Binary toxin expression by Clostridioides difficile is associated with worse disease

Background The incidence of Clostridioides difficile infection (CDI) has increased over the past two decades and is considered an urgent threat by the Centers for Disease Control. Hypervirulent strains such as ribotype 027, that possess genes for the additional toxin C. difficile binary toxin (CDT), are contributing to increased morbidity and mortality. Methods We retrospectively tested stool from 215 CDI patients for CDT by enzyme-linked immunosorbent assay (ELISA). Stratifying patients by CDT status, we assessed if disease severity and clinical outcomes correlated with CDT positivity. Additionally, we completed qPCR DNA extracted from patient stool to detect cdtB gene. Lastly, we performed 16 S rRNA gene sequencing to examine if CDT positive samples had an altered fecal microbiota. Results We found that patients with CdtB, the pore forming component of CDT, detected in their stool by ELISA were more likely to have severe disease with a higher 90-day mortality. CDT positive patients also had higher C. difficile bacterial burden and white blood cell counts. There was no significant difference in gut microbiome diversity between CDT positive and negative patients. Conclusions Patients with fecal samples that were positive for CDT had increased disease severity and worse clinical outcomes. Utilization of PCR and C. difficile Toxins A and B testing may not reveal the entire picture when diagnosing CDI, with the detection of CDT-expressing strains valuable in identifying patients at risk of more severe disease.

Clostridioides difficile Toxin CDT Induces Cytotoxic Responses in Human Mucosal-Associated Invariant T (MAIT) Cells

Clostridioides difficile is the major cause of antibiotic-associated colitis (CDAC) with increasing prevalence in morbidity and mortality. Severity of CDAC has been attributed to hypervirulent C. difficile strains, which in addition to toxin A and B (TcdA, TcdB) produce the binary toxin C. difficile transferase (CDT). However, the link between these toxins and host immune responses as potential drivers of immunopathology are still incompletely understood. Here, we provide first experimental evidence that C. difficile toxins efficiently activate human mucosal-associated invariant T (MAIT) cells. Among the tested toxins, CDT and more specifically, the substrate binding and pore-forming subunit CDTb provoked significant MAIT cell activation resulting in selective MAIT cell degranulation of the lytic granule components perforin and granzyme B. CDT-induced MAIT cell responses required accessory immune cells, and we suggest monocytes as a potential CDT target cell population. Within the peripheral blood mononuclear cell fraction, we found increased IL-18 levels following CDT stimulation and MAIT cell response was indeed partly dependent on this cytokine. Surprisingly, CDT-induced MAIT cell activation was found to be partially MR1-dependent, although bacterial-derived metabolite antigens were absent. However, the role of antigen presentation in this process was not analyzed here and needs to be validated in future studies. Thus, MR1-dependent induction of MAIT cell cytotoxicity might be instrumental for hypervirulent C. difficile to overcome cellular barriers and may contribute to pathophysiology of CDAC.

Antibiotic Resistances and Molecular Characteristics of Clostridioides difficile in ICUs in a Teaching Hospital From Central South China

Clostridioides (C.) difficile is a major healthcare-associated pathogen inducing infectious diarrhea. Approximately 25–33% of patients with antibiotic-associated diarrhea (AAD) and 90% of patients with pseudomembranous enteritis are caused by C. difficile infection (CDI). Stool samples were collected from hospitalized adults with presumptive AAD in four nonneonatal intensive care units (ICUs). Diagnosis of CDI was based on both clinical symptoms and laboratory results. The stool specimens were transferred onto CDIF (C. difficile agar), and C. difficile was finally confirmed by the latex agglutination test. Toxin-producing genes tcdA (A), tcdB (B), and cdt (CDT) were detected by PCR, and all isolates were performed multilocus sequence typing analysis. The antibiotic susceptibility of C. difficile isolates was assessed by the agar dilution method. A total of 184 C. difficile were isolated from 857 specimens in our study, the isolation rate of C. difficile was 21.5% (184/857). The 184 C. difficile were isolated from 179 patients, among these 115 patients were toxin-positive, giving the incidence of CDI being 58.0/10,000 patient days in the four ICUs. Among these 115 toxin-positive C. difficile isolates, 100 (87.0%) isolates produced two toxins (A+B+CDT-), three (2.6%) isolates were A+B+ with binary toxin-producing (A+B+CDT+), and 12 (10.4%) isolates only produced one toxin (A-B+CDT-). A total of 27 sequencing types (STs) were obtained. The most prevalent was ST3 (34 isolates), followed by ST39 (27 isolates), ST54 (19 isolates), ST26 (16 isolates), ST35 (15 isolates), and ST2 (13 isolates). All the ST26 isolates were nontoxigenic. Meanwhile, five STs were newly discovered. Although multidrug resistance was present in ≥50% of these C. difficile isolates, all of them were susceptible to tigecycline, fidaxomicin, metronidazole, and vancomycin. In conclusion, C. difficile isolates producing two toxins (A+B+CDT-) were dominant in our hospital. The most prevalent was ST3, and all ST26 isolates were NTCD. Although multidrug resistance was present in ≥50% of the C. difficile isolates, metronidazole, tigecycline, fidaxomicin, and vancomycin were still effective treatments for CDI in our hospital.

Genome analysis of Lysinibacillus sphaericus isolate 6.2 pathogenic to Culex quinquefasciatus Say, 1823 (Diptera: Culicidae)

Viersanova A, Purwanto H. 2021. Genome analysis of Lysinibacillus sphaericus isolate 6.2 pathogenic to Culex quinquefasciatus Say, 1823 (Diptera: Culicidae). Biodiversitas 22: 5211-5222. Lysinibacillus sphaericus is an entomopathogenic bacteria that is specific to vector mosquitoes, especially Culex spp., and Anopheles spp., so it has been widely used as a bioinsecticide. L. sphaericus has a wide variation of toxicity efficiencies, which have led to continuous exploration of new isolates with higher toxicity and a new toxin to deal with resistance problems. This study aimed to identify the genomic characteristics and toxin characteristics of isolate 6.2 based on whole genome analysis and analyze the identification of isolate 6.2. Isolate 6.2 was previously obtained from rhizosphere in Yogyakarta. To analyze the genome and toxins, the NGS technique was used and then the analysis was carried out using a couple of freely available bioinformatics tools. Molecular identification was carried out with the 16SrRNA gene and the relationship was analyzed by reconstructing the phylogenetic tree using Neighbours-Joining. The genomic analysis of isolate 6.2 showed good results with G+C content and genome size that matched the reference genome of L. sphaericus. The result of the 16SrRNA gene blasting showed that the closest related gene of isolate 6.2 is L. fusiformis (NR_042072.1). However, the reconstructed phylogenetic tree did not show the formation of clusters according to the species. Toxin analysis indicates that isolate 6.2 has Mtx, s-layer protein, hemolysin, and chitin-binding protein genes. All of which are known to be associated with the toxicity of L. sphaericus to binary toxin resistant population of Culex quinquefasciatus.

Cryo-EM structures of the translocational binary toxin complex CDTa-bound CDTb-pore from Clostridioides difficile

Besides two large cytotoxins (TcdA and TcdB), certain Clostridioides difficile strains also produce a binary toxin, called C. difficile toxin (CDT) composed of an enzymatic subunit involved in actin ADP-ribosylation (CDTa) and translocation pore (CDTb) that delivers CDTa into host cells through receptor-mediated endocytosis. CDTb is proposed to be a di-heptamer, but its physiological heptameric structure has not been reported to date. Here, we report the CDTa-bound CDTb-pore (heptamer) as a physiological complexes using cryo-EM. The high-resolution structure of the CDTa-bound CDTb-pore at 2.56-Å resolution revealed that CDTa binding to CDTb-pore induces partial unfolding and tilting of the first CDTa a-helix, and the translocation. In the CDTb-pore, the NSS-loop exists in “in” and “out” conformations, suggesting their involvement in substrate translocation through formation of weak, non-specific interactions. This structural information provides insights into drug design against hypervirulent C. difficile strains.

Molecular Epidemiology of Community-Onset Clostridioides Difficile Infections At A Tertiary Hospital In Mainland China: A Ten-Year (2010-2019) Retrospective Study

Background: Clostridioides difficile infection (CDI) is an increasingly common disease in healthcare facilities and community settings. However, there are limited reports of community-onset CDI (CO-CDI) in China. We retrospectively analyzed the molecular epidemiology of CO-CDI at a tertiary hospital over a period of 10 years. A total of 1307 stool samples from 1213 outpatients were tested by culturing. The presence of toxin genes (tcdA, tcdB, cdtA, and cdtB) were confirmed by PCR. Toxigenic strains were typed using multilocus sequence typing (MLST). Susceptibility to 9 antimicrobials was evaluated using the E-test.Results: Eighty-nine of 1213 outpatients (7.3%) had CO-CDI, 4 of these patients (4.5%) had one or more recurrence, and there were 95 strains of toxigenic C. difficile. Among these strains, 82 (86.3%) had the tcdA and tcdB genes (A+B+) and 5 of these 82 strains were positive for the binary toxin genes (cdtA and cdtB); the other 13 strains (13.7%) had the tcdB gene only (A−B+). There were 15 different STs, and the most prevalent were ST-54 (23.2%), ST-35 (16.8%), and ST-2 (13.7%). All strains were susceptible to metronidazole and vancomycin, and had low resistance to moxifloxacin and tetracycline, but had high resistance to ciprofloxacin, clindamycin, and erythromycin. Twenty-three isolates (24.2%) were multidrug-resistant.Conclusions: Outpatients with CDI were common during this period in our hospital. The C. difficile isolates had high genetic diversity. All isolates were susceptible to metronidazole and vancomycin, and nearly one quarter of all isolates had multidrug resistance.

Cathepsin Release from Lysosomes Promotes Endocytosis of Clostridium perfringens Iota-Toxin

Iota-toxin from Clostridium perfringens type E is a binary toxin composed of two independent proteins: actin-ADP-ribosylating enzyme component, iota-a (Ia), and binding component, iota-b (Ib). Ib binds to target cell receptors and mediates the internalization of Ia into the cytoplasm. Extracellular lysosomal enzyme acid sphingomyelinase (ASMase) was previously shown to facilitate the internalization of iota-toxin. In this study, we investigated how lysosomal cathepsin promotes the internalization of iota-toxin into target cells. Cysteine protease inhibitor E64 prevented the cytotoxicity caused by iota-toxin, but aspartate protease inhibitor pepstatin-A and serine protease inhibitor AEBSF did not. Knockdown of lysosomal cysteine protease cathepsins B and L decreased the toxin-induced cytotoxicity. E64 suppressed the Ib-induced ASMase activity in extracellular fluid, showing that the proteases play a role in ASMase activation. These results indicate that cathepsin B and L facilitate entry of iota-toxin via activation of ASMase.

Molecular epidemiology of community-onset Clostridioides difficile infections at a tertiary hospital in mainland China: A ten-year (2010-2019) retrospective study

Background Clostridioides difficile infection (CDI) is an increasingly common disease in healthcare facilities and community settings. However, there are limited reports of community-onset CDI (CO-CDI) in China. We retrospectively analyzed the molecular epidemiology of CO-CDI at a tertiary hospital over a period of 10 years. Methods A total of 1307 stool samples from 1213 outpatients were tested by culturing. The presence of toxin genes (tcd A, tcd B, cdtA and cdtB) were confirmed by PCR. Toxigenic strains were typed using multilocus sequence typing (MLST). Susceptibility to 9 antimicrobials was evaluated using the E-test. Results Eighty-nine of 1213 outpatients (7.3%) had CO-CDI, 4 of these patients (4.5%) had one or more recurrence, and there were 95 strains of toxigenic C. difficile. Among these strains, 82 (86.3%) had the tcdA and tcdB genes (A + B+) and 5 of these 82 strains were positive for the binary toxin genes (cdtA and cdtB); the other 13 strains (13.7%) had the tcdB gene only (A-B+). There were 15 different STs and the most prevalent were ST-54 (23.2%), ST-35 (16.8%), and ST-2 (13.7%). All strains were susceptible to metronidazole and vancomycin, and had low resistance to moxifloxacin and tetracycline, but had high resistance to ciprofloxacin, clindamycin, and erythromycin. Twenty-three isolates (24.2%) were multidrug-resistant. Conclusions Outpatients with CDI were common during this period in our hospital. The C. difficile isolates had high genetic diversity. All isolates were susceptible to metronidazole and vancomycin, and nearly one quarter of all isolates had multidrug resistance.

The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

Clostridioides difficile is a major cause of nosocomial infection worldwide causing antibiotic-associated diarrhea and some cases are leading to pseudomembranous colitis. The main virulence factors are toxin A and toxin B. Hypervirulent strains of C. difficile are linked to higher mortality rates and most of these strains produce additionally the C. difficile binary toxin (CDT) that possesses two subunits, CDTa and CDTb. The latter is responsible for binding and transfer of CDTa into the cytoplasm of target cells; CDTa is an ADP ribosyltransferase catalyzing the modification of actin fibers that disturbs the actin vs microtubule balance and induces microtubule-based protrusions of the cell membrane increasing the adherence of C. difficile. The underlying mechanisms remain elusive. Thus, we performed a screening experiment using MS-based proteomics and phosphoproteomics techniques. Epithelial Hep-2 cells were treated with CDTa and CDTb in a multiplexed study for 4 and 8 h. Phosphopeptide enrichment was performed using affinity chromatography with TiO2 and Fe-NTA; for quantification, a TMT-based approach and DDA measurements were used. More than 4,300 proteins and 5,600 phosphosites were identified and quantified at all time points. Although only moderate changes were observed on proteome level, the phosphorylation level of nearly 1,100 phosphosites responded to toxin treatment. The data suggested that CSNK2A1 might act as an effector kinase after treatment with CDT. Additionally, we confirmed ADP-ribosylation on Arg-177 of actin and the kinetic of this modification for the first time.