Laboratory diagnosis of Clostridioides (Clostridium) difficile infection in domestic animals: A short review.

Despite the known importance of Clostridioides (Clostridium) difficile infection (CDI) in animals, there are no published guidelines for the diagnosis of CDI. The performance of the available commercial methods, all standardized for human stool samples, can vary according to the animal species. Thus, the aim of the present study was to review the literature on the detection of C. difficile in pigs, horses, and dogs. The detection of toxins A and B using enzyme immunoassays seems to have low performance in piglet and dog samples, while it shows high sensitivity for the diagnosis of CDI in foals. On the other hand, tests for the detection of glutamate dehydrogenase (GDH) have a high sensitivity towards detection of C. difficile in animal samples, suggesting that it can be an adequate screening method. A few studies have evaluated real-time PCR or nucleic acid amplification tests in animal samples and, so far, these methods have also shown a low performance for the detection of C. difficile in animals. Although the intestinal lesions caused by CDI can vary among animal species, histopathology can be a useful auxiliary tool for postmortem diagnosis in animals.

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant Pichia pastoris.

BACKGROUND: Virus infected killer strains of the baker's yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/ß heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants. RESULTS: In this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the ß-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding. CONCLUSION: Successful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

[Diagnosis of infections caused by Clostridium difficile in the Czech Republic: availability, possibilities, and interpretation of laboratory results]. / Diagnostika infekcí vyvolaných Clostridium difficile v Ceské republice--dostupnost, moznosti, interpretace laboratorních nálezu.

OBJECTIVE: To assess the availability of the laboratory diagnosis of infections caused by C. difficile in the Czech Republic (CR), including the range of tests used, possible combinations, and adequate interpretation of model results. MATERIAL AND METHODS: Data were collected through a web questionnaire survey with the participation of representatives of 61 public and private microbiological laboratories. The questionnaire addressed the use of diagnostic test kits and culture media in the diagnosis of C. difficile infection (CDI). In addition, the respondents were asked to interpret a glutamate dehydrogenase (GDH) positive and, at the same time, toxin A/B negative result, without or with laboratory confirmation if available. RESULTS: In the CR, the most commonly used test in the diagnosis of CDI is the C. DIFF Quik Chek Complete® test (Alere) for the detection of GDH and A/B toxins, as reported by 50 (82%) laboratories. Anaerobic culture is performed by 43 (70.5%) laboratories, 21 (48.8%) of which use selective agar (Oxoid). Direct detection of DNA of toxigenic C. difficile is feasible in 17 (27.9%) laboratories, with most of them (15 laboratories) using the closed system Xpert® C. difficile (Cepheid). The diagnosis based only on the detection of GDH and A/B toxins is used by 13 (21.3%) laboratories. Two (3.3%) laboratories detect A/B toxins alone and three (4.9%) laboratories carry out the detection of A/B toxins followed by anaerobic culture. A three step scheme: detection of GDH and A/B toxins with subsequent anaerobic culture is used by 26 (42.6%) laboratories. The detection of GDH and A/B toxins along with a PCR assay are provided by three (4.9%) laboratories. A complete diagnostic scheme for CDI (detection of GDH and A/B toxins, direct detection of DNA, and aerobic culture) is feasible in 14 (23%) laboratories. CONCLUSION: This questionnaire survey study identified 24 different testing algorithms to be in use within the study period (April to July 2014) in the CR. Five (8.2%) laboratories have no highly sensitive screening test such as the detection of GDH or nucleic acid amplification test (NAAT) included in their testing algorithm. Thirteen (21.3%) laboratories perform the detection of GDH and A/B toxins but have no confirmation method to be used if only one test turns out positive. In the case of GDH positivity and A/B toxin negativity, the result should be provided with a supplementary comment on further possibilities for the laboratory detection of CDI and the claimed sensitivity of the test used. If no confirmation test is available, the result should be considered as epidemiologically and clinically significant, once other possible causes of diarrhoea are ruled out.

Solution Structures of Bacillus anthracis Protective Antigen Proteins Using Small Angle Neutron Scattering and Protective Antigen 63 Ion Channel Formation Kinetics.

We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the Staphylococcus aureus α-hemolysin (α-HL) channel in its functional state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the Bacillus anthracis protective antigen 63 (PA63) channel, locate where B. anthracis lethal factor and edema factor toxins (LF and EF, respectively) bind to it, and determine how certain small molecules can inhibit the interaction of LF and EF with the channel. We report here the solution structures of channel-forming PA63 and its precursor PA83 (which does not form channels) obtained with small angle neutron scattering. At near neutral pH, PA83 is a monomer and PA63 a heptamer. The latter is compared to two cryo-electron microscopy structures. We also show that although the α-HL and PA63 channels have similar structural features, unlike α-HL, PA63 channel formation in lipid bilayer membranes ceases within minutes of protein addition, which currently precludes the use of NR for elucidating the interactions between PA63, LF, EF, and potential therapeutic agents.

Detection of toxigenic Clostridium difficile in paediatric patients. / Detección de Clostridium difficile toxigénico en pediatría.

INTRODUCTION: Our main objective was a revision of clinical, microbiological and epidemiological results of Clostridium difficile-associated infection in paediatric patients (2010-2015). We compared the diagnoses performed by detection of toxins in feces and those performed by real-time PCR. METHODS: This retrospective study included 82 paediatric patients. Detection of toxigenic C. difficile was performed sequentially, in diarrheal feces and under clinical request. RESULTS: A total of 39% of the patients were attended at Haematology-oncology Unit and >50% of them had previously received cephalosporins. Fever associated with diarrhea was more frequent in the group of toxin detection, whereas not receiving specific antibiotic treatment was more frequent in the group of positive PCR, without statistically significant differences. CONCLUSIONS: We highlight the presence of C. difficile infection in children under 2years old. A diagnostic testing in selected paediatric patients would be advisable when there is clinical suspicion of infection.

T3SS-Independent Uptake of the Short-Trip Toxin-Related Recombinant NleC Effector of Enteropathogenic Escherichia coli Leads to NF-κB p65 Cleavage.

Effector proteins secreted by the type 3 secretion system (T3SS) of pathogenic bacteria have been shown to precisely modulate important signaling cascades of the host for the benefit of the pathogens. Among others, the non-LEE encoded T3SS effector protein NleC of enteropathogenic Escherichia coli (EPEC) is a Zn-dependent metalloprotease and suppresses innate immune responses by directly targeting the NF-κB signaling pathway. Many pathogenic bacteria release potent bacterial toxins of the A-B type, which-in contrast to the direct cytoplasmic injection of T3SS effector proteins-are released first into the environment. In this study, we found that NleC displays characteristics of bacterial A-B toxins, when applied to eukaryotic cells as a recombinant protein. Although lacking a B subunit, that typically mediates the uptake of toxins, recombinant NleC (rNleC) induces endocytosis via lipid rafts and follows the endosomal-lysosomal pathway. The conformation of rNleC is altered by low pH to facilitate its escape from acidified endosomes. This is reminiscent of the homologous A-B toxin AIP56 of the fish pathogen Photobacterium damselae piscicida (Phdp). The recombinant protease NleC is functional inside eukaryotic cells and cleaves p65 of the NF-κB pathway. Here, we describe the endocytic uptake mechanism of rNleC, characterize its intracellular trafficking and demonstrate that its specific activity of cleaving p65 requires activation of host cells e.g., by IL1ß. Further, we propose an evolutionary link between some T3SS effector proteins and bacterial toxins from apparently unrelated bacteria. In summary, these properties might suggest rNleC as an interesting candidate for future applications as a potential therapeutic against immune disorders.

Investigating pertussis toxin and its impact on vaccination.

Whooping cough, caused by Bordetella pertussis, remains a major global health problem. Each year around 40 million of pertussis cases resulting in 200,000-400,000 annual deaths occur worldwide. Pertussis toxin is a major virulence factor of B. pertussis. Murine studies have shown its importance in bacterial colonization and in immunomodulation to evade innate or adaptive immunity. The toxin is composed of an A protomer expressing ADP-ribosyltransferase activity and a B oligomer, responsible for toxin binding to target cells. The toxin is also a major protective antigen in all currently available vaccines. However, vaccine escape mutants with altered toxin expression have recently been isolated in countries with high vaccination coverage illustrating the need for improved pertussis vaccines.

Detection of A/B toxin and isolation of Clostridium difficile and Clostridium perfringens from foals.

REASONS FOR PERFORMING THE STUDY: Toxin detection and screening could contribute to knowledge of the transmission patterns, risk factors and epidemiology of Clostridium difficile and Clostridium perfringens. OBJECTIVE: To isolate C. difficile and C. perfringens and to detect A/B toxins in faecal samples from diarrhoeic and nondiarrhoeic foals. STUDY DESIGN: Cross-sectional observational study. METHODS: A total of 153 samples from foals were collected: 139 samples from farms and 14 samples from diarrhoeic foals admitted to a veterinary hospital. The A/B toxins were detected by cytotoxicity assay. All suspected colonies of C. perfringens were subjected to polymerase chain reaction for detection of the major toxin genes (α, ß, ε and ι) and for detection of ß2-, NetB- and enterotoxin-encoding genes. Furthermore, C. difficile and C. perfringens isolates were evaluated for in vitro antimicrobial susceptibility. RESULTS: Seven of 153 (4.6%) samples, all from diarrhoeic foals, were positive for C. difficile A/B toxin. Of these, 5 of 14 (35.7%) were from hospitalised foals, and only 2 of 63 (3.2%) diarrhoeic foal samples were from farms (P = 0.002). Clostridium perfringens was isolated from 31 (20.3%) foals, of which 21 of 76 (27.6%) were diarrhoeic and 10 of 76 (13.2%) were nondiarrhoeic, demonstrating a difference between these 2 groups (P = 0.045). Only 4 strains were positive for the ß2-encoding gene (cpb2). All C. difficile and C. perfringens isolates were susceptible to metronidazole and vancomycin. CONCLUSIONS: The present report highlights the need for laboratory diagnostics to differentiate C. difficile-associated infection in foals from other causes of diarrhoea to facilitate adequate antimicrobial therapy. POTENTIAL RELEVANCE: More studies are needed to clarify the role of C. perfringens as a primary agent of diarrhoea in foals.