Genomic study of European Clostridioides difficile ribotype 002/sequence type 8.

Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.

Clostridioides difficile infection epidemiology during the COVID-19 pandemic in Greece.

Aim: The aim was to highlight the incidence and epidemiology of C. difficile infections (CDI) in a tertiary Greek hospital during the COVID-19 pandemic.Methods: A single-center prospective observational cohort study was conducted (October 2021 until April 2022). 125 C. difficile isolates were cultured from hospitalized patients stool samples and screened by PCR for toxin A (tcdA), toxin B (tcdB), binary toxin (cdtA and cdtB) genes and the regulating gene of tcdC.Results: The incidence of CDI increased to 13.1 infections per 10,000 bed days. The most common PCR ribotypes identified included hypervirulent RT027-related RT181 (73.6%), presumably hypervirulent RT126 (8.0%) and toxin A negative RT017 (7.2%).Conclusion: Although the incidence of CDI increased significantly, the CDI epidemiology remained stable.

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Combined analysis of metabolomics and 16S rRNA sequencing for ankylosing spondylitis patients before and after secukinumab therapy.

OBJECTIVE: Alterations in gut microbiota have been implicated in the pathogenesis of ankylosing spondylitis (AS), but the underlying mechanisms remain elusive. This study aims to investigate changes in gut microbiota and metabolites in individuals with AS before and after treatment with secukinumab, to identify the biological characteristics specific to AS patients and investigate the potential biomarkers, for optimizing therapeutic strategies more effectively. METHODS: Fecal microbiome data were collected from 30 AS patients before and after secukinumab therapy and compared with data from 40 healthy controls (HC). Additionally, we analyzed the metabolic profile of both groups from plasma. RESULTS: Findings indicated that the treatment-induced changes in the composition of several crucial bacterial groups, including Megamonas, Prevotella_9, Faecalibacterium, Roseburia, Bacteroides, and Agathobacter. Post-treatment, these groups exhibited a distribution more akin to that of the healthy populations compared with their pretreatment status. We identified three gut microbial taxa, namely Prevotellaceae_bacterium_Marseille_P2831, Prevotella_buccae, and Elusimicrobiota, as potential biomarkers for diagnosing individuals at a higher risk of developing AS and assessing disease outcomes. Plasma metabolomics analysis revealed 479 distinct metabolites and highlighted three disrupted metabolic pathways. Integration of microbiome and metabolomics datasets demonstrated a significant degree of correlation, underscoring the impact of the microbiome on metabolic activity. CONCLUSION: Secukinumab can restore the balance of the gut microbiome and metabolites in AS patients, rendering them more similar to those found in the healthy population. The analysis of microbiome and metabolomics data have unveiled some candidate biomarkers capable of evaluating treatment efficacy.

Significant Correlation Between Cutaneous Abundance of Streptococcus and Psoriasis Severity in Patients with FBXL19 Gene Variants.

Psoriasis results from both genetic predisposition and environmental triggers, such as Streptococcal infections. This study aimed to explore the correlation between the abundance of the Streptococcus genus on the skin and psoriasis severity in individuals carrying specific psoriasis-associated genetic variants. Studying 39 chronic plaque psoriasis patients, the elbow skin microbiome and 49 psoriasis-related single nucleotide polymorphisms (SNPs) were analysed using a MiSeq instrument for 16S rDNA sequencing, and CLC Genomic Workbench for processing and analysis. Through multivariate linear regression analysis, a positive correlation was found between Streptococcus genus abundance and psoriasis severity in patients with certain FBXL19 gene-related heterozygous SNPs (rs12924903, rs10782001, rs12445568). Conversely, a negative association was observed in patients with homozygous genotypes. Moreover, we identified an association between Streptococcus abundance and psoriasis severity in patients with genetic variants related to IL-22, ERAP1, NOS2, and ILF3. This is the first study highlighting a positive association between Streptococcus skin colonization and psoriasis severity in patients with heterozygous genotypes within the FBXL19 gene region. FXBL19 targets the IL-33/IL1RL1 axis, crucial in infectious diseases and innate immunity promotion. These novel results suggests an intricate interaction among host genetics, Streptococcus skin colonization, and psoriasis inflammation, offering potential avenues for novel treatment approaches.

Molecular epidemiology and antimicrobial resistance in Clostridioides difficile strains isolated from children and adolescents in a tertiary referral pediatric hospital in Fortaleza, Brazil.

BACKGROUND: C. difficile has been increasingly reported as a cause of gastrointestinal disease in children, ranging from mild self-limiting diarrhea to severe conditions such as pseudomembranous colitis and toxic megacolon. Only two pediatric research groups reported the presence of C. difficile infection in Brazilian children, but no previous research has examined C. difficile infection among children in northeastern Brazil. This prospective cross-sectional study investigated the molecular epidemiology and antimicrobial resistance of C. difficile strains isolated from children and adolescents with diarrhea referred to a tertiary pediatric hospital in Brazil while exploring the associated risk factors. RESULTS: Toxin positivity or C. difficile isolation was found in 30.4 % (17/56) samples. C. difficile was isolated from 35 % (6/17) samples. Four toxigenic strains were identified (tpi+, tcdA+, tcdB+, cdtB-, without tcdC deletions) belonging to PCR ribotypes and PFGE-pulsotypes: 046 (new pulsotype 1174), 106 (NAP11), 002 (new pulsotype 1274), 012 (new pulsotype NML-1235). Two of the six isolates belonging to ribotypes 143 and 133 were non-toxigenic. All toxigenic strains were sensitive to metronidazole and vancomycin. Regarding the clinical manifestation, diarrhea lasted an average of 11 days, ranging from 3 to 50 days and was often associated with mucus and/or blood. All six patients from whom the C. difficile was isolated had a chronic disease diagnosis, with these comorbidities as the main risk factors. CONCLUSION: Our study enhances our understanding of the present epidemiological landscape of C. difficile-associated diarrhea (CDI) among children in northeastern Brazil, reveling a substantial CDI frequency of 30.4 %, with toxigenic strains detected in 76.4 % of cases, highlighting a higher prevalence compared to earlier Brazilian studies. In the globalized world, an understanding of disease-generating strains, the associated risk factors, clinical manifestation, and antimicrobial sensitivity has fundamental epidemiological importance and draws attention to preventive measures, allowing for more decisive action.

Increasing correlation between oral and gastric microbiota during gastric carcinogenesis.

BACKGROUND/AIMS: Recent research has increasingly focused on the role of the gastric microbiome in the development of gastric cancer. We aimed to investigate the changes in the microbiome during gastric carcinogenesis in structural and functional aspects, with a specific focus on the association between oral and gastric microbiomes. METHODS: We collected saliva, gastric juice, and gastric tissue samples from 141 patients at different stages of gastric carcinogenesis and processed them for microbiome analysis using 16S rRNA gene profiling. The alpha and beta diversities were analyzed, and the differences in microbiome composition and function profiles were analyzed among the groups, as well as the correlation between changes in the oral and gastric microbiomes during carcinogenesis. RESULTS: We observed significant differences in microbial diversity and composition between the disease and control groups, primarily in the gastric juice. Specific bacterial strains, including Schaalia odontolytica, Streptococcus cristatus, and Peptostreptococcus stomatis, showed a significant increase in abundance in the gastric juice in the low-grade dysplasia and gastric cancer groups. Notably, the correlation between the oral and gastric microbiota compositions, increased as the disease progressed. Predictive analysis of the metagenomic functional profiles revealed changes in functional pathways that may be associated with carcinogenesis (ABC transport and two-component systems). CONCLUSION: During gastric carcinogenesis, the abundance of oral commensals associated with cancer increased in the stomach. The similarity in microbial composition between the stomach and oral cavity also increased, implying a potential role of oral-gastric bacterial interactions in gastric cancer development.

Fecal shedding of Clostridioides difficile in calves in Sao Paulo state, Brazil.

OBJECTIVE: This study aimed to evaluate the fecal shedding of C. difficile in calves on farms in Sao Paulo State, Brazil. MATERIALS AND METHODS: Fecal samples (n = 300) were collected from diarrheic (n = 78) and nondiarrheic (n = 222) calves less than 60 days of age from 20 farms. Fecal samples were inoculated into enrichment broth supplemented with taurocholate and cultured under anaerobic conditions. Colonies suspected to be C. difficile were harvested for DNA extraction and then multiplex PCR for the detection of genes encoding toxins A and B and binary toxins. All toxigenic isolates were ribotyped and tested for antimicrobial susceptibility, and five selected strains were subjected to whole-genome sequencing to determine their sequence type. RESULTS AND DISCUSSION: C. difficile was isolated from 29.3 % (88/300) of the samples. All toxigenic isolates (17/88, 19.3 %) were classified as ribotypes RT046 (13/17-79.47 %, A+B+ CDT-) and RT126 (4/17 = 20.53 %, A+B+ CDT+). The sequenced strains from RT046 were classified as ST35 (Clade 1), while those from RT126 were classified as ST11 (Clade 5). No associations between the epidemiological factors in any of the groups and C. difficile isolation were observed. Most of the toxigenic isolates (16/17 = 94.41 %) were classified as multidrug-resistant. Calves can be an important source of toxigenic C. difficile strains, including multidrug-resistant isolates from ribotypes commonly observed in humans.

Defining the phylogenetics and resistome of the major Clostridioides difficile ribotypes circulating in Australia.

Clostridioides difficile infection (CDI) remains a significant public health threat globally. New interventions to treat CDI rely on an understanding of the evolution and epidemiology of circulating strains. Here we provide longitudinal genomic data on strain diversity, transmission dynamics and antimicrobial resistance (AMR) of C. difficile ribotypes (RTs) 014/020 (n=169), 002 (n=77) and 056 (n=36), the three most prominent C. difficile strains causing CDI in Australia. Genome scrutiny showed that AMR was uncommon in these lineages, with resistance-conferring alleles present in only 15/169 RT014/020 strains (8.9 %), 1/36 RT056 strains (2.78 %) and none of 77 RT002 strains. Notably, ~90 % of strains were resistant to MLSB agents in vitro, but only ~5.9 % harboured known resistance alleles, highlighting an incongruence between AMR genotype and phenotype. Core genome analyses revealed all three RTs contained genetically heterogeneous strain populations with limited evidence of clonal transmission between CDI cases. The average number of pairwise core genome SNP (cgSNP) differences within each RT group ranged from 23.3 (RT056, ST34, n=36) to 115.6 (RT002, ST8, n=77) and 315.9 (RT014/020, STs 2, 13, 14, 49, n=169). Just 19 clonal groups (encompassing 40 isolates), defined as isolates differing by ≤2 cgSNPs, were identified across all three RTs (RT014/020, n=14; RT002, n=3; RT056, n=2). Of these clonal groups, 63 % (12/19) comprised isolates from the same Australian State and 37 % (7/19) comprised isolates from different States. The low number of plausible transmission events found for these major RTs (and previously documented populations in animal and environmental sources/reservoirs) points to widespread and persistent community sources of diverse C. difficile strains as opposed to ongoing nationwide healthcare outbreaks dominated by a single clone. Together, these data provide new insights into the evolution of major lineages causing CDI in Australia and highlight the urgent need for enhanced surveillance, and for public health interventions to move beyond the healthcare setting and into a One Health paradigm to effectively combat this complex pathogen.

Association of short-chain fatty acids and the gut microbiome with type 2 diabetes: Evidence from the Henan Rural Cohort.

BACKGROUND AND AIMS: Human studies about short-chain fatty acids (SCFAs), the gut microbiome, and Type 2 diabetes (T2DM) are limited. Here we explored the association between SCFAs and T2DM and the effects of gut microbial diversity on glucose status in rural populations. METHODS AND RESULTS: We performed a cross-sectional study from the Henan Rural Cohort and collected stool samples. Gut microbiota composition and faecal SCFA concentrations were measured by 16S rRNA and GC-MS. The population was divided based on the tertiles of SCFAs, and logistic regression models assessed the relationship between SCFAs and T2DM. Generalized linear models tested the interactions between SCFAs and gut microbial diversity on glucose indicators (glucose, HbAlc and insulin). Compared to the lowest tertile of total SCFA, acetate and butyrate, the highest tertile exhibited lower T2DM prevalence, with ORs and 95% CIs of 0.291 (0.085-0.991), 0.160 (0.044-0.574) and 0.171 (0.047-0.620), respectively. Restricted cubic spline demonstrated an approximately inverse S-shaped association. We also noted interactions of the ACE index with the highest tertile of valerate on glucose levels (P-interaction = 0.022) and the Shannon index with the middle tertile of butyrate on insulin levels (P-interaction = 0.034). Genus Prevotella_9 and Odoribacter were inversely correlated with T2DM, and the genus Blautia was positively associated with T2DM. These bacteria are common SCFA-producing members. CONCLUSIONS: Inverse S-shaped associations between SCFAs (total SCFA, acetate, and butyrate) and T2DM were observed. Valerate and butyrate modify glucose status with increasing gut microbial diversity.

Is shorter also better in the treatment of Clostridioides difficile infection?

OBJECTIVES: To assess the effectiveness of shortened regimens of vancomycin or fidaxomicin in the treatment of Clostridioides difficile infection (CDI). METHODS: Adult patients with CDI hospitalized from January 2022 to May 2023 were included in this observational study. In patients with CDI treated with vancomycin or fidaxomicin, antibiotic treatment was discontinued after either 5 or 7 days of vancomycin or 5 days of fidaxomicin if there was a clinical response and improvement in laboratory parameters. The control cohort was treated with the standard 10 day regimen of either vancomycin or fidaxomicin. The follow-up was 60 days. Causative C. difficile strains were characterized by ribotyping and toxin gene detection when available. RESULTS: Twenty-five patients (median age 76 years) received shortened treatment with vancomycin (n = 21), or fidaxomicin (n = 4). Five cases fulfilled the criteria for severe CDI. Twenty-three patients completed follow-up; two died from causes other than CDI, and two developed recurrent CDI (8.0%). Ribotypes (RTs) 001 and 014 were the most prevalent with 20% each. In two C. difficile isolates, binary toxin genes were detected (RTs 078 and 023). In the control group of 22 patients recurrent CDI developed in 5 patients (22.7%). No statistically significant differences were found between the groups. CONCLUSIONS: Shortened treatment regimens for CDI with vancomycin and fidaxomicin were shown to be effective in our cohort of patients compared with 10 days of treatment. The recurrence rate was lower in the study group. A larger, prospective, double-blind, randomized, multicentre study is needed to support our findings.

Reduced Vancomycin Susceptibility in Clostridioides difficile Is Associated With Lower Rates of Initial Cure and Sustained Clinical Response.

BACKGROUND: Epidemiologic studies have shown decreasing vancomycin susceptibility among clinical Clostridioides difficile isolates, but the impact on patient outcomes is unknown. We hypothesized that reduced vancomycin susceptibility would be associated with decreased rates of sustained clinical response (SCR). METHODS: This multicenter cohort study included adults with C. difficile infection (CDI) treated with oral vancomycin between 2016 and 2021. Clostridioides difficile isolates underwent agar dilution vancomycin susceptibility testing, ribotyping, and Sanger sequencing of the vancomycin resistance vanR gene. Reduced susceptibility was defined as vancomycin minimum inhibitory concentration (MIC) >2 µg/mL. The primary outcome was 30-day SCR; secondary outcomes were 14-day initial cure, 30-day recurrence, and 30-day mortality. Exploratory analysis assessed the association between the VanR Thr115Ala polymorphism, susceptibility, and outcomes. RESULTS: A high proportion (34% [102/300]) of C. difficile isolates exhibited reduced vancomycin susceptibility (range, 0.5-16 µg/mL; MIC50/90 = 2/4 µg/mL). Ribotype 027 accounted for the highest proportion (77.4% [41/53]) of isolates with reduced vancomycin susceptibility. Overall, 83% (249) of patients achieved 30-day SCR. Reduced vancomycin susceptibility was associated with lower rates of 30-day SCR (76% [78/102]) than vancomycin-susceptible strains (86% [171/198]; P = .031). A significantly lower rate of 14-day initial cure was also observed among individuals infected with strains with reduced vancomycin susceptibility (89% vs 96%; P = .04). Reduced susceptibility remained an independent predictor of 30-day SCR in multivariable modeling (odds ratio, 0.52 [95% confidence interval, .28-.97]; P = .04). CONCLUSIONS: Reduced vancomycin susceptibility in C. difficile was associated with decreased odds of 30-day SCR and lower 14-day initial cure rates in the studied patient cohort.

Heart failure-induced microbial dysbiosis contributes to colonic tumour formation in mice.

AIMS: Heart failure (HF) and cancer are the leading causes of death worldwide. Epidemiological studies revealed that HF patients are prone to develop cancer. Preclinical studies provided some insights into this connection, but the exact mechanisms remain elusive. In colorectal cancer (CRC), gut microbial dysbiosis is linked to cancer progression and recent studies have shown that HF patients display microbial dysbiosis. This current study focussed on the effects of HF-induced microbial dysbiosis on colonic tumour formation. METHODS AND RESULTS: C57BL/6J mice were subjected to myocardial infarction (MI), with sham surgery as control. After six weeks faeces were collected, processed for 16 s rRNA sequencing, and pooled for faecal microbiota transplantation. CRC tumour growth was provoked in germ-free mice by treating them with Azoxymethane/Dextran sodium sulphate. The CRC mice were transplanted with faeces from MI or sham mice. MI-induced HF resulted in microbial dysbiosis, characterized by a decreased α-diversity and microbial alterations on the genus level, several of which have been associated with CRC. We then performed faecal microbiota transplantation with faeces from HF mice in CRC mice, which resulted in a higher endoscopic disease score and an increase in the number of tumours in CRC mice. CONCLUSION: We demonstrated that MI-induced HF contributes to colonic tumour formation by altering the gut microbiota composition, providing a mechanistic explanation for the observed association between HF and increased risk for cancer. Targeting the microbiome may present as a tool to mitigate HF-associated co-morbidities, especially cancer.

The burden of Clostridioides difficile infections in South-East Asia and the Western Pacific: A narrative review.

BACKGROUND: Clostridioides difficile (formerly Clostridium difficile) is well-documented in Europe and North America to be a common cause of healthcare-associated gastrointestinal tract infections. In contrast, C difficile infection (CDI) is infrequently reported in literature from Asia, which may reflect a lack of clinician awareness. We conducted a narrative review to better understand CDI burden in Asia. METHODS: We searched the PubMed database for English language articles related to C difficile, Asia, epidemiology, and molecular characteristics (eg, ribotype, antimicrobial resistance). RESULTS: Fifty-eight articles that met eligibility criteria were included. C difficile prevalence ranged from 7.1% to 45.1 % of hospitalized patients with diarrhea, and toxigenic strains among all C difficile in these patients ranged from 68.2% to 91.9 % in China and from 39.0% to 60.0 % outside of China. Widespread C difficile ribotypes were RT017, RT014/020, RT012, and RT002. Recurrence in patients with CDI ranged from 3.0% to 17.2 %. Patients with CDI typically had prior antimicrobial use recently. High rates of resistance to ciprofloxacin, clindamycin, and erythromycin were frequently reported. CONCLUSION: The regional CDI burden in Asia is still incompletely documented, seemingly due to low awareness and limited laboratory testing. Despite this apparent under recognition, the current CDI burden highlights the need for broader surveillance and for application of preventative measures against CDI in Asia.

The anti-inflammatory and anti-apoptotic effects of Achillea millefolium L. extracts on Clostridioides difficile ribotype 001 in human intestinal epithelial cells.

BACKGROUND: Clostridioides difficile infection (CDI) is one of the most common health care-acquired infections. The dramatic increase in antimicrobial resistance of C. difficile isolates has led to growing demand to seek new alternative medicines against CDI. Achillea millefolium L. extracts exhibit strong biological activity to be considered as potential therapeutic agents. In this work, the inhibitory effects of A. millefolium, its decoction (DEC) and ethanol (ETOH) extracts, were investigated on the growth of C. difficile RT001 and its toxigenic cell-free supernatant (Tox-S) induced inflammation and apoptosis. METHODS: Phytochemical analysis of extracts was performed by HPLC and GC analysis. The antimicrobial properties of extracts were evaluated against C. difficile RT001. Cell viability and cytotoxicity of Caco-2 and Vero cells treated with various concentrations of extracts and Tox-S were examined by MTT assay and microscopy, respectively. Anti-inflammatory and anti-apoptotic effects of extracts were assessed in Tox-S stimulated Caco-2 cells by RT-qPCR. RESULTS: Analysis of the phytochemical profile of extracts revealed that the main component identified in both extracts was chlorogenic acid. Both extracts displayed significant antimicrobial activity against C. difficile RT001. Moreover, both extracts at concentration 50 µg/mL had no significant effect on cell viability compared to untreated cells. Pre-treatment of cells with extracts (50 µg/mL) significantly reduced the percentage of Vero cells rounding induced by Tox-S. Also, both pre-treatment and co-treatment of Tox-S stimulated Caco-2 cells with extracts significantly downregulated the gene expression level of IL-8, IL-1ß, TNF-α, TGF-ß, iNOS, Bax, caspase-9 and caspase-3 and upregulated the expression level of Bcl-2. CONCLUSION: The results of the present study for the first time demonstrate the antimicrobial activity and protective effects of A. millefolium extracts on inflammatory response and apoptosis induced by Tox-S from C. difficile RT001 clinical strain in vitro. Further research is needed to evaluate the potential application of A. millefolium extracts as supplementary medicine for CDI prevention and treatment in clinical setting.

Ribotypes and New Virulent Strains Across Europe.

Clostridioides (formerly Clostridium) difficile is a major bacterial cause of post-antibiotic diarrhoea. The epidemiology of C. difficile infections (CDIs) has dramatically changed since the early 2000s, with an increasing incidence and severity across Europe. This trend is partly due to the emergence and rapid worldwide spread of the hypervirulent and epidemic PCR ribotype 027. Profiles of patients with CDI have also evolved, with description of community-acquired (CA) infections in patients with no traditional risk factors for CDI. However, epidemiological studies indicated that some European countries have successfully controlled the dissemination of the 027 clone whereas other countries reported the emergence of other virulent or unusual strains. The aims of this review are to summarize the current European CDI epidemiology and to describe the new virulent C. difficile strains circulating in Europe, as well as other potential emerging strains described elsewhere. Standardized typing methods and surveillance programmes are mandatory for a better understanding and monitoring of CDI in Europe.

Ribotypes and antimicrobial susceptibility profiles of clinical Clostridioides difficile isolates: A multicenter, laboratory-based surveillance in Taiwan, 2019-2021.

BACKGROUND: The clinical burden of Clostridioides difficile infections (CDIs) remains substantial globally. This study aimed to investigate the ribotypes (RTs) and antimicrobial susceptibility of C. difficile isolates collected in Taiwan. METHODS: C. difficile isolates were prospectively collected from four medical centers in Taiwan from 2019 to 2021. In a reference laboratory, in vitro susceptibility to clindamycin, moxifloxacin, metronidazole, vancomycin, fidaxomicin, and rifaximin were tested, and ribotyping was conducted to determine their genetic diversity. RESULTS: A total of 568 C. difficile isolates were included. Metronidazole resistance was not observed, and the susceptibility rate of vancomycin was 99.5 %. Clindamycin showed poor activity against these isolates, with a resistance rate of 74.8 %. Fidaxomicin exhibited potent activity and 97.4 % of isolates were inhibited at 0.25 µg/mL. Rifaximin MIC90 increased from 0.015 µg/mL in 2019 to 0.03 µg/mL in 2020 and 2021. Of 40 RTs identified, two predominant RTs were RT 078/126 (78, 14 %) and 014/020 (76, 13 %). RT 017, traditional harboring truncated tcdA, accounted for 3 % (20 isolates) and there was no isolate belonging to RT 027. The proportions of RT 078 increased from 11.2 % in 2019 to 17.1 % in 2021, and the predominance of RT 078/126 was more evident in central Taiwan. CONCLUSIONS: Vancomycin, fidaxomicin, and metronidazole remain in vitro effective against clinical C. difficile isolates in Taiwan. The reservoirs and genetic relatedness of two major RTs with zoonotic potentials, RT 078/126 and 014/020, warrant further investigations.

Increased Prevalence of Clostridioides difficile Infection Among Pediatric Oncology Patients: Risk Factors for Infection and Complications.

BACKGROUND: Pediatric oncology patients, who are typically immunosuppressed, exposed to medications associated with increased Clostridioides difficile infection (CDI) risk and hospitalized, are expected to be at substantial risk for infection and complications. Although certain C. difficile ribotypes have been associated with more severe infection in adults, such an association has not been described in children. METHODS: To characterize CDI epidemiology, including risk factors and complications among pediatric oncology patients, we retrospectively reviewed charts of patients 1-18 years old treated at a designated cancer center during 2000-2017. We used fluorescence-based polymerase chain reaction to identify ribotypes causing disease at our institution. RESULTS: In 11,366 total patients, we identified 207 CDI cases during the study period. CDI prevalence in our pediatric oncology population was 18 cases per 1000 patients. CDI was highest among patients with acute myeloid leukemia, neuroblastoma, and desmoplastic small round cell tumor (105, 66 and 111 cases per 1000 patients, respectively; P < 0.01). Fever, leukocytosis, elevated creatinine and abdominal radiation and fluoroquinolone exposure concurrent with treatment of CDI were associated with complications. Patients with severe CDI experienced increased mortality. Ribotypes previously associated with severe infection were observed infrequently and were not associated with mortality. CONCLUSIONS: This is the largest study of CDI in pediatric oncology patients to date. The study identifies specific oncologic diagnoses with increased CDI risk and factors predictive of poor outcomes. As CDI treatment guidelines are developed for this population, these data will be useful for risk stratification of patients in need of early, aggressive treatment.

Clostridioides difficile PCR ribotypes 001 and 084 can trigger autophagy process in human intestinal Caco-2 cells.

Autophagy is a homeostatic process that can promote cell survival or death. However, the exact role of autophagy in Clostridioides difficile infection (CDI) is still not precisely elucidated. Here, we investigate the role of distinct C. difficile ribotypes (RTs) in autophagy induction using Caco-2 cells. The expression analysis of autophagy-associated genes and related miRNAs were examined following treatment of Caco-2 cells with C. difficile after 4 and 8 h using RT-qPCR. Toxin production was assessed using enzyme-linked immunosorbent assay (ELISA). Immunofluorescence analysis was performed to detect MAP1LC3B/LC3B, followed by an autophagic flux analysis. C. difficile significantly reduced the viability of Caco-2 cells in comparison with untreated cells. Elevated levels of LC3-II and SQSTM1/p62 by C. difficile RT001 and RT084 in the presence of E64d/leupeptin confirmed the induction of autophagy activity. Similarly, the immunofluorescence analysis demonstrated that C. difficile RT001 and RT084 significantly increased the amount of LC3-positive structures in Caco-2 cells. The induction of autophagy was further demonstrated by increased levels of LC3B, ULK1, ATG12, PIK3C3/VPS34, BECN1 (beclin 1), ATG5, and ATG16L1 transcripts and reduced levels of AKT and MTOR gene expression. The expression levels of MIR21 and MIR30B, microRNAs that suppress autophagy, were differentially affected by C. difficile. In conclusion, the present work revealed that C. difficile bacteria can induce autophagy through both toxin-dependent and -independent mechanisms. Also, our results suggest the potential role of other C. difficile virulence factors in autophagy modulation using intestinal cells in vitro.

Biogeographic distribution and molecular epidemiology of Clostridioides (Clostridium) difficile in Western Australian soils.

Clostridioides (Clostridium) difficile is a leading cause of infectious diarrhea in humans and production animals and can be found in a variety of environmental sources. The prevalence and diversity of multi-locus sequence type clade 5 strains of C. difficile in Australian production animals suggest Australia might be the ancestral home of this lineage of One Health importance. To better understand the role of the environment in the colonization of humans and animals in Australia, it is important to investigate these endemic sources. This study describes the prevalence, molecular epidemiology, and biogeographic distribution of C. difficile in soils of Western Australia. A total of 321 soil samples from remote geographical locations across the eight health regions of Western Australia were screened for C. difficile and isolates characterized by PCR ribotyping and toxin gene profiling. C. difficile was isolated from 31.15% of samples, with the highest prevalence in the Perth Metropolitan Health Region (49.25%, n = 33/67). Overall, 52 different strains [PCR ribotypes (RTs)] were identified, with 14 being novel, and 38% (38/100) of isolates being toxigenic, the most common of which was RT014/020. Five unique novel isolates showed characteristics similar to C. difficile clade 5. This is the first study of C. difficile isolated from soils in Australia. The high prevalence and heterogeneity of C. difficile strains recovered suggest that soils play a role in the survival and environmental dissemination of this organism, and potentially its transmission among native wildlife and production animals, and in community and hospital settings.IMPORTANCEClostridium difficile is a pathogen of One Health importance. To better understand the role of the environment in human and animal colonization/infection, it is critical that autochthonous reservoirs/sources of C. difficile be investigated. This is the first study of C. difficile isolated from soils of Western Australia (WA). Here, the ecology of C. difficile in WA is described by examining the geographic distribution, molecular epidemiology, and diversity of C. difficile isolated from soils across WA.

Novel ribotype/sequence type associations and diverse CRISPR-Cas systems in environmental Clostridioides difficile strains from northern Iraq.

The environment is a natural reservoir of Clostridioides difficile, and here, we aimed to isolate the pathogen from seven locations in northern Iraq. Four of the sites yielded thirty-one isolates (ten from soils, twenty-one from sediments), which together represent ribotypes (RTs) 001 (five), 010 (five), 011 (two), 035 (two), 091 (eight), and 604 (nine). Twenty-five of the isolates (∼81%) are non-toxigenic, while six (∼19%) encode the toxin A and B genes. The genomes of eleven selected isolates represent six sequence types (STs): ST-3 (two), ST-15 (one), ST-107 (five), ST-137 (one), ST-177 (one), and ST-181 (one). Five novel RT/ST associations: RT011/ST-137, RT035/ST-107, RT091/ST-107, RT604/ST-177, and RT604/ST-181 were identified, and the first three are linked to RTs previously uncharacterized by multilocus sequence typing (MLST). Nine of the genomes belong to Clade 1, and two are closely related to the cryptic C-I clade. Diverse multiple prophages and CRISPR-Cas systems (class 1 subtype I-B1 and class 2 type V CRISPR-Cas systems) with spacers identical to other C. difficile phages and plasmids were detected in the genomes. Our data show the broader diversity that exists within environmental C. difficile strains from a much less studied location and their potential role in the evolution and emergence of new strains.