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1.
Braz J Infect Dis ; 28(3): 103767, 2024.
Article in English | MEDLINE | ID: mdl-38843868

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents , Clostridioides difficile , Clostridium Infections , Hospitals, Pediatric , Microbial Sensitivity Tests , Tertiary Care Centers , Humans , Clostridioides difficile/genetics , Clostridioides difficile/drug effects , Clostridioides difficile/isolation & purification , Child , Adolescent , Female , Male , Brazil/epidemiology , Cross-Sectional Studies , Prospective Studies , Tertiary Care Centers/statistics & numerical data , Child, Preschool , Anti-Bacterial Agents/pharmacology , Clostridium Infections/epidemiology , Clostridium Infections/microbiology , Risk Factors , Infant , Molecular Epidemiology , Diarrhea/microbiology , Diarrhea/epidemiology , Ribotyping , Drug Resistance, Bacterial/genetics
2.
Microbiol Spectr ; 12(7): e0394723, 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38864670

ABSTRACT

Clostridioides difficile (C. difficile) is widely distributed in the intestinal tract of humans, animals, and in the environment. It is the most common cause of diarrhea associated with the use of antimicrobials in humans and among the most common healthcare-associated infections worldwide. Its pathogenesis is mainly due to the production of toxin A (TcdA), toxin B (TcdB), and a binary toxin (CDT), whose genetic variants may be associated with disease severity. We studied genetic diversity in 39 C. difficile isolates from adults and children attended at two Mexican hospitals, using different gene and genome typing methods and investigated their association with in vitro expression of toxins. Whole-genome sequencing in 39 toxigenic C. difficile isolates were used for multilocus sequence typing, tcdA, and tcdB typing sequence type, and phylogenetic analysis. Strains were grown in broth media, and expression of toxin genes was measured by real-time PCR and cytotoxicity in cell-culture assays. Clustering of strains by genome-wide phylogeny matched clade classification, forming different subclusters within each clade. The toxin profile tcdA+/tcdB+/cdt+ and clade 2/ST1 were the most prevalent among isolates from children and adults. Isolates presented two TcdA and three TcdB subtypes, of which TcdA2 and TcdB2 were more prevalent. Prevalent clades and toxin subtypes in strains from children differed from those in adult strains. Toxin gene expression or cytotoxicity was not associated with genotyping or toxin subtypes. In conclusion, genomic and phenotypic analysis shows high diversity among C. difficile isolates from patients with healthcare-associated diarrhea. IMPORTANCE: Clostridioides difficile is a toxin-producing bacterial pathogen recognized as the most common cause of diarrhea acquired primarily in healthcare settings. This bacterial species is diverse; its global population has been divided into five different clades using multilocus sequence typing, and strains may express different toxin subtypes that may be related to the clades and, importantly, to the severity and progression of disease. Genotyping of children strains differed from adults suggesting toxins might present a reduced toxicity. We studied extensively cytotoxicity, expression of toxins, whole genome phylogeny, and toxin typing in clinical C. difficile isolates. Most isolates presented a tcdA+/ tcdB+/cdt+ pattern, with high diversity in cytotoxicity and clade 2/ST1 was the most prevalent. However, they all had the same TcdA2/TcdB2 toxin subtype. Advances in genomics and bioinformatics tools offer the opportunity to understand the virulence of C. difficile better and find markers for better clinical use.


Subject(s)
Bacterial Toxins , Clostridioides difficile , Clostridium Infections , Cross Infection , Diarrhea , Genetic Variation , Multilocus Sequence Typing , Phylogeny , Humans , Clostridioides difficile/genetics , Clostridioides difficile/classification , Clostridioides difficile/isolation & purification , Diarrhea/microbiology , Diarrhea/epidemiology , Mexico/epidemiology , Child , Bacterial Toxins/genetics , Adult , Clostridium Infections/microbiology , Clostridium Infections/epidemiology , Cross Infection/microbiology , Cross Infection/epidemiology , Bacterial Proteins/genetics , Enterotoxins/genetics , Male , Child, Preschool , Female , Prevalence , Adolescent , Whole Genome Sequencing , Phenotype , Genome, Bacterial/genetics , Infant , Middle Aged , Genomics
3.
Microb Biotechnol ; 17(6): e14478, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38850267

ABSTRACT

Clostridioides difficile (CD) infections are defined by toxins A (TcdA) and B (TcdB) along with the binary toxin (CDT). The emergence of the 'hypervirulent' (Hv) strain PR 027, along with PR 176 and 181, two decades ago, reshaped CD infection epidemiology in Europe. This study assessed MALDI-TOF mass spectrometry (MALDI-TOF MS) combined with machine learning (ML) and Deep Learning (DL) to identify toxigenic strains (producing TcdA, TcdB with or without CDT) and Hv strains. In total, 201 CD strains were analysed, comprising 151 toxigenic (24 ToxA+B+CDT+, 22 ToxA+B+CDT+ Hv+ and 105 ToxA+B+CDT-) and 50 non-toxigenic (ToxA-B-) strains. The DL-based classifier exhibited a 0.95 negative predictive value for excluding ToxA-B- strains, showcasing accuracy in identifying this strain category. Sensitivity in correctly identifying ToxA+B+CDT- strains ranged from 0.68 to 0.91. Additionally, all classifiers consistently demonstrated high specificity (>0.96) in detecting ToxA+B+CDT+ strains. The classifiers' performances for Hv strain detection were linked to high specificity (≥0.96). This study highlights MALDI-TOF MS enhanced by ML techniques as a rapid and cost-effective tool for identifying CD strain virulence factors. Our results brought a proof-of-concept concerning the ability of MALDI-TOF MS coupled with ML techniques to detect virulence factor and potentially improve the outbreak's management.


Subject(s)
Clostridioides difficile , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Virulence Factors , Clostridioides difficile/genetics , Clostridioides difficile/classification , Clostridioides difficile/chemistry , Clostridioides difficile/pathogenicity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Virulence Factors/genetics , Virulence Factors/analysis , Humans , Clostridium Infections/microbiology , Clostridium Infections/diagnosis , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacterial Toxins/genetics , Machine Learning , Deep Learning , Sensitivity and Specificity , Enterotoxins/analysis , Enterotoxins/genetics
4.
mSphere ; 9(6): e0008124, 2024 Jun 25.
Article in English | MEDLINE | ID: mdl-38837404

ABSTRACT

In a healthy colon, the stratified mucus layer serves as a crucial innate immune barrier to protect the epithelium from microbes. Mucins are complex glycoproteins that serve as a nutrient source for resident microflora and can be exploited by pathogens. We aimed to understand how the intestinal pathogen, Clostridioides difficile, independently uses or manipulates mucus to its benefit, without contributions from members of the microbiota. Using a 2-D primary human intestinal epithelial cell model to generate physiologic mucus, we assessed C. difficile-mucus interactions through growth assays, RNA-Seq, biophysical characterization of mucus, and contextualized metabolic modeling. We found that host-derived mucus promotes C. difficile growth both in vitro and in an infection model. RNA-Seq revealed significant upregulation of genes related to central metabolism in response to mucus, including genes involved in sugar uptake, the Wood-Ljungdahl pathway, and the glycine cleavage system. In addition, we identified differential expression of genes related to sensing and transcriptional control. Analysis of mutants with deletions in highly upregulated genes reflected the complexity of C. difficile-mucus interactions, with potential interplay between sensing and growth. Mucus also stimulated biofilm formation in vitro, which may in turn alter the viscoelastic properties of mucus. Context-specific metabolic modeling confirmed differential metabolism and the predicted importance of enzymes related to serine and glycine catabolism with mucus. Subsequent growth experiments supported these findings, indicating mucus is an important source of serine. Our results better define responses of C. difficile to human gastrointestinal mucus and highlight flexibility in metabolism that may influence pathogenesis. IMPORTANCE: Clostridioides difficile results in upward of 250,000 infections and 12,000 deaths annually in the United States. Community-acquired infections continue to rise, and recurrent disease is common, emphasizing a vital need to understand C. difficile pathogenesis. C. difficile undoubtedly interacts with colonic mucus, but the extent to which the pathogen can independently respond to and take advantage of this niche has not been explored extensively. Moreover, the metabolic complexity of C. difficile remains poorly understood but likely impacts its capacity to grow and persist in the host. Here, we demonstrate that C. difficile uses native colonic mucus for growth, indicating C. difficile possesses mechanisms to exploit the mucosal niche. Furthermore, mucus induces metabolic shifts and biofilm formation in C. difficile, which has potential ramifications for intestinal colonization. Overall, our work is crucial to better understand the dynamics of C. difficile-mucus interactions in the context of the human gut.


Subject(s)
Biofilms , Clostridioides difficile , Gene Expression Regulation, Bacterial , Mucus , Clostridioides difficile/genetics , Clostridioides difficile/physiology , Clostridioides difficile/metabolism , Biofilms/growth & development , Humans , Mucus/microbiology , Mucus/metabolism , Epithelial Cells/microbiology , Intestinal Mucosa/microbiology , Intestinal Mucosa/metabolism , Clostridium Infections/microbiology
5.
Nan Fang Yi Ke Da Xue Xue Bao ; 44(5): 998-1003, 2024 May 20.
Article in Chinese | MEDLINE | ID: mdl-38862459

ABSTRACT

OBJECTIVE: To investigate the characteristics of Clostridioides difficile infection (CDI) in patients hospitalized for diarrhea and analyze the risk factors for CDI. METHODS: Stool samples were collected from 306 patients with diarrhea hospitalized in 3 university hospitals in a mid-south city of China from October to December, 2020. C. difficile was isolated by anaerobic culture, and qRT-PCR was used to detect the expressions of toxin A (tcdA) and B (tcdB) genes and the binary toxin genes (cdtA and cdtB). Multilocus sequence typing (MLST) was performed for the isolated strains without contaminating strains as confirmed by 16S rDNA sequencing. Etest strips were used to determine the drug resistance profiles of the isolated strains, and the risk factors of CDI in the patients were analyzed. RESULTS: CDI was detected in 25 (8.17%) out of the 306 patients. All the patients tested positive for tcdA and tcdB but negative for the binary toxin genes. Seven noncontaminated C. difficile strains with 5 ST types were isolated, including 3 ST54 strains and one strain of ST129, ST98, ST53, and ST631 types each, all belonging to clade 1 and sensitive to metronidazole and vancomycin. Hospitalization within the past 6 months (OR= 3.675; 95% CI: 1.405-9.612), use of PPIs (OR=7.107; 95% CI: 2.575-19.613), antibiotics for ≥1 week (OR=7.306; 95% CI: 2.274-23.472), non-steroidal anti-inflammatory drugs (OR=4.754; 95% CI: 1.504-15.031) in the past month, and gastrointestinal disorders (OR=5.050; 95% CI: 1.826-13.968) were all risk factors for CDI in the patients hospitalized for diarrhea. CONCLUSION: The CDI rate remains low in the hospitalized patients with diarrhea in the investigated hospitals, but early precaution measures are recommended when exposure to the risk factors is reported to reduce the risk of CDI in the hospitalized patients.


Subject(s)
Clostridioides difficile , Clostridium Infections , Diarrhea , Hospitals, University , Multilocus Sequence Typing , Humans , Diarrhea/microbiology , Diarrhea/epidemiology , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Risk Factors , Clostridium Infections/microbiology , Clostridium Infections/epidemiology , China/epidemiology , Bacterial Toxins/genetics , Feces/microbiology , Anti-Bacterial Agents/pharmacology , Hospitalization , Bacterial Proteins/genetics , Enterotoxins/genetics , Male , Female , Middle Aged
6.
Toxins (Basel) ; 16(6)2024 May 24.
Article in English | MEDLINE | ID: mdl-38922136

ABSTRACT

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI.


Subject(s)
Bacterial Toxins , Clostridioides difficile , Host Microbial Interactions , Clostridioides difficile/genetics , Clostridioides difficile/pathogenicity , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Bacterial Toxins/immunology , Clostridium Infections/drug therapy , Clostridium Infections/microbiology , Clostridium Infections/pathology , Gene Order , Inflammation/pathology , Humans , Animals
7.
J Vet Med Sci ; 86(7): 769-776, 2024 Jul 02.
Article in English | MEDLINE | ID: mdl-38797681

ABSTRACT

The pathogenicity of Clostridioides difficile in piglets remains controversial. It is unknown whether C. difficile control helps protect piglet health. To clarify the association between C. difficile presence and piglet diarrhea, isolates were obtained from piglets with and without diarrhea. In addition, to determine the genetic relationship of C. difficile from pigs and humans, we performed whole-genome sequencing (WGS) of C. difficile isolates. Diarrheal and non-diarrheal stool samples were collected from neonatal piglets from five farms in Japan in 2021. To clarify the relationship between C. difficile derived from pigs and those from human clinical cases, WGS of C. difficile isolates was performed. Toxin-positive C. difficile were significantly more prevalent in piglets with diarrhea, although the overall frequency of C. difficile did not differ between piglets with and without diarrhea. This observation indicates an association between toxin-positive C. difficile and diarrhea in piglets. However, further studies are needed to establish a direct causal relationship and to explore other contributing factors to diarrhea in piglets. WGS results showed that C. difficile sequence type (ST) 11 including the hypervirulent PCR ribotype 078 isolates derived from Japanese pigs were closely related to ST11 of overseas strains (human clinical and animal-derived) and a Japanese human clinical strain. Toxin-positive C. difficile may cause diarrhea in piglets and hypervirulent C. difficile are spreading among pigs and human populations worldwide.


Subject(s)
Bacterial Toxins , Clostridioides difficile , Clostridium Infections , Diarrhea , Swine Diseases , Animals , Swine , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Swine Diseases/microbiology , Swine Diseases/transmission , Diarrhea/veterinary , Diarrhea/microbiology , Clostridium Infections/veterinary , Clostridium Infections/microbiology , Clostridium Infections/transmission , Humans , Bacterial Toxins/genetics , Japan/epidemiology , Whole Genome Sequencing , Animals, Newborn/microbiology , Feces/microbiology
8.
Microbiol Spectr ; 12(6): e0035424, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38709085

ABSTRACT

Paeniclostridium sordellii hemorrhagic toxin (TcsH) and Clostridioides difficile toxin A (TcdA) are two major members of the large clostridial toxin (LCT) family. These two toxins share ~87% similarity and are known to cause severe hemorrhagic pathology in animals. Yet, the pathogenesis of their hemorrhagic toxicity has been mysterious for decades. Here, we examined the liver injury after systemic exposure to different LCTs and found that only TcsH and TcdA induce overt hepatic hemorrhage. By investigating the chimeric and truncated toxins, we demonstrated that the enzymatic domain of TcsH alone is not sufficient to determine its potent hepatic hemorrhagic toxicity in mice. Likewise, the combined repetitive oligopeptide (CROP) domain of TcsH/TcdA alone also failed to explain their strong hemorrhagic activity in mice. Lastly, we showed that disrupting the first two short repeats of CROPs in TcsH and TcdA impaired hemorrhagic toxicity without causing overt changes in cytotoxicity and lethality. These findings lead to a deeper understanding of toxin-induced hemorrhage and the pathogenesis of LCTs and could be insightful in developing therapeutic avenues against clostridial infections. IMPORTANCE: Paeniclostridium sordellii and Clostridioides difficile infections often cause hemorrhage in the affected tissues and organs, which is mainly attributed to their hemorrhagic toxins, TcsH and TcdA. In this study, we demonstrate that TcsH and TcdA, but not other related toxins. including Clostridioides difficile toxin B and TcsL, induce severe hepatic hemorrhage in mice. We further determine that a small region in TcsH and TcdA is critical for the hemorrhagic toxicity but not cytotoxicity or lethality of these toxins. Based on these results, we propose that the hemorrhagic toxicity of TcsH and TcdA is due to an uncharacterized mechanism, such as the presence of an unknown receptor, and future studies to identify the interactive host factors are warranted.


Subject(s)
Bacterial Toxins , Clostridioides difficile , Enterotoxins , Hemorrhage , Animals , Mice , Bacterial Toxins/toxicity , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Clostridioides difficile/genetics , Clostridioides difficile/pathogenicity , Enterotoxins/toxicity , Enterotoxins/genetics , Enterotoxins/metabolism , Liver/pathology , Clostridium Infections/microbiology , Humans , Female
9.
Mol Microbiol ; 121(6): 1182-1199, 2024 06.
Article in English | MEDLINE | ID: mdl-38690761

ABSTRACT

The dipeptide D-Ala-D-Ala is an essential component of peptidoglycan and the target of vancomycin. Most Clostridioides difficile strains possess the vanG operon responsible for the synthesis of D-Ala-D-Ser, which can replace D-Ala-D-Ala in peptidoglycan. The C. difficile vanG operon is regulated by a two-component system, VanRS, but is not induced sufficiently by vancomycin to confer resistance to this antibiotic. Surprisingly, in the absence of the VanS histidine kinase (HK), the vanG operon is still induced by vancomycin and also by another antibiotic, ramoplanin, in a VanR-dependent manner. This suggested the cross-regulation of VanR by another HK or kinases that are activated in the presence of certain lipid II-targeting antibiotics. We identified these HKs as CD35990 and CD22880. However, mutations in either or both HKs did not affect the regulation of the vanG operon in wild-type cells suggesting that intact VanS prevents the cross-activation of VanR by non-cognate HKs. Overproduction of VanR in the absence of VanS, CD35990, and CD22880 led to high expression of the vanG operon indicating that VanR can potentially utilize at least one more phosphate donor for its activation. Candidate targets of CD35990- and CD22880-mediated regulation in the presence of vancomycin or ramoplanin were identified by RNA-Seq.


Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Clostridioides difficile , Gene Expression Regulation, Bacterial , Histidine Kinase , Operon , Vancomycin Resistance , Vancomycin , Operon/genetics , Clostridioides difficile/genetics , Clostridioides difficile/drug effects , Clostridioides difficile/metabolism , Histidine Kinase/metabolism , Histidine Kinase/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Vancomycin/pharmacology , Vancomycin Resistance/genetics , Anti-Bacterial Agents/pharmacology , Depsipeptides/pharmacology , Transcription Factors
10.
Vet Microbiol ; 294: 110129, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38810364

ABSTRACT

OBJECTIVE: To conduct molecular prevalence and genetic polymorphism analysis of 24 Swine Farm associated C. difficile ST11 strains, in addition to other representative sequenced ST strains. METHODS: The collected C. difficile strains underwent whole genome sequencing and bioinformatic analysis using the illumina NovaSeq platform, SPAdes, Prokka, MOB-suite, and FastTree. Virulence and antibiotic resistance genes were identified through NCBI Pathogen Database. Cytotoxicity tests were conducted on HT-29 cells and Vero cells to verify the function of toxin A and toxin B. RESULTS: The most prevalent resistance genes in ST11 were found to be against ß-lactamases, aminoglycosides, and tetracycline. A C. difficile isolate (strain 27) with tcdA deletion and high antibiotic resistance genes was far apart from other swine farm associated ST11 isolates in the phylogenetic branch. The remarkable genetic similarity between animal and human C. difficile strains suggests potential transmission of ST11 strains between animals and humans. The plasmid replicon sequences repUS43 were identified in all ST11 strains except one variant (strain 27), and 91.67% (22/24) of these were assessed by MOB-typer as having mobilizable plasmids. CONCLUSION: Swine farm associated C. difficile ST11 carried fewer virulence genes than ST11 strains collected from NCBI database. It is critical to monitor the evolution of C. difficile strains to understand their changing characteristics, host-switching, and develop effective control and prevention strategies.


Subject(s)
Clostridioides difficile , Clostridium Infections , Farms , Phylogeny , Swine Diseases , Animals , Clostridioides difficile/genetics , Clostridioides difficile/classification , Swine , Swine Diseases/microbiology , Swine Diseases/epidemiology , Clostridium Infections/veterinary , Clostridium Infections/microbiology , Clostridium Infections/epidemiology , Whole Genome Sequencing , Anti-Bacterial Agents/pharmacology , Virulence/genetics , Vero Cells , Humans , Chlorocebus aethiops , Drug Resistance, Bacterial/genetics , Plasmids/genetics , Virulence Factors/genetics
12.
Microbiol Res ; 285: 127739, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38763016

ABSTRACT

Clostridioides difficile may have a negative impact on gut microbiota composition in terms of diversity and abundance, thereby triggering functional changes supported by the differential presence of genes involved in significant metabolic pathways, such as short-chain fatty acids (SCFA). This work has evaluated shotgun metagenomics data regarding 48 samples from four groups classified according to diarrhea acquisition site (community- and healthcare facility-onset) and positive or negative Clostridioides difficile infection (CDI) result. The metagenomic-assembled genomes (MAGs) obtained from each sample were taxonomically assigned for preliminary comparative analysis concerning differences in composition among groups. The predicted genes involved in metabolism, transport, and signaling remained constant in microbiota members; characteristic patterns were observed in MAGs and genes involved in SCFA butyrate and acetate metabolic pathways for each study group. A decrease in genera and species, as well as relative MAG abundance with the presence of the acetate metabolism-related gene, was evident in the HCFO/- group. Increased antibiotic resistance markers (ARM) were observed in MAGs along with the genes involved in acetate metabolism. The results highlight the need to explore the role of acetate in greater depth as a potential protector of the imbalances produced by CDI, as occurs in other inflammatory intestinal diseases.


Subject(s)
Acetates , Clostridioides difficile , Clostridium Infections , Gastrointestinal Microbiome , Metagenome , Metagenomics , Clostridioides difficile/genetics , Acetates/metabolism , Humans , Clostridium Infections/microbiology , Fatty Acids, Volatile/metabolism , Genome, Bacterial , Butyrates/metabolism , Metabolic Networks and Pathways/genetics , Feces/microbiology , Diarrhea/microbiology
13.
Microb Genom ; 10(5)2024 May.
Article in English | MEDLINE | ID: mdl-38717815

ABSTRACT

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.


Subject(s)
Clostridioides difficile , Clostridium Infections , Phylogeny , Ribotyping , Clostridioides difficile/genetics , Clostridioides difficile/classification , Clostridioides difficile/drug effects , Clostridioides difficile/isolation & purification , Australia/epidemiology , Humans , Clostridium Infections/microbiology , Clostridium Infections/epidemiology , Clostridium Infections/transmission , Genome, Bacterial , Drug Resistance, Bacterial/genetics , Anti-Bacterial Agents/pharmacology , Polymorphism, Single Nucleotide , Genotype
14.
Microbiol Spectr ; 12(6): e0400523, 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38709045

ABSTRACT

Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCEClostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.


Subject(s)
Bacterial Proteins , Biofilms , Clostridioides difficile , Clostridium Infections , Gene Deletion , Clostridioides difficile/genetics , Clostridioides difficile/pathogenicity , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Humans , Clostridium Infections/microbiology , Biofilms/growth & development , Anti-Bacterial Agents/pharmacology , Virulence/genetics , CRISPR-Cas Systems , Bacterial Adhesion/genetics , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism
15.
PLoS Pathog ; 20(5): e1012224, 2024 May.
Article in English | MEDLINE | ID: mdl-38739653

ABSTRACT

Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile. In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile, we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, 3D structural analyses of Spo0E revealed specific and exclusive interactions between Spo0E and binding partners in C. difficile and B. subtilis that provide insight into the conservation of this regulatory mechanism among different species.


Subject(s)
Bacterial Proteins , Clostridioides difficile , Gene Expression Regulation, Bacterial , Spores, Bacterial , Clostridioides difficile/pathogenicity , Clostridioides difficile/genetics , Clostridioides difficile/metabolism , Spores, Bacterial/genetics , Virulence , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Animals , Mice , Clostridium Infections/microbiology
16.
Clin Chim Acta ; 559: 119728, 2024 Jun 01.
Article in English | MEDLINE | ID: mdl-38750779

ABSTRACT

BACKGROUND AND AIMS: The incidence of Clostridioides difficile infection and the prevalence of hypervirulent ST1 (BI/NAP1/027)strain are increasing, especially in developing countries. We aimed to develop a new PCR assay for the identification of hypervirulent ST1 strains and toxigenic C. difficile in stool samples. MATERIALS AND METHODS: We established a quadruplex TaqMan real-time PCR (pilW_4-plex PCR) assay targeting the pilW, a ST1-specific type Ⅳ minor pilin gene, and three C. difficile genes including cdtB, tcdB, and hsp. The sensitivity and specificity of the assay was tested using 403C. difficile isolates and 180 unformed stool sample. The results were compared with anaerobic culture-based conventional PCR method and MLST. RESULTS: The pilW_4-plex PCR identified toxigenic C. difficile in 333 (82.6%, 333/403) isolates with 100% sensitivity and specificity, and in 78 (43.3%, 78/180) stool samples with the sensitivity and specificity of 94.7% and 93.3%, respectively. Hypervirulent ST1 were detected in 21 strains and nine stool samples by the pilW_4-plex PCR. The pilW_4-plex PCR assay has no cross-reaction with non-toxigenic C. difficile or other bacteria. CONCLUSION: The pilW_4-plex PCR assay is an accurate and rapid method with high sensitivity and specificity for identification of ST1 and detection of toxigenic C. difficile in stool.


Subject(s)
Clostridioides difficile , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Humans , Real-Time Polymerase Chain Reaction , Feces/microbiology , Polymerase Chain Reaction/methods , Clostridium Infections/diagnosis , Clostridium Infections/microbiology , Virulence/genetics , Sensitivity and Specificity
17.
BMC Infect Dis ; 24(1): 512, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38778271

ABSTRACT

AIM: Diarrhea is a common disease in immunocompromised patients and can be associated with greater morbidity and even mortality. Therefore, the present study was designed to determine the prevalence of Aeromonas spp., Campylobacter spp., and C. difficile among immunocompromised children. METHODS: This study was conducted on 130 stool samples from patients with diarrhea who had defects in the immune system and were referred to Hazrat Masoumeh Children's Hospital in Qom. Demographic information, clinical symptoms, immune status, and duration of chemotherapy were also recorded for each child. DNAs were extracted from the stool, and then direct PCR assays were done by specific primers for the detection of Aeromonas spp., Campylobacter spp., and toxigenic C. difficile, including tcdA/B and cdtA/B genes. Co-infection in patients was also evaluated. RESULTS: 60.8% and 39.2% were male and female, respectively, with a m ± SD age of 56.72 ± 40.49 months. Most cases of immunocompromised states were related to Acute Lymphocytic Leukemia (77.7%) and Non-Hodgkin Lymphoma (14.6%). 93.1% of patients were undergoing chemotherapy during the study. Among patients, most clinical symptoms were related to bloody diarrhea (98.5%) and fever (92.3%). Based on PCR, 14.6, 9.2, and 1.5% were positive for Aeromonas spp., C. difficile, and C. jejuni, respectively. Among the C. difficile-positive cases, the tcdA gene was only detected in one patient. In total, three co-infections were identified, which included Aeromonas spp./C. difficile (tcdA+), C. jejuni/C. difficile, and C. jejuni/Aeromonas spp. CONCLUSIONS: This is the first study in Iran to investigate the simultaneous prevalence of some pathogens in immunocompromised children with diarrhea. Because Aeromonas spp., Campylobacter spp., and C. difficile are not routinely detected in some laboratories, infections caused by them are underappreciated in the clinic. Our results showed that these pathogens are present in our region and can cause gastroenteritis in children, especially those with underlying diseases. Therefore, increasing the level of hygiene in some areas and controlling bacterial diarrheal diseases should be given more attention by health officials.


Subject(s)
Aeromonas , Campylobacter , Clostridioides difficile , Clostridium Infections , Diarrhea , Feces , Immunocompromised Host , Humans , Female , Male , Child, Preschool , Diarrhea/microbiology , Diarrhea/epidemiology , Child , Aeromonas/isolation & purification , Aeromonas/genetics , Prevalence , Clostridioides difficile/genetics , Clostridioides difficile/isolation & purification , Campylobacter/isolation & purification , Campylobacter/genetics , Infant , Feces/microbiology , Clostridium Infections/epidemiology , Clostridium Infections/microbiology , Gram-Negative Bacterial Infections/epidemiology , Gram-Negative Bacterial Infections/microbiology , Adolescent , Campylobacter Infections/epidemiology , Campylobacter Infections/microbiology , Coinfection/microbiology , Coinfection/epidemiology
18.
BMC Microbiol ; 24(1): 177, 2024 May 23.
Article in English | MEDLINE | ID: mdl-38783194

ABSTRACT

BACKGROUND: Clostridioides difficile is the main pathogen of antimicrobial-associated diarrhoea and health care facility-associated infectious diarrhoea. This study aimed to investigate the prevalence, toxin genotypes, and antibiotic resistance of C. difficile among hospitalized patients in Xi'an, China. RESULTS: We isolated and cultured 156 strains of C. difficile, representing 12.67% of the 1231 inpatient stool samples collected. Among the isolates, tcdA + B + strains were predominant, accounting for 78.2% (122/156), followed by 27 tcdA-B + strains (27/156, 17.3%) and 6 binary toxin gene-positive strains. The positive rates of three regulatory genes, tcdC, tcdR, and tcdE, were 89.1% (139/156), 96.8% (151/156), and 100%, respectively. All isolates were sensitive to metronidazole, and the resistance rates to clindamycin and cephalosporins were also high. Six strains were found to be resistant to vancomycin. CONCLUSION: Currently, the prevalence rate of C. difficile infection (CDI) in Xi'an is 12.67% (156/1231), with the major toxin genotype of the isolates being tcdA + tcdB + cdtA-/B-. Metronidazole and vancomycin were still effective drugs for the treatment of CDI, but we should pay attention to antibiotic management and epidemiological surveillance of CDI.


Subject(s)
Anti-Bacterial Agents , Bacterial Toxins , Clostridioides difficile , Clostridium Infections , Feces , Genotype , Hospitals , Clostridioides difficile/genetics , Clostridioides difficile/drug effects , Clostridioides difficile/isolation & purification , Clostridioides difficile/classification , Humans , China/epidemiology , Anti-Bacterial Agents/pharmacology , Clostridium Infections/microbiology , Clostridium Infections/epidemiology , Bacterial Toxins/genetics , Hospitals/statistics & numerical data , Feces/microbiology , Drug Resistance, Bacterial/genetics , Prevalence , Microbial Sensitivity Tests , Female , Middle Aged , Male , Aged , Adult , Bacterial Proteins/genetics , Diarrhea/microbiology , Diarrhea/epidemiology , Metronidazole/pharmacology , Young Adult , Enterotoxins/genetics , Adolescent , Vancomycin/pharmacology , Clindamycin/pharmacology , Aged, 80 and over
19.
Biochem Biophys Res Commun ; 715: 149957, 2024 Jun 30.
Article in English | MEDLINE | ID: mdl-38688057

ABSTRACT

Clostridioides difficile endolysin (Ecd09610) consists of an unknown domain at its N terminus, followed by two catalytic domains, a glucosaminidase domain and endopeptidase domain. X-ray structure and mutagenesis analyses of the Ecd09610 catalytic domain with glucosaminidase activity (Ecd09610CD53) were performed. Ecd09610CD53 was found to possess an α-bundle-like structure with nine helices, which is well conserved among GH73 family enzymes. The mutagenesis analysis based on X-ray structures showed that Glu405 and Asn470 were essential for enzymatic activity. Ecd09610CD53 may adopt a neighboring-group mechanism for a catalytic reaction in which Glu405 acted as an acid/base catalyst and Asn470 helped to stabilize the oxazolinium ion intermediate. Structural comparisons with the newly identified Clostridium perfringens autolysin catalytic domain (AcpCD) in the P1 form and a zymography analysis demonstrated that AcpCD was 15-fold more active than Ecd09610CD53. The strength of the glucosaminidase activity of the GH73 family appears to be dependent on the depth of the substrate-binding groove.


Subject(s)
Catalytic Domain , Clostridioides difficile , Endopeptidases , Clostridioides difficile/enzymology , Clostridioides difficile/genetics , Crystallography, X-Ray , Endopeptidases/chemistry , Endopeptidases/metabolism , Endopeptidases/genetics , Models, Molecular , Hexosaminidases/chemistry , Hexosaminidases/genetics , Hexosaminidases/metabolism , Mutagenesis , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mutagenesis, Site-Directed , Protein Domains
20.
J Antimicrob Chemother ; 79(6): 1413-1417, 2024 Jun 03.
Article in English | MEDLINE | ID: mdl-38661207

ABSTRACT

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.


Subject(s)
Anti-Bacterial Agents , Clostridioides difficile , Clostridium Infections , Fidaxomicin , Ribotyping , Vancomycin , Humans , Clostridium Infections/drug therapy , Clostridium Infections/microbiology , Aged , Male , Female , Clostridioides difficile/genetics , Clostridioides difficile/drug effects , Clostridioides difficile/classification , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/administration & dosage , Vancomycin/therapeutic use , Vancomycin/administration & dosage , Fidaxomicin/therapeutic use , Fidaxomicin/administration & dosage , Middle Aged , Aged, 80 and over , Treatment Outcome
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