Pore-Forming Toxin-Driven Recovery of Peroxidase-Mimicking Activity in Biomass Channels for Label-Free Electrochemical Bacteria Sensing.

The development of sensitive, selective, and rapid methods to detect bacteria in complex media is essential to ensuring human health. Virulence factors, particularly pore-forming toxins (PFTs) secreted by pathogenic bacteria, play a crucial role in bacterial diseases and serve as indicators of disease severity. In this study, a nanochannel-based label-free electrochemical sensing platform was developed for the detection of specific pathogenic bacteria based on their secreted PFTs. In this design, wood substrate channels were functionalized with a Fe-based metal-organic framework (FeMOF) and then protected with a layer of phosphatidylcholine (PC)-based phospholipid membrane (PM) that serves as a peroxidase mimetic and a channel gatekeeper, respectively. Using Staphylococcus aureus (S. aureus) as the model bacteria, the PC-specific PFTs secreted by S. aureus perforate the PM layer. Now exposed to the FeMOF, uncharged 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) molecules in the electrolyte undergo oxidation to cationic products (ABTS•+). The measured transmembrane ionic current indicates the presence of S. aureus and methicillin-resistant S. aureus (MRSA) with a low detection limit of 3 cfu mL-1. Besides excellent specificity, this sensing approach exhibits satisfactory performance for the detection of target bacteria in the complex media of food.

Comparative evaluation of the STANDARD M10 and Xpert C. difficile assays for detection of toxigenic Clostridioides difficile in stool specimens.

This study compared the performance of two commercial molecular assays, the STANDARD M10 Clostridioides difficile assay (M10) and the Xpert C. difficile assay (Xpert), for detecting toxigenic C. difficile in stool specimens. A total of 487 consecutive stool specimens submitted for routine C. difficile testing between June and November 2023 were included. Following routine testing using C. DIFF QUIK CHEK COMPLETE (QCC), M10 and Xpert were tested in parallel, alongside toxigenic culture (reference standard). Additionally, two-step algorithms, using QCC on the first step and either M10 or Xpert on the second step, were assessed. Both M10 and Xpert demonstrated a sensitivity and negative predictive value (NPV) of 100%. M10 exhibited significantly higher specificity and positive predictive value (PPV; 91.9% and 64.2%, respectively) than Xpert (90.3% and 59.8%, respectively). Both two-step algorithms showed a sensitivity and NPV of 98.4% and 99.8%, respectively. The specificity and PPV of the two-step algorithm using M10 (95.2% and 75.0%, respectively) were slightly higher than those of the one using Xpert (94.8% and 73.2%, respectively), without statistical significance. Receiver operating characteristic curve analysis, assessing the predictive ability of cycle threshold (Ct) values for the detection of free toxin, exhibited an area under the curve of 0.825 for M10 and 0.843 for Xpert. This indicates the utility of Ct values as predictors for the detection of free toxin in both assays. In conclusion, M10 proves to be an effective diagnostic tool with performance comparable to Xpert, whether utilized independently or as part of a two-step algorithm.

What, How, When, and Where: Spatiotemporal Water Quality Hazards of Cyanotoxins in Subtropical Eutrophic Reservoirs.

Though toxins produced during harmful blooms of cyanobacteria present diverse risks to public health and the environment, surface water quality surveillance of cyanobacterial toxins is inconsistent, spatiotemporally limited, and routinely relies on ELISA kits to estimate total microcystins (MCs) in surface waters. Here, we employed liquid chromatography tandem mass spectrometry to examine common cyanotoxins, including five microcystins, three anatoxins, nodularin, cylindrospermopsin, and saxitoxin in 20 subtropical reservoirs spatially distributed across a pronounced annual rainfall gradient. Probabilistic environmental hazard analyses identified whether water quality values for cyanotoxins were exceeded and if these exceedances varied spatiotemporally. MC-LR was the most common congener detected, but it was not consistently observed with other toxins, including MC-YR, which was detected at the highest concentrations during spring with many observations above the California human recreation guideline (800 ng/L). Cylindrospermopsin was also quantitated in 40% of eutrophic reservoirs; these detections did not exceed a US Environmental Protection Agency swimming/advisory level (15,000 ng/L). Our observations have implications for routine water quality monitoring practices, which traditionally use ELISA kits to estimate MC levels and often limit collection of surface samples during summer months near reservoir impoundments, and further indicate that spatiotemporal surveillance efforts are necessary to understand cyanotoxins risks when harmful cyanobacteria blooms occur throughout the year.

Clinical outcomes and treatment necessity in patients with toxin-negative Clostridioides difficile stool samples.

PURPOSE: The clinical significance of negative toxin enzyme immunoassays (EIA) for Clostridioides difficile infections (CDIs) is unclear. Our study aimed to investigate the significance of toxin EIA-negative in the diagnosis and prognosis of CDI. METHODS: All stool specimens submitted for C. difficile toxin EIA testing were cultured to isolate C. difficile. In-house PCR for tcdA, tcdB, cdtA, and cdtB genes were performed using C. difficile isolates. Stool specimens were tested with C. difficile toxins A and B using EIA kit (RIDASCREEN Clostridium difficile toxin A/B, R-Biopharm AG, Darmstadt, Germany). Characteristics and subsequent CDI episodes of toxin EIA-negative and -positive patients were compared. RESULTS: Among 190 C. difficile PCR-positive patients, 83 (43.7%) were toxin EIA-negative. Multivariate analysis revealed independent associations toxin EIA-negative results and shorter hospital stays (OR = 0.98, 95% CI 0.96-0.99, p = 0.013) and less high-risk antibiotic exposure in the preceding month (OR = 0.38, 95% CI 0.16-0.94, p = 0.035). Toxin EIA-negative patients displayed a significantly lower white blood cell count rate (11.0 vs. 35.4%, p < 0.001). Among the 54 patients who were toxin EIA-negative and did not receive CDI treatment, three (5.6%) were diagnosed with CDI after 7-21 days without complication. CONCLUSION: Our study demonstrates that toxin EIA-negative patients had milder laboratory findings and no complications, despite not receiving treatment. Prolonged hospitalisation and exposure to high-risk antibiotics could potentially serve as markers for the development of toxin EIA-positive CDI.

Cyanotoxin Occurrence and Diversity in 98 Cyanobacterial Blooms from Swedish Lakes and the Baltic Sea.

The Drinking Water Directive (EU) 2020/2184 includes the parameter microcystin LR, a cyanotoxin, which drinking water producers need to analyze if the water source has potential for cyanobacterial blooms. In light of the increasing occurrences of cyanobacterial blooms worldwide and given that more than 50 percent of the drinking water in Sweden is produced from surface water, both fresh and brackish, the need for improved knowledge about cyanotoxin occurrence and cyanobacterial diversity has increased. In this study, a total of 98 cyanobacterial blooms were sampled in 2016-2017 and identified based on their toxin production and taxonomical compositions. The surface water samples from freshwater lakes throughout Sweden including brackish water from eight east coast locations along the Baltic Sea were analyzed for their toxin content with LC-MS/MS and taxonomic composition with 16S rRNA amplicon sequencing. Both the extracellular and the total toxin content were analyzed. Microcystin's prevalence was highest with presence in 82% of blooms, of which as a free toxin in 39% of blooms. Saxitoxins were found in 36% of blooms in which the congener decarbamoylsaxitoxin (dcSTX) was detected for the first time in Swedish surface waters at four sampling sites. Anatoxins were most rarely detected, followed by cylindrospermopsin, which were found in 6% and 10% of samples, respectively. As expected, nodularin was detected in samples collected from the Baltic Sea only. The cyanobacterial operational taxonomic units (OTUs) with the highest abundance and prevalence could be annotated to Aphanizomenon NIES-81 and the second most profuse cyanobacterial taxon to Microcystis PCC 7914. In addition, two correlations were found, one between Aphanizomenon NIES-81 and saxitoxins and another between Microcystis PCC 7914 and microcystins. This study is of value to drinking water management and scientists involved in recognizing and controlling toxic cyanobacteria blooms.

Blue revolution turning green? A global concern of cyanobacteria and cyanotoxins in freshwater aquaculture: A literature review.

To enhance productivity, aquaculture is intensifying, with high-density fish ponds and increased feed input, contributing to nutrient load and eutrophication. Climate change further exacerbates cyanobacterial blooms and cyanotoxin production that affect aquatic organisms and consumers. A review was conducted to outline this issue from its inception - eutrophication, cyanobacterial blooms, their harmful metabolites and consequential effects (health and economic) in aquacultures. The strength of evidence regarding the relationship between cyanobacteria/cyanotoxins and potential consequences in freshwater aquacultures (fish production) globally were assessed as well, while identifying knowledge gaps and suggesting future research directions. With that aim several online databases were searched through June 2023 (from 2000), and accessible publications conducted in aquacultures with organisms for human consumption, reflecting cyanotoxin exposure, were selected. Data on cyanobacteria/cyanotoxins in aquacultures and its products worldwide were extracted and analyzed. Selected 63 papers from 22 countries were conducted in Asia (48%), Africa (22%), America (22%) and Europe (8%). Microcystis aeruginosa was most frequent, among over 150 cyanobacterial species. Cyanobacterial metabolites (mostly microcystins) were found in aquaculture water and fish from 18 countries (42 and 33 papers respectively). The most affected were small and shallow fish ponds, and omnivorous or carnivorous fish species. Cyanotoxins were detected in various fish organs, including muscles, with levels exceeding the tolerable daily intake in 60% of the studies. The majority of research was done in developing countries, employing less precise detection methods, making the obtained values estimates. To assess the risk of human exposure, the precise levels of all cyanotoxins, not just microcystins are needed, including monitoring their fate in aquatic food chains and during food processing. Epidemiological research on health consequences, setting guideline values, and continuous monitoring are necessary as well. Further efforts should focus on methods for elimination, prevention, and education.

Cyanobacterial Harmful Algal Mats (CyanoHAMs) in tropical rivers of central Mexico and their potential risks through toxin production.

Cyanobacteria inhabiting lotic environments have been poorly studied and characterized in Mexico, despite their potential risks from cyanotoxin production. This article aims to fill this knowledge gap by assessing the importance of benthic cyanobacteria as potential cyanotoxin producers in central Mexican rivers through: (i) the taxonomic identification of cyanobacteria found in these rivers, (ii) the environmental characterization of their habitats, and (iii) testing for the presence of toxin producing genes in the encountered taxa. Additionally, we introduce and discuss the use of the term "CyanoHAMs" for lotic water environments. Populations of cyanobacteria were collected from ten mountain rivers and identified using molecular techniques. Subsequently, these taxa were evaluated for genes producing anatoxins and microcystins via PCR. Through RDA analyses, the collected cyanobacteria were grouped into one of three categories based on their environmental preferences for the following: (1) waters with high ionic concentrations, (2) cold-temperate waters, or (3) waters with high nutrient enrichment. Populations from six locations were identified to genus level: Ancylothrix sp., Cyanoplacoma sp., and Oxynema sp. The latter was found to contain the gene that produces anatoxins and microcystins in siliceous rivers, while Oxynema tested positive for the gene that produces microcystins in calcareous rivers. Our results suggest that eutrophic environments are not necessarily required for toxin-producing cyanobacteria. Our records of Compactonostoc, Oxynema, and Ancylothrix represent the first for Mexico. Four taxa were identified to species level: Wilmottia aff. murrayi, Nostoc tlalocii, Nostoc montejanii, and Dichothrix aff. willei, with only the first testing positive using PCR for anatoxin and microcystin-producing genes in siliceous rivers. Due to the differences between benthic growths with respect to planktonic ones, we propose the adoption of the term Cyanobacterial Harmful Algal Mats (CyanoHAMs) as a more precise descriptor for future studies.

Epidemiology and diagnostic accuracy of Clostridium perfringens toxins in the intestinal contents of camels, sheep, and cattle: a cross-sectional study in Dakahlia governorate, Egypt.

This study aimed to establish an accurate epidemiological surveillance tool for the detection of different C. perfringens types from 76 diseased and 34 healthy animals in Dakhalia Governorate, Egypt. A total of 110 intestinal content samples were randomly collected from camels, sheep, and cattle. C. perfringens was isolated and biochemically identified by the VITEK2 system. Toxinotyping and genotyping of C. perfringens isolates were specified by a multiscreen ELISA and real-time qPCR (rt-qPCR). The occurrence of C. perfringens was highest among camels (20% in healthy and 25% in diseased) and was lowest in cattle (23.1% and 14.7%). The cpa toxin was detected in all isolates by rt-qPCR and in 7 isolates by ELISA, ext toxin was detected in 7 isolates by rt-qPCR and in 6 isolates by ELISA, and cpb toxin was detected in 2 isolates by both rt-qPCR and ELISA. Four types of C. perfringens were identified by rt-qPCR, type A (65.2%), B (4.3%), C (4.3%), and D (26.1%), and three types by ELISA, type D (17.4%), A (8.7%) and C (4.3%). Our study indicated the prevalence of infection in Dakahlia by C. perfringens type A and D, particularly camels, and recommends adopting an appropriate vaccination strategy among the studied animals.

Alternative boronic acids in the detection of Mycolactone A/B using the thin layer chromatography (f-TLC) method for diagnosis of Buruli ulcer.

BACKGROUND: Mycobacterium ulcerans is the causative agent of Buruli ulcer. The pathology of M. ulcerans disease has been attributed to the secretion of a potent macrolide cytotoxin known as mycolactone which plays an important role in the virulence of the disease. Mycolactone is a biomarker for the diagnosis of BU that can be detected using the fluorescent-thin layer chromatography (f-TLC) technique. The technique relies on the chemical derivatization of mycolactone A/B with 2-naphthylboronic acid (BA) which acts as a fluorogenic chemosensor. However, background interferences due to co-extracted human tissue lipids, especially with clinical samples coupled with the subjectivity of the method call for an investigation to find an alternative to BA. METHODS: Twenty-six commercially available arylboronic acids were initially screened as alternatives to BA using the f-TLC experiment. UV-vis measurements were also conducted to determine the absorption maximum spectra of mycolactone A/B and myco-boronic acid adducts followed by an investigation of the fluorescence-enhancing ability of the boronate ester formation between mycolactone A/B and our three most promising boronic acids (BA15, BA18, and BA21). LC-MS technique was employed to confirm the adduct formation between mycolactone and boronic acids. Furthermore, a comparative study was conducted between BA18 and BA using 6 Polymerase Chain Reaction (PCR) confirmed BU patient samples. RESULTS: Three of the boronic acids (BA15, BA18, and BA21) produced fluorescent band intensities superior to BA. Complexation studies conducted on thin layer chromatography (TLC) using 0.1 M solution of the three boronic acids and various volumes of 10 ng/µL of synthetic mycolactone ranging from 1 µL - 9 µL corresponding to 10 ng - 90 ng gave similar results with myco-BA18 adduct emerging with the most visibly intense fluorescence bands. UV-vis absorption maxima (λmax) for the free mycolactone A/B was observed at 362 nm, and the values for the adducts myco-BA15, myco-BA18, and myco-BA21 were at 272 nm, 270 nm, and 286 nm respectively. The comparable experimental λmax of 362 nm for mycolactone A/B to the calculated Woodward-Fieser value of 367 nm for the fatty acid side chain of mycolactone A/B demonstrate that even though 2 cyclic boronates were formed, only the boronate of the southern side chain with the chromophore was excited by irradiation at 365 nm. Fluorescence experiments have demonstrated that coupling BA18 to mycolactone A/B along the 1,3-diols remarkably enhanced the fluorescence intensity at 537 nm. High-Resolution Mass Spectrometer (HR-MS) was used to confirm the formation of the myco-BA15 adduct. Finally, f-TLC analysis of patient samples with BA18 gave improved BA18-adduct intensities compared to the original BA-adduct. CONCLUSION: Twenty-six commercially available boronic acids were investigated as alternatives to BA, used in the f-TLC analysis for the diagnosis of BU. Three (3) of them BA15, BA18, and BA21 gave superior fluorescence band intensity profiles. They gave profiles that were easier to interpret after the myco-boronic acid adduct formation and in experiments with clinical samples from patients with BA18 the best. BA18, therefore, has been identified as a potential alternative to BA and could provide a solution to the challenge of background interference of co-extracted human tissue lipids from clinical samples currently associated with the use of BA.

Clostridioides difficile PCR Tcdb Cycle Threshold predicts toxin EIA positivity but not severity of infection.

BACKGROUND: Diagnosis of Clostridioides difficile Infection (CDI) entails compatible clinical presentation and laboratory findings. We evaluated real-time polymerase chain reaction (qPCR) cycle threshold (CT) as a predictor for disease severity and TcdB enzyme immunoassay (EIA) results. METHODS: Inpatients or emergency department patients who tested positive for tcdB gene by PCR were evaluated. Patients' stools underwent testing for GDH and TcdA/B by EIA. Medical health records were reviewed for demographic, clinical presentation, laboratory, treatment and outcome data. Severity of CDI was calculated using various severity score indexes. RESULTS: The median CT of cases was 32.05 ± 5.45. The optimal cut-off for predicting toxin EIA positivity and severe CDI based on chart review was 32.6 and 29.8, respectively, with the area under the receiver operator characteristics curve (AUC) of 0.74 and 0.60 respectively. CONCLUSION: CT value was an acceptable predictor for EIA toxin but less so for clinical severity. Our study potentially supports a diagnostic algorithm including CT value to reduce the number of EIA toxin assays performed.

VE303, a Defined Bacterial Consortium, for Prevention of Recurrent Clostridioides difficile Infection: A Randomized Clinical Trial.

Importance: The effect of rationally defined nonpathogenic, nontoxigenic, commensal strains of Clostridia on prevention of Clostridioides difficile infection (CDI) is unknown. Objective: To determine the efficacy of VE303, a defined bacterial consortium of 8 strains of commensal Clostridia, in adults at high risk for CDI recurrence. The primary objective was to determine the recommended VE303 dosing for a phase 3 trial. Design, Setting, and Participants: Phase 2, randomized, double-blind, placebo-controlled, dose-ranging study conducted from February 2019 to September 2021 at 27 sites in the US and Canada. The study included 79 participants aged 18 years or older who were diagnosed with laboratory-confirmed CDI with 1 or more prior CDI episodes in the last 6 months and those with primary CDI at high risk for recurrence (defined as aged ≥75 years or ≥65 years with ≥1 risk factors: creatinine clearance <60 mL/min/1.73 m2, proton pump inhibitor use, remote [>6 months earlier] CDI history). Interventions: Participants were randomly assigned to high-dose VE303 (8.0 × 109 colony-forming units [CFUs]) (n = 30), low-dose VE303 (1.6 × 109 CFUs) (n = 27), or placebo capsules (n = 22) orally once daily for 14 days. Main Outcomes and Measures: The primary efficacy end point was the proportion of participants with CDI recurrence at 8 weeks using a combined clinical and laboratory definition. The primary efficacy end point was analyzed in 3 prespecified analyses, using successively broader definitions for an on-study CDI recurrence: (1) diarrhea consistent with CDI plus a toxin-positive stool sample; (2) diarrhea consistent with CDI plus a toxin-positive, polymerase chain reaction-positive, or toxigenic culture-positive stool sample; and (3) diarrhea consistent with CDI plus laboratory confirmation or (in the absence of a stool sample) treatment with a CDI-targeted antibiotic. Results: Baseline characteristics were similar across the high-dose VE303 (n = 29; 1 additional participant excluded from efficacy analysis), low-dose VE303 (n = 27), and placebo (n = 22) groups. The participants' median age was 63.5 years (range, 24-96); 70.5% were female; and 1.3% were Asian, 1.3% Black, 2.6% Hispanic, and 96.2% White. CDI recurrence rates through week 8 (using the efficacy analysis 3 definition) were 13.8% (4/29) for high-dose VE303, 37.0% (10/27) for low-dose VE303, and 45.5% (10/22) for placebo (P = .006, high-dose VE303 vs placebo). Conclusions and Relevance: Among adults with laboratory-confirmed CDI with 1 or more prior CDI episodes in the last 6 months and those with primary CDI at high risk for recurrence, high-dose VE303 prevented recurrent CDI compared with placebo. A larger, phase 3 study is needed to confirm these findings. Trial Registration: ClinicalTrials.gov Identifier: NCT03788434.

Clostridioides difficile: Characterization of the circulating toxinotypes in an Argentinean public hospital.

Clostridioides difficile is a spore-forming anaerobe microorganism associated to nosocomial diarrhea. Its virulence is mainly associated with TcdA and TcdB toxins, encoded by their respective tcdA and tcdB genes. These genes are part of the pathogenicity locus (PaLoc). Our aim was to characterize relevant C. difficile toxinotypes circulating in the hospital setting. The tcdA and tcdB genes were amplified and digested with different restriction enzymes: EcoRI for tcdA; HincII and AccI for tcdB. In addition, the presence of the cdtB (binary toxin) gene, TcdA and TcdB toxins by dot blot and the cytotoxic effect of culture supernatants on Vero cells, were evaluated. Altogether, these studies revealed three different circulating toxinotypes according to Rupnik's classification: 0, I and VIII, being the latter the most prevalent one. Even though more studies are certainly necessary (e.g. sequencing analysis), it is worth noting that the occurrence of toxinotype I could be related to the introduction of bacteria from different geographical origins. The multivariate analysis conducted on the laboratory values of individuals infected with the most prevalent toxinotype (VIII) showed that the isolates associated with fatal outcomes (GCD13, GCD14 and GCD22) are located in regions of the biplots related to altered laboratory values at admission. In other patients, although laboratory values at admission were not correlated, levels of urea, creatinine and white blood cells were positively correlated after the infection was diagnosed. Our study reveals the circulation of different toxinotypes of C. difficile strains in this public hospital. The variety of toxinotypes can arise from pre-existing microorganisms as well as through the introduction of bacteria from other geographical regions. The existence of microorganisms with different pathogenic potential is relevant for the control, follow-up, and treatment of the infections.

[Evaluation of the Clinical Validity of the Clostridioides difficile Nucleic Acid Detection Kit "Smart Gene® CD ToxinB"].

Clostridioides difficile is the most common anaerobic bacterium that causes healthcare-associated infections, and prompt diagnosis and infection control are important because it causes C. difficile infection (CDI). In this evaluation, the C. difficile nucleic acid detection reagent, Smart Gene CD Toxin B (Mizuho Medy Co., Ltd., hereinafter referred to as the "evaluation reagent") was evaluated for its clinical performance in comparison with real-time PCR and toxigenic culture (TC). Measurement of evaluation reagents and real-time PCR were performed on 157 residual stool specimens from suspected CDI patients. For TC, stool culture was performed, and colonies in which C. difficile was identified by a mass spectrometer (MALDI Biotyper) were checked for toxin production using a rapid antigen diagnostic kit. The results of the evaluation reagents showed a high concordance rate; 100% sensitivity (81/81) and 100% specificity (76/76) with real-time PCR, 89.8% sensitivity (79/88), and 97.1% specificity (67/69) with TC. The evaluation reagent enables a simple nucleic acid amplification test (NAAT) in a short time and is thought to be useful in CDI treatment, which requires rapid diagnosis and infection control.

Higher In Vivo Fecal Concentrations of Clostridioides difficile Toxins A and B in Patients With North American Pulsed-Field Gel Electrophoresis Type 1/Ribotype 027 Strain Infection.

Ultrasensitive, quantitative Clostridioides difficile stool toxin measurement demonstrated significantly higher concentrations of toxins A and B in patients infected with the North American pulsed-field gel electrophoresis type 1/ribotype 027 (NAP-1/027) strain compared with other strains, providing in vivo confirmation of the in vitro association between NAP-1/027 and elevated toxin production.

Clinical Outcomes of Treated and Untreated C. difficile PCR-Positive/Toxin-Negative Adult Hospitalized Patients: a Quasi-Experimental Noninferiority Study.

Clostridioides difficile infection (CDI) is routinely diagnosed by PCR, with or without toxin enzyme immunoassay testing. The role of therapy for positive PCR and negative toxin remains unclear. The objective of this study was to determine whether clinical outcomes of PCR+/cycle threshold-based toxin (CT-toxin)- individuals vary by result reporting and treatment strategy. We performed a quasiexperimental noninferiority study comparing clinical outcomes of PCR+/CT-toxin- individuals by reporting PCR result only (most patients treated) with reporting CT-toxin result only (most patients untreated) in a single-center, tertiary academic hospital. The primary outcome was symptomatic PCR+/CT-toxin+ conversion at 8 weeks. Secondary outcomes included 7-day diarrhea resolution, hospital length of stay, and 30-day all-cause mortality. A total of 663 PCR+/CT-toxin- test results were analyzed from 632 individuals with a median age of 61 years (interquartile range [IQR], 44 to 72) and 50.4% immunocompromised. Individuals in the preintervention group were more likely to have received CDI therapy than those in the intervention group (91.5 versus 15.1%; P < 0.001). Symptomatic toxin conversion at 8 weeks and hospital length of stay failed to establish the predefined thresholds for noninferiority. Lack of diarrhea resolution at 7 days and 30-day all-cause mortality was similar and established noninferiority (20.0 versus 13.7%; adjusted odds ratio [aOR], 0.57; 90% confidence interval [CI], 0.32 to 1.01; P = 0.1; and 8.6 versus 6.5%; aOR, 0.46; 90% CI, 0.20 to 1.04; P = 0.12). These data support the safety of withholding antibiotics for selected hospitalized individuals with suspected CDI but negative toxin.

Presence of Clostridioides difficile in cattle feces, carcasses, and slaughterhouses: Molecular characterization and antibacterial susceptibility of the recovered isolates.

The aims of this study were to isolate and identify Clostridioides difficile from cattle feces and carcasses, and slaughterhouse samples, and to determine the molecular characteristics and antibacterial susceptibility of the recovered isolates. A total of 220 samples, including 100 cattle fecal samples, 100 cattle carcass surface samples, and 20 slaughterhouse samples were used as the study material. In total, 12 (5.45%) samples, including 11 (11%) cattle fecal samples and 1 (5%) slaughterhouse sample, were found to be positive for C. difficile. On the other hand, all of the carcass samples were negative for C. difficile. A total of 11 (91.66%) isolates, including 10 fecal isolates and 1 slaughterhouse wastewater isolate, were found to be positive for the presence of the toxin genes tcdA and tcdB, whilst 1 fecal isolate was found to be negative for both genes. In addition, 3 different ERIC-PCR profiles were identified in the 11 fecal isolates. The ERIC-PCR profile of the slaughterhouse wastewater isolate was found to be similar to one of the ERIC-PCR profiles obtained from the fecal isolates. All of the isolates were resistant to ciprofloxacin and levofloxacin. Considering that the agent is a spore-forming bacterium shed in feces, the detection of C. difficile isolates of different genotypes, some carrying toxin genes, suggests that feces and slaughterhouse wastewater carrying this bacterium may pose a risk for the contamination of carcasses. The current study revealed that hygiene conditions should be performed to the maximum extent in slaughterhouses.

Clostridium perfringens from fresh water fish of Kashmir Himalaya and their aquatic environment: Toxinotyping and phylogenetic analysis.

Clostridium perfringens is a common anaerobic foodborne pathogen known to produce >20 toxins. In nature, this bacterium has 7 different toxinotypes (A-G) based on the presence of its 6 main toxins. The present study examined the occurrence of different toxinotypes of this bacterium in the ichthyofauna and aquatic environments of Kashmir Himalayan lakes, India. A total of 510 samples (210 water; 150 each of common carp and snow trout) were collected from 3 different lacustrine habitats (Dal, Anchar and Nigeen Lakes) of the region. By performing 16S rRNA PCR test, it was observed that all 210 water samples and 80 (26.66%) of 300 fish samples tested for this specific bacterial species were positive. Then by using multiplex-PCR targeting six virulence genes of C. perfringens, it was confirmed that all the 290 isolates from water and fish samples were positive for Toxinotype A, as only cpa toxin gene was amplified. Phylogenetic analysis of the amplified gene and its amino-acid sequences revealed 95%-98% homology with analogous sequences of this bacterial strain reported from China, Egypt and India. The study documents the existence of C. perfringens toxinotype A in the ichthyofauna of Kashmiri Himalayan lakes, entailing that fish can likely act as transmission medium for C. perfringens food poisoning to humans via food.

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.

Isolation, toxinotyping and antimicrobial susceptibility testing of Clostridium perfringens isolated from Pakistan poultry.

BACKGROUND AND OBJECTIVES: Clostridium perfringens is a causative agent of enteric infections in animals including poultry by producing twenty different types of toxins. A single strain produces only a subset of these toxins, which form the basis of its classification into seven toxinotypes (A-G). C. perfringens toxinotype A is a widespread cause of necrotic enteritis (NE) in poultry. The current study was conducted to determine the prevalence of different toxins and antimicrobial susceptibility of C. perfringens isolated from Pakistan NE affected poultry. METHODS: A total of 134 intestinal samples of the diseased birds were collected postmortem and processed for isolation of C. perfringens using tryptose sulphite cycloserine (TSC) agar supplemented with d-cycloserine. Isolates were confirmed by Gram's staining, biochemical and molecular analyses. Toxinotyping was performed by multiplex PCR. Antimicrobial susceptibility profile of isolates was performed by Kirby Bauer disc diffusion method. RESULTS: A total of 34 strains of C. perfringens were isolated from 134 samples with prevalence rate of 25.37%. All the isolated strains were toxinotype A, as they were positive for alpha toxin (CPA) and negative for other tested toxins such as beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), toxin perfringens large (TpeL) and necrotic B-like toxin (NetB). Interestingly, all the isolated strains of C. perfringens were multidrug resistant. The highest resistance was observed against Neomycin, Trimethoprim, Tetracycline and Lincomycin which are routinely used at Pakistan poultry production. CONCLUSION: C. perfringens toxinotype A is prevalent in Pakistan poultry. Incidence of C. perfringens with prevalence rate of 25.37% can pose serious threat to Pakistan's poultry industry given that all the isolated strains were multidrug resistant. Our findings highlight the need for new antibiotics and antibiotic alternatives to overcome multidrug resistance.

Detection of Bacterial Rhamnolipid Toxin by Redox Liposome Single Impact Electrochemistry.

The detection of Rhamnolipid virulence factor produced by Pseudomonas aeruginosa involved in nosocomial infections is reported by using the redox liposome single impact electrochemistry. Redox liposomes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine as a pure phospholipid and potassium ferrocyanide as an encapsulated redox content are designed for using the interaction of the target toxin with the lipid membrane as a sensing strategy. The electrochemical sensing principle is based on the weakening of the liposomes lipid membrane upon interaction with Rhamnolipid toxin which leads upon impact at an ultramicroelectrode to the breakdown of the liposomes and the release/electrolysis of its encapsulated redox probe. We present as a proof of concept the sensitive and fast sensing of a submicromolar concentration of Rhamnolipid which is detected after less than 30 minutes of incubation with the liposomes, by the appearing of current spikes in the chronoamperometry measurement.