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.

The Importance of Therapeutically Targeting the Binary Toxin from Clostridioides difficile

Novel therapeutics are needed to treat pathologies associated with the Clostridioides difficile binary toxin (CDT), particularly when C. difficile infection (CDI) occurs in the elderly or in hospitalized patients having illnesses, in addition to CDI, such as cancer. While therapies are available to block toxicities associated with the large clostridial toxins (TcdA and TcdB) in this nosocomial disease, nothing is available yet to treat toxicities arising from strains of CDI having the binary toxin. Like other binary toxins, the active CDTa catalytic subunit of CDT is delivered into host cells together with an oligomeric assembly of CDTb subunits via host cell receptor-mediated endocytosis. Once CDT arrives in the host cell’s cytoplasm, CDTa catalyzes the ADP-ribosylation of G-actin leading to degradation of the cytoskeleton and rapid cell death. Although a detailed molecular mechanism for CDT entry and host cell toxicity is not yet fully established, structural and functional resemblances to other binary toxins are described. Additionally, unique conformational assemblies of individual CDT components are highlighted herein to refine our mechanistic understanding of this deadly toxin as is needed to develop effective new therapeutic strategies for treating some of the most hypervirulent and lethal strains of CDT-containing strains of CDI.

High levels of toxigenic Clostridioides difficile contamination of hospital environments: a hidden threat in hospital-acquired infections in Kenya

Introduction. The contribution of Clostridioides difficile (formerly Clostridium difficile ) to the burden of hospital-associated infections (HAIs) remains undetermined in many African countries. Aim. This study aimed to identify a sensitive and readily adaptable C. difficile detection assay and to evaluate the C. difficile HAI risk in Kenya. Methodology. Sterile swabs in neutralizing buffer were used to sample equipment or surfaces that patients and clinical staff touched frequently. These swabs were either plated directly on chromogenic agar or cultured in an enrichment broth before plating. The swab suspensions, enrichment broth and plate cultures were screened by quantitative PCR (qPCR) to determine the most efficient detection method. The HAI risk was evaluated by testing the C. difficile -positive samples by qPCR for the A, B and binary toxins. Results. C. difficile was detected on 4/57 (7.0 %) equipment and surfaces by direct culture. The additional enrichment step increased the detection rate 10-fold to 43/57 (75.4 %). In total, 51/57 (89.5 %) environmental samples were positive for C. difficile detected through either culture or qPCR. The genes encoding the primary toxins, tcdA and tcdB, were detected on six surfaces, while the genes encoding the binary toxins, cdtA and cdtB, were detected on 2/57 (3.5 %) and 3/57 (5.3 %) surfaces, respectively. Different C. difficile toxin gene profiles were detected: the tcdA+/tcdB− gene profile on 4/10 (40 %) high-touch surfaces, tcdA−/tcdB+ on 3/10 (30 %) surfaces, tcdA+/tcdB+/cdtA+/cdtB+ on 2/10 (20 %) surfaces and tcdA−/tcdB+/cdtB+ on one high-touch surface. Conclusion. The widespread contamination of hospital environments by toxigenic C. difficile gives a strong indication of the high risk of C. difficile infections (CDIs). The two-step culture process described can easily be adapted for monitoring hospital environment contamination by C. difficile .

1HN, 13C, and 15N resonance assignments of the Clostridioides difficile receptor binding domain 2 (CDTb, residues 757–876)

Clostridioides difficile is a bacterial pathogen responsible for the majority of nosocomial infections in the developed world. C. difficile infection (CDI) is difficult to treat in many cases because hypervirulent strains have evolved that contain a third toxin, termed the C. difficile toxin (CDT), in addition to the two enterotoxins TcdA and TcdB. CDT is a binary toxin comprised of an enzymatic, ADP-ribosyltransferase (ART) toxin component, CDTa, and a pore-forming or delivery subunit, CDTb. In the absence of CDTa, CDTb assembles into two distinct di-heptameric states, a symmetric and an asymmetric form with both states having two surface-accessible host cell receptor-binding domains, termed RBD1 and RBD2. RBD1 has a unique amino acid sequence, when aligned to other well-studied binary toxins (i.e., anthrax), and it contains a novel Ca2+-binding site important for CDTb stability. The other receptor binding domain, RBD2, is critically important for CDT toxicity, and a domain such as this is missing altogether in other binary toxins and shows further that CDT is unique when compared to other binary toxins. In this study, the 1H, 13C, and 15N backbone and sidechain resonances of the 120 amino acid RBD2 domain of CDTb (residues 757–876) were assigned sequence-specifically and provide a framework for future NMR-based drug discovery studies directed towards targeting the most virulent strains of CDI.

Acute Hepatopancreatic Necrosis Disease-Causing Vibrio parahaemolyticus Strains Maintain an Antibacterial Type VI Secretion System with Versatile Effector Repertoires

ABSTRACT Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus that have acquired a “selfish plasmid” encoding the deadly binary toxins PirAvp/PirBvp. To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus, we conducted a comparative analysis using the genome sequences of the clinical strain RIMD2210633 and of environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus. Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1), which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer to AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we found highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control. IMPORTANCE Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio parahaemolyticus. Although the plasmid-encoded binary toxins PirAvp/PirBvp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND, and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to what was seen with the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections.

A new tubRZ operon involved in the maintenance of the Bacillus sphaericus mosquitocidal plasmid pBsph

pBsph is a mosquitocidal plasmid first identified from Bacillus sphaericus, encoding binary toxins (Bin toxins) that are highly toxic to mosquito larvae. This plasmid plays an important role in the maintenance and evolution of the bin genes in B. sphaericus. However, little is known about its replication and partitioning. Here, we identified a 2.4 kb minimal replicon of pBsph plasmid that contained an operon encoding TubR-Bs and TubZ-Bs, each of which was shown to be required for plasmid replication. TubR-Bs was shown to be a transcriptional repressor of tubRZ-Bs genes and could bind cooperatively to the replication origin of eleven 12 bp degenerate repeats in three blocks, and this binding was essential for plasmid replication. TubZ-Bs exhibited GTPase activities and interacted with TubR-Bs : DNA complex to form a ternary nucleoprotein apparatus. Electron and fluorescence microscopy revealed that TubZ-Bs assembled filaments both in vitro and in vivo, and two point mutations in TubZ-Bs (T114A and Y260A) that severely impaired the GTPase and polymerization activities were found to be defective for plasmid maintenance. Further investigation demonstrated that overproduction of TubZ-Bs-GFP or its mutant forms significantly reduced the stability of pBsph. Taken together, these results suggested that TubR-Bs and TubZ-Bs are involved in the replication and probably in the partitioning of pBsph plasmid, increasing our understanding of the genetic particularity of TubZ systems.