RT078 clinical isolates; their phylogenic relationship was closely related to the whole gene sequences of tcdB, thus suggesting zoonotic potential for C. difficile infection in Taiwan., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Clostridium difficile is a human and animal pathogen. Recently, the incidence of community-acquired C. difficile infection has increased, and many studies have indicated that C. difficile might be food-borne. The correlation between C. difficile infection in humans and in animals has been a topic of debate. The objective of this study was to determine the genetic relatedness of C. difficile from human and pigs in Taiwan. We investigated the molecular epidemiology of C. difficile in healthy humans and pigs from 2011 to 2015. The isolation rate of C. difficile from pigs in 13 commercial farms was 49% (100/204), and a high proportion of hypervirulent (C. difficile carrying tcdA, tcdB, and cdtA/B genes and a 39-bp deletion in the tcdC gene) ribotype 078 lineage isolates (90%, 90/100; including 078, 126, 127, and 066-like isolates) were identified. In addition, the C. difficile ribotype 127 isolates from pigs typically exhibited moxifloxacin resistance (37/43; 86%). In healthy humans, the isolation rate was 4.3% (3/69), and all healthy human isolates were non-toxigenic. In particular, we compared the porcine isolates with two patient strains (ribotype 127) obtained from two hospitals in central Taiwan. The multilocus variable number tandem repeat analysis revealed a high genetic relatedness between ribotype 127 from patients and pigs. This study indicated that isolates of the ribotype 078 lineage, and especially ribotype 127, were widely distributed in pig farms and showed a high frequency of moxifloxacin resistance. The closely related ribotype 127 from patients and pigs may have had a common origin or low diversity. In conclusion, C. difficile ribotype 127 is a noteworthy pathogen in pigs and poses a potential public health threat., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Background: Clostridium difficile is currently the leading cause of infectious diarrhea in hospitalized patients. In addition to the infection due to toxigenic C. difficile in the gastrointestinal tract of susceptible hosts, other predisposing factors for C. difficile infection (CDI) are identified, including advanced age, a prolonged hospital stay, and use of acid-suppressive drugs. Of note, exposure to gastric acid-reducing agents, such as H2 blockers and proton pump inhibitors (PPIs), remains a controversial risk factor, and has been associated with CDI in some studies but not in others. A mouse model of antibiotic-associated clostridial colitis was established to examine the role of PPIs for CDI., Materials and Methods: A mouse model of antibiotic-associated clostridial colitis was set up. NF-κB reporter mice were used to address the in vivo spatial and temporal inflammatory patterns of C. difficile-associated colitis. Serum levels of lipopolysaccharide and dextran-FITC were measured to reflect the barrier permeability of affected intestines., Results: Mice with CDI that were exposed to PPI exhibited greater losses of stool consistency and body and cecal weights than those that were not exposed to PPI. Further, more neutrophilic infiltrations, epithelial damage, and inflammatory cytokine expression were noted in colon specimens of the mice with PPI exposure. More-evident inflammatory responses were detected by in vivo imaging of NF-κB reporter mice with CDI that were exposed to PPI. Gut barrier permeability was increased to a greater extent, as reflected by higher serum levels of lipopolysaccharide and dextran-FITC in mice with CDI that were exposed to PPI., Conclusions: Our mouse model demonstrates that PPI exposure increases the severity of intestinal inflammation in mice with C. difficile-associated colitis., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Clostridium difficile can cause antibiotic-associated diarrhea in hospitalized patients. Asymptomatic colonization by C. difficile is common during the neonatal period and early infancy, ranging from 21% to 48%, and in childhood. The colonization rate of C. difficile in adult hospitalized patients shows geographic variation, ranging from 4.4% to 23.2%. Asymptomatic carriage in neonates caused no further disease in many studies, whereas adult patients colonized with toxigenic C. difficile were prone to the subsequent development of C. difficile-associated diarrhea (CDAD). However, the carriage of nontoxigenic C. difficile strains appears to prevent CDAD in hamsters and humans. Risk factors for C. difficile colonization include recent hospitalization, exposure to antimicrobial agents or gastric acid-suppressing drugs (such as proton-pump inhibitors and H2 blockers), a history of CDAD or cytomegalovirus infection, the presence of an underlying illness, receipt of immunosuppressants, the presence of antibodies against toxin B, and Toll-like receptor 4 polymorphisms. Asymptomatic C. difficile carriers are associated with significant skin and environmental contamination, similar to those with CDAD, and contact isolation and hand-washing practices should therefore be employed as infection control policies for the prevention of C. difficile spread. Treating patients with asymptomatic C. difficile colonization with metronidazole or vancomycin is not suggested by the currently available evidence. In conclusion, asymptomatic C. difficile colonization may lead to skin and environmental contamination by C. difficile, but more attention should be paid to the clinical impact of those with C. difficile colonization., (Copyright © 2014. Published by Elsevier B.V.)

Background: Patients with toxigenic Clostridium difficile colonization (tCDC) are at risk of developing C. difficile-associated diarrhea (CDAD). However, the risk factors of hospitalized patients with tCDC developing CDAD are not clear., Methods: We conducted an 18-month prospective study at a medical ward in a district hospital in southern Taiwan. Within 48 hours of admission, weekly stool samples from asymptomatic hospitalized patients were obtained to detect fecal CDC. A polymerase chain reaction for tcdB was performed to determine toxigenic isolates. CDAD was diagnosed if the patient had diarrhea and toxigenic C. difficile present in a stool sample., Results: A total 483 patients with stool samples were eligible for the study. Eighty-six (17.8%) patients had tCDC after screening, of whom 14 (16.3%) developed CDAD during follow-up. Among those with tCDC, patients with subsequent CDAD were more likely to have diabetes mellitus (p = 0.01) and to have received piperacillin-tazobactam (p = 0.04), or proton-pump inhibitors (PPIs; p = 0.04) than those without developing CDAD. The variables were statistically significant as determined by multivariate analysis. However, the 60-day crude mortality rates among tCDC patients with and without subsequent development of CDAD were similar., Conclusion: Diabetes mellitus and recent receipt of piperacillin-tazobactam or PPIs are independent risk factors for the development of CDAD among hospitalized patients with tCDC., (Copyright © 2013. Published by Elsevier B.V.)

Introduction: Several virulent Clostridium difficile clones, designated as polymerase chain reaction (PCR) ribotypes 017, 027, or 078, are well recognized in western countries. However, the ribotype distribution of clinical C. difficile isolates in Taiwan remains unclear., Method: Between 2010 and 2012, we identified three patients with C. difficile-associated diarrhea (CDAD) at a hospital in southern Taiwan. The C. difficile strains isolated from these patients were further characterized by PCR detection of tcdA, tcdB, tcdC, cdtA, and cdtB, toxinotyping, multilocus sequence typing, ribotyping and repetitive-based PCR., Results: Three C. difficile strains harbored tcdCΔ39 and belonged to multilocus sequence typing 11 (ST11), toxinotype V, and ribotype 126 (a ribotype 078-like clone). Notably, one patient developed pseudomembranous colitis and recurrent CDAD. These three isolates were noted between January 2012 and June 2012 and were identical, as evidenced by repetitive sequence-based PCR, suggestive of case clustering., Conclusion: A hypervirulent C. difficile clone, ribotype 126, causing pseudomembranous colitis and recurrent CDAD, is present in southern Taiwan., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: This study is to investigate the significance and risk factors of fecal toxigenic (tCdC) or non-toxigenic Clostridium difficile colonization (ntCdC) among hospitalized patients., Methods: Adults admitted to medical wards in a district hospital between January 2011 and June 2012 were enrolled, and those with a history of colectomy, C. difficile fecal colonization or infection or receipt of either metronidazole or oral vancomycin within 3 months, were excluded. Stools collected within 48 hours after admission and every week during hospitalization were cultured for C. difficile., Findings: Among the 441 enrolled patients, 84 (20.0%) had CdC at initial screening, including 58 (13.2%) with tCdC and 26 (6.8%) with ntCdC. Among patients with initial negative fecal screening for CdC, it took an average of 70.6 days or 66.5 days to develop tCdC or ntCdC during the study period. Finally 78 (17.7%) had tCdC and 34 (7.7%) had ntCdC. During the follow-up period, the patients with tCdC had a higher risk of CDAD (11/79, 14.1%) than those without CdC (3/328, 0.9%) and those with ntCdC (0/34, 0%) (P<0.001). In multivariate analysis, the TLR4 rs1927914 polymorphism (GG genotype) (odds ratio [OR] 4.4, 95% confidence interval [CI] 1.6-11.8, P = 0.003) and recent cefepime therapy (OR 5.3, 95% CI 2.1-13.2, P<0.001) were independently associated with tCdC, whereas recent cefuroxime (OR 11.7, 95% CI 2.3-60.2, P = 0.003) and glycopeptide therapy (OR 10.9, CI: 2.1-57.2, P = 0.005) associated with ntCdC., Conclusion: The incidence of CDAD is highest in patients with tCdC and lowest in patients with ntCdC, and the TLR4 rs1927914 polymorphism GG genotype and recent cefepime therapy were independently associated with tCdC.

Background: The impact of toxigenic Clostridium difficile colonization (tCDC) in hospitalized patients is not clear., Aim: To study the significance of tCDC in hospitalized patients., Methods: A prospective study in the medical wards of a regional hospital was performed from January to June 2011. Fecal samples collected from patients at the time of admission were tested for tcdB by real-time polymerase chain reaction (PCR) and cultured for C. difficile. The patients were followed up weekly or when they developed diarrhea during hospitalization. If C. difficile was isolated, tcdA and tcdB would be tested by multiplex PCR. The primary outcome was the development of C. difficile-associated diarrhea (CDAD)., Findings: Of 168 patients enrolled, females predominated (87, 51.8%), and the mean patient age was 75.4 years old. Approximately 70% of the patients were nursing home residents, and one third had a recent hospitalization within the prior three months. Twenty-eight (16.7%) patients had tCDC, including 16 (9.5%) patients with tCDC at the time of admission and 12 (7.2%) with tCDC during the follow-up period. With regard to the medications taken during hospitalization, the patients were more likely to have tCDC if they had received more than one class of antibiotics than if they had received monotherapy (odds ratio [OR] 6.67, 95% confidence interval [CI] 1.41-31.56, P = 0.01), particularly if they received a glycopeptide in combination with a cephalosporin or penicillin or a cephalosporin and a carbapenem. More patients with tCDC developed CDAD than those without tCDC (17.9%, 5/28 vs. 1.4%, 2/140, P = 0.002). Overall 7 (4.2%) of the 168 patients developed CDAD, and crude mortality rate of those with and without tCDC was similar (21.4%, 6/28 vs. 19.4%, 27/140, P = 0.79)., Conclusion: Recent use of glycopeptides and β-lactam antibiotics is associated with toxigenic C. difficile colonization, which is a risk factor for developing C. difficile-associated diarrhea.

The antimicrobial susceptibility and virulence factors of Clostridium difficile clinical isolates in Taiwan have not previously been reported. One hundred and thirteen isolates were collected from two major teaching hospitals in Taiwan from 2001 to 2009. Molecular typing was performed by an automated repetitive extragenic palindromic sequence-based PCR (rep-PCR) method (DiversiLab; Bacterial Barcodes, Inc., Athens, GA) and PCR ribotyping. Detection of tcdA, tcdB, cdtA, and cdtB genes was performed using a multiplex PCR assay, and gyrA and gyrB genes of moxifloxacin-nonsusceptible isolates were sequenced. All isolates were susceptible to vancomycin and metronidazole. Ninety-five (84%) isolates were susceptible to moxifloxacin, and the MIC(90) for nemonoxacin was 4 μg/ml. Tigecycline showed favorable antibacterial activity (MIC(90) of 0.06 μg/ml). Thirteen rep-PCR types were identified as a predominant rep-PCR type (type A; non-North American pulsed-field gel electrophoresis type 1 [NAP1], -NAP7, or -NAP8) accounting for 52.2% (59 isolates). Nine of 18 moxifloxacin-nonsusceptible isolates belonged to the rep-PCR type A. The rep-PCR type A and C isolates were distinct from NAP1 (ribotype 027) and NAP8 (ribotype 078) as determined by PCR ribotyping. Seventy-four (65%) isolates harbored tcdA and tcdB, and 15 (13%) harbored cdtAB encoding binary toxin. Eleven isolates had a gene deletion in tcdC, including a 39-bp deletion (9 isolates) and an 18-bp deletion (2). In conclusion, dissemination of a predominant C. difficile clone in southern and northern Taiwan was noted. However, no NAP1 (ribotype 027) isolate could be discovered in this study.

Ching-Chi Lee,1,2,&ast; Jen-Chieh Lee,2,&ast; Chun-Wei Chiu,3 Pei-Jane Tsai,4– 6 Wen-Chien Ko,2,7 Yuan-Pin Hung2,3,7,8 1Clinical Medicine Research Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan; 2Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan; 3Department of Internal Medicine, Tainan Hospital, Ministry of Health and Welfare, Tainan, 700, Taiwan; 4Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan; 5Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 6Centers of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan; 7Department of Medicine, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan; 8Department of Microbiology & Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan&ast;These authors contributed equally to this workCorrespondence: Yuan-Pin Hung, Department of Internal Medicine, Tainan Hospital, Ministry of Health and Welfare, Tainan, 700, Taiwan, Email yuebin16@yahoo.com.tw Wen-Chien Ko, Department of Medicine, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan, Email winston3415@gmail.comIntroduction: The influence of corticosteroid therapy before or after the onset of Clostridioides difficile infections (CDIs) on the clinical outcomes of adults with hospital-onset CDIs was investigated.Materials and Methods: A clinical study was conducted on the medical wards of a teaching hospital between January 2013 and April 2020. Adults (aged ≥ 20 years) with hospital-onset CDIs (ie, symptom onset at least 48 hours after hospitalization) were included. “Corticosteroid therapy during acute CDIs” was defined as the receipt of a corticosteroid at the prednisolone equivalent (PE) dose of ≥ 10 mg for at least 48 hours within one week after the CDI diagnosis. “Prior corticosteroid exposure” was defined as the receipt of a corticosteroid at the PE dose of ≥ 5 mg PE for at least 48 hours within one month before the CDI diagnosis.Results: Of the 243 adults with hospital-onset CDIs, patients (44, 18.1%) who received corticosteroid therapy during acute CDIs were more likely to have prior corticosteroid exposure (86.4% vs 11.9%, P < 0.001) and CDI episodes in intensive care units (31.8% vs 10.8%, P =0.001). Of note, a crucial association between corticosteroid therapy during acute CDIs and CDI recurrence was evidenced (13.6% vs 1.5%, P =0.002). Prior corticosteroid exposure was not associated with favorable CDI outcomes in terms of successful treatment (78.3% vs 74.9%, P =0.89), in-hospital crude mortality (17.4% vs 24.0%, P =0.61), or CDI recurrence (4.3% vs 5.3%, P = 1.00). However, for 177 patients without prior corticosteroid exposure, corticosteroid therapy during acute CDIs was linked to a higher proportion of CDI recurrence (33.3% vs 5.3%, P =0.046).Conclusion: Corticosteroid therapy during acute CDIs might impact the recurrence of CDIs, particularly in those with a lack of prior corticosteroid exposure.Keywords: steroid, prednisolone, Clostridioides difficile, diarrhea, recurrence, intensive care unit, immunosuppression

Ching-Chi Lee,1,2,&ast; Jen-Chieh Lee,2,&ast; Chun-Wei Chiu,3 Pei-Jane Tsai,4– 6 Wen-Chien Ko,2,7 Yuan-Pin Hung2,3,7 1Clinical Medicine Research Center, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan; 2Department of Internal Medicine, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan; 3Department of Internal Medicine, Tainan Hospital, Ministry of Health and Welfare, Tainan, 700, Taiwan; 4Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan; 5Department of Pathology, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 6Centers of Infectious Disease and Signaling Research, National Cheng Kung University, Tainan, Taiwan; 7Department of Medicine, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan&ast;These authors contributed equally to this workCorrespondence: Yuan-Pin Hung, Department of Internal Medicine, Tainan Hospital, Ministry of Health and Welfare, Tainan, 700, Taiwan, Email yuebin16@yahoo.com.tw Wen-Chien Ko, Department of Medicine, College of Medicine, National Cheng Kung University, Tainan, 704, Taiwan, Email winston3415@gmail.comIntroduction: A leukocyte count ≥ 15,000 cells/mL and serum creatinine of > 1.5 mg/dL have been reported as two important predictors of severe CDI. However, the association of the differential ratios of blood leukocytes, and the prognosis of Clostridioides difficile infection (CDI) is not clear.Materials and Methods: A clinical study was conducted at medical wards of Tainan Hospital, Ministry of Health and Welfare in southern Taiwan between January 2013 and April 2020. Hospitalized adults (aged ≥ 20 years) with hospital-onset CDI (ie, symptom onset after at least 48 hours of admission) were included.Results: A total of 235 adults with an average age of 75.7 years and female predominance (51.5%), including 146 (62%) adults with non-severe CDI and 87 (38%) severe CDI, were included for analysis. Patients with severe CDI had a higher crude in-hospital mortality rate than patients with non-severe CDI (35.6% vs 18.5%, P = 0.005). Multivariate analysis revealed no association between a leukocyte count > 15,000 cell/mL at the onset of CDI and in-hospital mortality (odds ratio [OR] 1.66, P = 0.21). In contrast, a neutrophil ratio > 75% (OR 2.65, P = 0.02), serum creatinine > 1.5 mg/L (OR 3.42, P = 0.03), and CDI caused by isolates harboring the tcdC gene (OR 3.54, P = 0.02) were independently associated with in-hospital mortality. Patients with a neutrophil ratio > 85%, 80– 85%, or 75– 80% of serum leukocytes had a higher mortality rate (34.8%, 30.3%, or 34.4%, respectively) than patients with a neutrophil ratio of 70– 75% or ≤ 75% (12.5% or 13.9%, respectively).Conclusion: Serum creatinine > 1.5 mg/L, a high neutrophil ratio of blood leukocytes (> 75%), and the causative C. difficile harboring the tcdC gene was independent prognostic predictors in hospitalized adults with CDI.Keywords: Clostridioides difficile, leukocyte, differential ratio, neutrophil, prognosis, mortality