Hypervirulent Strains Research Articles

Australian Meningococcal Surveillance Programme Annual Report, 2020.

Invasive meningococcal disease (IMD) is a notifiable disease in Australia, and both probable and laboratory-confirmed cases of IMD are reported to the National Notifiable Diseases Surveillance System (NNDSS). In 2020, there were 90 notifications of IMD, the lowest number documented since records began in the NNDSS in 1991. Of these, 97% (87/90) were laboratory-confirmed cases, with 70% (61/87) confirmed by bacterial culture and 30% (26/87) by nucleic acid amplification testing. The serogroup was determined for 85/87 laboratory-confirmed cases of IMD: serogroup B (MenB) accounted for 64% of infections (54/85); MenW for 19% (16/85); MenY for 16% (14/85); and MenC 1.2% (1/85). Fine typing was available on 60/85 (71%) of cases with serogroup determined; of the typed MenW, all were PorA antigen type P1.5,2 and sequence type 11, the hypervirulent strain reported in recent outbreaks in Australia and overseas. The primary peaks of IMD notifications in Australia in 2020 were observed in infants less than 1 year (16/87, 18%) and in adults aged 45-64 years (14/87, 16%). MenB infections predominated in those aged less than 5 years and 15-19 years; MenW and MenY infections predominated in those aged 45 years or more. All 61 IMD isolates were tested for antimicrobial susceptibility: none were penicillin resistant; however, 56/61 (92%) had decreased susceptibility to penicillin. All isolates were susceptible to ceftriaxone, ciprofloxacin and rifampicin.

Comparative clinical outcomes evaluation of hospitalized patients infected with Clostridioides difficile ribotype 106 vs. other toxigenic strains

BackgroundAlthough Clostridioides difficile surveillance often identifies emerging strains, clinical outcome evaluations are rarely performed. Ribotype (RT) 106 is a commonly isolated C. difficile strain worldwide; however, studies investigating RT 106 clinical outcomes are limited. The purpose of this study was to investigate clinical outcomes of RT 106 infections compared with two other endemic strains of varying virulence. MethodsThis multicenter study evaluated adults hospitalized with C. difficile infection (CDI). C. difficile samples underwent PCR ribotyping and patients infected with RT 106 were compared to patients infected with a known hypervirulent strain (RT 027) and a strain associated with less virulence (RT 014–020). Electronic medical records were reviewed by blinded investigators to assess the primary outcome of poor clinical outcome (composite of initial clinical failure, discharge to a higher level of care, 90-day CDI recurrence, and CDI-contributable mortality). ResultsA total of 396 patients with CDI were identified (RT 106, 32.3%; RT 027, 29.3%; RT 014–020, 38.3%). Patients infected with RT 014–020 less often experienced a poor clinical outcome (40%) compared with RT 106 (56%) and RT 027 (65%) infection (P < 0.0001). After controlling for covariates and using RT 014–020 as a comparator, patients infected with RT 106 (OR, 2.25; 95% CI, 1.36–3.73) or RT 027 (OR, 2.56; 95% CI, 1.52–4.31) had higher odds of poor clinical outcome. Using RT 027 as the comparator, only RT 014–020 was associated with lower odds of poor clinical outcome (OR, 0.42; 95% CI, 0.27–0.65). ConclusionThis study demonstrated that the emergent C. difficile RT 106 was associated with increased rates of poor clinical outcomes compared to RT 014–020 and comparable poor clinical outcomes to RT 027. These findings can help to better understand the clinical significance of this and future emerging ribotypes.

Potential therapeutic targets of Klebsiella pneumoniae: a multi-omics review perspective.

The multidrug resistance developed in many organisms due to the prolonged use of antibiotics has been an increasing global health crisis. Klebsiella pneumoniae is a causal organism for various infections, including respiratory, urinary tract and biliary diseases. Initially, immunocompromised individuals are primarily affected by K. pneumoniae. Due to the emergence of hypervirulent strains recently, both healthy and immunocompetent individuals are equally susceptible to K. pneumoniae infections. The infections caused by multidrug-resistant and hypervirulent K. pneumoniae strains are complicated to treat, illustrating an urgent need to develop novel and more practical approaches to combat the pathogen. We focused on the previously performed high-throughput analyses by other groups to discover several novel enzymes that may be considered attractive drug targets of K. pneumoniae. These targets qualify most of the selection criteria for drug targeting, including an absence of its homolog's gene in the host. The capsule, lipopolysaccharide, fimbriae, siderophores and essential virulence factors facilitate the pathogen entry, infection and survival inside the host. This review discusses K. pneumoniae pathophysiology, including its virulence determinants and further the potential drug targets that might facilitate the discovery of novel drugs and effective treatment regimens shortly.

Global surveillance of antimicrobial resistance and hypervirulence in Klebsiella pneumoniae from LMICs: An in-silico approach

The global emergence of antimicrobial resistance (AMR) among pathogens is a major public health crisis that might soon lead to a post-antibiotic era. Klebsiella pneumoniae, included in the World Health Organization list of critical priority pathogens, is an emerging threat to global health owing to the rapid rise of convergent AMR and hypervirulent strains. Comprehensive epidemiology of AMR-hypervirulent determinants in K. pneumoniae from Low-and Middle-Income Countries (LMICs) is still lacking. As part of rapid risk assessment, an in-silico approach was employed to characterize the AMR and hypervirulence determinants in K. pneumoniae from LMICs. Genomic data of 2432 K. pneumoniae strains were obtained from 33 LMICs representing 4 continents; Asia (n = 12), Africa (n = 18), N. America (n = 2) and Europe (n = 1). All the analysed strains were multiple-drug resistant and 13.6% of them were hypervirulent as well. The co-existence of antibiotic resistant genes conferring concomitant resistance towards life-saving drugs (carbapenems, colistin, fluoroquinolones and fosfomycin) were also observed among the strains, thereby challenging the efficacy of current treatment strategies. The emergence of convergent strains of K. pneumoniae carrying both multi-drug resistance and hypervirulence-associated genes in 12 LMICs is highlighted in this study. Asian region was identified as the major hotspot for convergent strains, especially being confined to 3 countries (India, Pakistan and Vietnam), further exacerbating the situation. This represents a major challenge to disease treatment and public health management in the LMICs. Regular surveillance for emergence of convergence and necessary interventions in the high-risk regions are strongly recommended.

Diagnostic Methods of Clostridioides difficile Infection and Clostridioides difficile Ribotypes in Studied Sample.

Background: Clostridioides (Clostridium) difficile is the most common nosocomial pathogen and antibiotic-related diarrhea in health-care facilities. Over the last few years, there was an increase in the incidence rate of C. difficile infection cases in Slovakia. In this study, the phenotypic (toxigenicity, antimicrobial susceptibility) and genotypic (PCR ribotypes, genes for binary toxins) patterns of C. difficile isolates from patients with CDI were analyzed, from July to August 2016, taken from hospitals in the Horne Povazie region of northern Slovakia. The aim of the study was also to identify hypervirulent strains (e.g., the presence of RT027 or RT176). Methods: The retrospective analysis of biological samples suspected of CDI were analyzed by GDH, anaerobic culture, enzyme immunoassay on toxins A/B, multiplex “real-time” PCR and PCR capillary-based electrophoresis ribotyping, and by MALDI TOF MS. Results: C. difficile isolates (n = 44) were identified by PCR ribotyping, which revealed five different ribotypes (RT001, 011, 017, 081, 176). The presence of hypervirulent RT027 was not identified. The C. difficile isolates (RT001, 011, 081, 176) were susceptible to metronidazole and vancomycin. One isolate RT017 had reduced susceptibility to vancomycin. A statistically significant difference between the most prevalent PCR ribotypes, RT001 and RT176, regarding variables such as albumin, CRP, creatinine, the length of hospitalization (p = 0.175), and glomerular filtration (p = 0.05) was not found. Conclusion: The results of PCR capillary-based electrophoresis ribotyping in the studied samples showed a high prevalence of RT176 and 001.

Host Defense against Klebsiella pneumoniae Pneumonia Is Augmented by Lung-Derived Mesenchymal Stem Cells.

Klebsiella pneumoniae is a common cause of Gram-negative pneumonia. The spread of antibiotic-resistant and hypervirulent strains has made treatment more challenging. This study sought to determine the immunomodulatory, antibacterial, and therapeutic potential of purified murine stem cell Ag-1+ (Sca-1+) lung mesenchymal stem cells (LMSCs) using in vitro cell culture and an in vivo mouse model of pneumonia caused by K pneumoniae. Sca-1+ LMSCs are plastic adherent, possess colony-forming capacity, express mesenchymal stem cell markers, differentiate into osteogenic and adipogenic lineages in vitro, and exhibit a high proliferative capacity. Further, these Sca-1+ LMSCs are morphologically similar to fibroblasts but differ ultrastructurally. Moreover, Sca-1+ LMSCs have the capacity to inhibit LPS-induced secretion of inflammatory cytokines by bone marrow-derived macrophages and neutrophils in vitro. Sca-1+ LMSCs inhibit the growth of K pneumoniae more potently than do neutrophils. Sca-1+ LMSCs also possess the intrinsic ability to phagocytize and kill K. pneumoniae intracellularly. Whereas the induction of autophagy promotes bacterial replication, inhibition of autophagy enhances the intracellular clearance of K. pneumoniae in Sca-1+ LMSCs during the early time of infection. Adoptive transfer of Sca-1+ LMSCs in K. pneumoniae-infected mice improved survival, reduced inflammatory cells in bronchoalveolar lavage fluid, reduced inflammatory cytokine levels and pathological lesions in the lung, and enhanced bacterial clearance in the lung and in extrapulmonary organs. To our knowledge, these results together illustrate for the first time the protective role of LMSCs in bacterial pneumonia.

Detection of carbapenemase-producing, hypervirulent Klebsiella spp. in wastewater and their potential transmission to river water and WWTP employees

Wastewater treatment plants (WWTPs) release drug-resistant microorganisms to water bodies (with effluents), and WWTP employees are exposed to bioaerosol emissions from the processed wastewater. Bacteria of the genus Klebsiella, in particular carbapenemase-producing (CP), hyper-virulent (Hvr) strains of Klebsiella pneumoniae, play a special role in this process. Klebsiella spp. strains isolated from wastewater, river water and the upper respiratory tract of WWTP employees were analyzed in this study. The isolated strains were identified as K. pneumoniae (K. pn) or K. non-pneumoniae (K. npn). The prevalence of nine types of genes encoding resistance to beta-lactams, nine genes encoding virulence factors and K1/K2 capsular serotypes, three genes encoding multi drug effluent pump systems, and the class 1 integron-integrase gene was determined by PCR. A total of 284 Klebsiella spp. isolates were obtained in the study: 270 environmental strains and 14 strains from the upper respiratory tract. Among environmental isolates 90.7% (245/270) harbored beta-lactam resistance genes, 17.4% (47/270) were classified as CP strains, 11.1% (30/270) were classified as Hvr strains, and 1.9% (5/270) were classified as CP-Hvr strains. CP-Hvr strains were also isolated from WWTP employees. Genes encoding β-lactamases (including carbapenemases), complete efflux pump systems and the K1 serotype were identified more frequently in K. pn strains. In turn, K. npn strains were characterized by a higher prevalence of blaSHV and intI1 genes and K2 serotype gene. The strains isolated from wastewater and river water also differed in the abundance of drug resistance and virulence genes. The results of the study indicate that CP-Hvr K. pn strains are possibly transmitted from wastewater via bioareosol to the upper respiratory tract of WWTP employees. blaGES-type carbapenemases significantly contributed to the spread of drug resistance in the environment.

What's a SNP between friends: The lineage of Clostridioides difficile R20291 can effect research outcomes

Clostridioides difficile R20291 is the most studied PCR-Ribotype 027 isolate. The two predominant lineages of this hypervirulent strain, however, exhibit substantive phenotypic differences and possess genomes that differ by a small number of nucleotide changes. It is important that the source of R20291 is taken into account in research outcomes.

Meningococcal Infection in Novosibirsk: Changes in Epidemiology and Prevention Problems

Relevance. Meningococcal disease is a relatively rare but still potentially fatal and disabling infection, affecting primarily healthy people of all age groups, and remains an important public health problem.Aims. The aim of the study was to analyze the long-term dynamics of the incidence of MI in the Novosibirsk region (NSO; identification and study of the factors that determined the change in the incidence of MI in 2019 in the region.Materials &amp; Methods. A retrospective comparative epidemiological analysis of the incidence of the population of the Novosibirsk region in 1992–2019 was carried out. using statistical reporting forms No. 2 «Information on infectious and parasitic diseases»; state reports «On the state of sanitary and epidemiological well-being ofthe population oftheRussian Federation» for 1998–2015, and according to the NSO for 2005–2019.Results. An analysis of the epidemiological patterns of MI in the NSO revealed the presence of significant fluctuations in the incidence rates with multiple replacements of the leading strains of meningococcus, in the absence of immunity to all the main pathogens of MI in the vast majority of the region's inhabitants (from 60% to 82.92% of the population in different years remained seronegative to meningococcus serogroup A, 100% of the examined are seronegative to serogroup C). Simultaneously with the change in the epidemic situation in MI in the NSO in 2019, a tendency towards an increase in the incidence of MI was registered during 2016–2019 and in all regions ofCentral Asia, from where the influx oflabor migrants with their families continues. As anti-epidemic measures in risk groups, vaccination was used mainly against meningococcus serogroup A in combination with chemoprophylaxis in contact persons.Conclusion. The NSO (Novosibirsk) is characterized by: dynamic changes in the circulating serogroups of meningococcus, the presence of refugees and migrants with a high risk of infection with local strains and the introduction of new ones, including hypervirulent strains from regions on the transit route; high risk of repeated outbreaks and further complication of the epidemic situation. It is necessary to form a regional vaccine prophylaxis program, taking into account the variability of the etiology of MI and the most effective prevention strategies.

Candida gut colonization, yeast species distribution, and biofilm production in Clostridioides difficile infected patients: a comparison between three populations in two different time periods

Candida gut colonization and yeast biofilm production capacity were investigated, by means of XTT reduction assay, in Clostridioides difficile infected (CDI) patients, in non-CDI diarrheic patients, and in healthy donors in two different time periods (2013–2015 and 2018–2019 respectively). Candida gut colonization was significantly (p < 0.001) associated to C. difficile infection, and to patients infected with hypervirulent C. difficile strains bearing the tcdC deletion at nucleotide 117 (p = 0.0003). Although there was not a prevalent yeast species in CDI patients, C. albicans was the species significantly (p < 0.001) associated to both the infections sustained by the non-hypervirulent C. difficile strains and those caused by the hypervirulent strain (p = 0.001). The biofilm production by the yeasts isolated from the CDI patients and from non-CDI diarrheic patients did not differ significantly. However, a significantly (p = 0.007) higher biofilm production was observed in the Candida strains, particularly C. albicans, isolated from healthy donors compared to that of the yeasts cultured from CDI patients. Seasonal occurrence was observed in the isolation rate of CDI and non-CDI diarrheic cases (p = 0.0019), peaking in winter for CDI patients and in spring for non-CDI diarrheic patients. Furthermore, seasonality emerged in the gut colonization by Candida of CDI patients in the winter. It seems, therefore, that the reduced capacity of biofilm production by Candida strains isolated from CDI patients might have a role in the development of C. difficile infection, probably facilitating the spread of the bacteria into the gut thus amplifying their pathogenic action.

Hypervirulent Klebsiella pneumoniae Endogenous Endophthalmitis-A Global Emerging Disease.

The review aims to document the new emerging hypervirulent Klebsiella pneumoniae (Kp) endogenous endophthalmitis (EKE) in terms of incidence, microbiological characterization of the pathogenic agent, associated risk factors, management, and outcomes. Hypervirulent (hv) strains of KP (hvKp) induce invasive liver abscesses (LA) with specific clinical features. Up to 80–90% of cases have hepatic liver abscess as a primary focus of infection, followed by renal or lung hvKp infections. However, the incidence of EKE in patients with KPLA varied between 3.4% (19) and 12.6% (13), with a total of 95 cases of endophthalmitis in 1455 cases of KPLA (6.5%). Severe visual loss was encountered in 75% of cases, with 25% bilateral involvement. Intravitreal antibiotics are the mainstay therapeutic approach. Pars plana vitrectomy is a subject of controversy. HvKp strains present mostly natural “wild-type” antibiotic resistance profile suggestive for community-acquired infections, being highly susceptive to the third and fourth generation of cephalosporins and carbapenems. Antimicrobial resistance in hypervirulent strains was recently documented via plasmid transfer and may result in extremely difficult to treat cases. Global dissemination of these strains is a major epidemiologic shift that should be considered in the diagnostic and therapeutic management of patients with endogenous endophthalmitis. Ophthalmologic screening in patients with KPLA and other hvKp infections and a multidisciplinary therapeutic approach is extremely important for early diagnosis and preservation of the visual function.

Does high adherence to contact precautions lead to low in-hospital transmission of multi-drug-resistant micro-organisms in the endemic setting?

Conflicting results have been published on the impact of contact precautions (CPs) on reduction of transmission of multi-drug-resistant micro-organisms (MDROs) in the endemic setting. Ambiguous definitions coupled with low adherence partly explain these differences. We prospectively monitored the level of adherence to CPs and aimed to relate it to in-hospital transmission of MDROs. Between January 2016 and March 2018, all patients under CPs underwent continuous monitoring of adherence to CPs by routine on-site visits on days 0, 3 and 7 after initiating CPs using a standardized checklist. The protocol included 10 interventions that were routinely checked such as CP sign at the door as well as wearing of gowns and gloves upon entry to the patient room. Patients requiring CPs were defined as colonized or infected with MDROs (meticillin-resistant Staphylococcus aureus (MRSA), non-Escherichia coli extended-spectrum beta lactamase (ESBL) Enterobacterales, vancomycin-resistant enterococci (VRE) and carbapenem-resistant Gram-negative micro-organisms (CRGN)) as well as patients infected with respiratory viruses, norovirus, scabies and hypervirulent strains of Clostridioides difficile. Overall, data from 13,756 CP records from 1378 visits of 812 patients were analysed. Adherence varied between 93% and 100% for each intervention, except for "separate space for contaminated material" with an adherence of 5.3-6.1%. The incidence of in-hospital transmission during the study period was extremely low for MRSA, VRE, non-E.coli ESBL Enterobacterales and CRGN with 0.00-0.064 cases/1000 patient days. High adherence coupled with continuous monitoring of CPs correlated with a very low in-hospital transmission rate. These results indicate that CPs are highly effective if routine monitoring of adherence is implemented.

The changing face of meningococcal infection.

Menigococcal infection is caused by Neisseria meningitidis, a Gram negative diplococci. Invasive meningococcal disease (IMD) is caused by 6 capsular groups. The spectrum of infection is broad, with meningitis and meningococcal sepsis associated with a case fatality of between 4 and 20%. The main burden of disease is felt in the under 1′s and this is where vaccination has been focused. Vaccination against MenC begun in 1999, with vaccines against Men ACWY and MenB added to the schedule in 2015. Over the last 1o years rates of IMD in the UK have fallen by over 50%. The impact of COVID-19 on cases has also been felt, with early data suggesting a significant drop in cases during the first wave in 2020. Despite the success of vaccination we need to remain vigilant. Clonal expansion of hypervirulent strains has been seen in epidemics and we have a significant proportion of the population who remain unvaccinated.

Whole Genome-Based Characterization of Listeria monocytogenes Isolates Recovered From the Food Chain in South Africa.

Listeria monocytogenes is an important foodborne pathogen which has the ability to adapt and survive in food and food processing facilities where it can persist for years. In this study, a total of 143 L. monocytogenes isolates in South Africa (SA) were characterized for their strain’s genetic relatedness, virulence profiles, stress tolerance and resistance genes associated with L. monocytogenes. The Core Genome Multilocus Sequence Typing (cgMLST) analysis revealed that the most frequent serogroups were IVb and IIa; Sequence Types (ST) were ST204, ST2, and ST1; and Clonal Complexes (CC) were CC204, CC1, and CC2. Examination of genes involved in adaptation and survival of L. monocytogenes in SA showed that ST1, ST2, ST121, ST204, and ST321 are well adapted in food processing environments due to the significant over-representation of Benzalkonium chloride (BC) resistance genes (bcrABC cassette, ermC, mdrL and Ide), stress tolerance genes (SSI-1 and SSI-2), Prophage (φ) profiles (LP_101, vB LmoS 188, vB_LmoS_293, and B054 phage), plasmids profiles (N1-011A, J1776, and pLM5578) and biofilm formation associated genes. Furthermore, the L. monocytogenes strains that showed hyper-virulent potential were ST1, ST2 and ST204, and hypo-virulent were ST121 and ST321 because of the presence and absence of major virulence factors such as LIPI-1, LIPI-3, LIPI-4 and the internalin gene family members including inlABCEFJ. The information provided in this study revealed that hyper-virulent strains ST1, ST2, and ST204 could present a major public health risk due to their association with meat products and food processing environments in SA.

Exoproteomic analysis of two MLST clade 2 strains of Clostridioides difficile from Latin America reveal close similarities

Clostridioides difficile BI/NAP1/ribotype 027 is an epidemic hypervirulent strain found worldwide, including in Latin America. We examined the genomes and exoproteomes of two multilocus sequence type (MLST) clade 2 C. difficile strains considered hypervirulent: ICC-45 (ribotype SLO231/UK[CE]821), isolated in Brazil, and NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica. C. difficile isolates were cultured and extracellular proteins were analyzed using high-performance liquid chromatography-tandem mass spectrometry. Genomic analysis revealed that these isolates shared most of the gene composition. Only 83 and 290 NAP1/027 genes were considered singletons in ICC-45 and NAP1/027, respectively. Exoproteome analysis revealed 197 proteins, of which 192 were similar in both strains. Only five proteins were exclusive to the ICC-45 strain. These proteins were involved with catalytic and binding functions and indirectly interacted with proteins related to pathogenicity. Most proteins, including TcdA, TcdB, flagellin subunit, and cell surface protein, were overrepresented in the ICC-45 strain; 14 proteins, including mature S-layer protein, were present in higher proportions in LIBA5756. Data are available via ProteomeXchange with identifier PXD026218. These data show close similarity between the genome and proteins in the supernatant of two strains with hypervirulent features isolated in Latin America and underscore the importance of epidemiological surveillance of the transmission and emergence of new strains.

Profile of Helicobacter pylori cagA &vacA genotypes and its association with the spectrum of gastroduodenal disease

PurposeGlobally, H. pylori virulence factors cagA and vacA genotypes and its variation is leading to the austere form of the gastroduodenal disease. Our objectives were to detect H. pylori in dyspeptic patients from biopsy samples with the validation of the various existing diagnostic tools and to screen the cagA, vacA genotypes profile from biopsy specimens and how it impacts in progression of gastroduodenal disease in southern India. Methods374 patients who attended endoscopy unit at Kasturba Hospital, Manipal with their consent obtained their biopsies. H. pylori were detected by HPE, Culture, RUT and PCR and its virulence gene were patterned with PCR. ResultsThe positive rate of H. pylori by HPE, RUT, Culture and PCR were 51.33%, 47.1%, 32.4% and 50.3% respectively and comparison by Bayesian LCMs analysis showed PCR is superior among them. The frequency of H. pylori virulence gene viz cagPAI (cagA) were 80.9%, and vacA alleles-s1m1 (42%), s1m2 (33%) and s2m2 (25%) genotypes by PCR respectively. Four combinations of cagA/vacA genotypes were noted, majority of strains harboured cagA+/vacA s1m1 genotypes (42.6%), interestingly this hyper-virulent strain more frequently seen in severe gastroduodenal disease whereas cagPAI negative strains as well as cagA−/vacA s2m2 combinations (19.1%) are seen most commonly in functional dyspepsia cases and depicted significant association by Chi-square test. ConclusionsThis study validates and compares the existing diagnostic methods for detecting H. pylori in biopsies. Also, it reveals some pattern of virulence gene combination will play a vital role in disease progression.

Community-acquired Klebsiella spp Meningitis/Invasive Infection in Filipino-descent Patients Living in Greece: A Case Series.

Klebsiella spp community-acquired meningitis caused by hypervirulent strains is well described as part of a distinct syndrome consisting of liver abscess and multiple septic metastatic lesions (Klebsiella pneumoniae invasive syndrome) occurring usually in diabetic, alcoholic, elderly or cancer patients, in Taiwan and other South-East Asian countries. In Western countries, these infections are very rare in natives and usually occur in patients of Asian origin. We report three cases of Filipino-origin patients, residents of Greece, with community-acquired invasive Klebsiella meningitis, who were treated in our ICU over a 10-year period.LEARNING POINTSCommunity-acquired Klebsiella spp meningitis has a very bad prognosis.A physician must suspect an invasive Klebsiella infection in patients of Asian origin, even though they are residents of Western countries and have not visited their homeland recently.

Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection

C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB–CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB.

Transition From PCR-Ribotyping to Whole Genome Sequencing Based Typing of Clostridioides difficile.

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not complete, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters, using cutoffs of 30-50 allele differences. The exceptions are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile.

Characterization and Pharmacological Inhibition of the Pore-Forming Clostridioides difficile CDTb Toxin.

The clinically highly relevant Clostridioides (C.) difficile releases several AB-type toxins that cause diseases such as diarrhea and pseudomembranous colitis. In addition to the main virulence factors Rho/Ras-glycosylating toxins TcdA and TcdB, hypervirulent strains produce the binary AB-type toxin CDT. CDT consists of two separate proteins. The binding/translocation B-component CDTb facilitates uptake and translocation of the enzyme A-component CDTa to the cytosol of cells. Here, CDTa ADP-ribosylates G-actin, resulting in depolymerization of the actin cytoskeleton. We previously showed that CDTb exhibits cytotoxicity in the absence of CDTa, which is most likely due to pore formation in the cytoplasmic membrane. Here, we further investigated this cytotoxic effect and showed that CDTb impairs CaCo-2 cell viability and leads to redistribution of F-actin without affecting tubulin structures. CDTb was detected at the cytoplasmic membrane in addition to its endosomal localization if CDTb was applied alone. Chloroquine and several of its derivatives, which were previously identified as toxin pore blockers, inhibited intoxication of Vero, HCT116, and CaCo-2 cells by CDTb and CDTb pores in vitro. These results further strengthen pore formation by CDTb in the cytoplasmic membrane as the underlying cytotoxic mechanism and identify pharmacological pore blockers as potent inhibitors of cytotoxicity induced by CDTb and CDTa plus CDTb.