Non-toxigenic Strains Research Articles

Clostridioides difficile aggravates dextran sulfate solution (DSS)-induced colitis by shaping the gut microbiota and promoting neutrophil recruitment

ABSTRACT Clostridioides difficile is a pathogen contributing to increased morbidity and mortality of patients with inflammatory bowel disease (IBD). To determine how C. difficile affects the severity of colitis, we constructed a dextran sulfate solution-induced colitis model challenged with C. difficile. Without antibiotic administration, C. difficile led to transient colonization in mice with colitis, but still significantly enhanced disease severity as assessed by weight loss, histopathological damages, and inflammatory cytokine concentrations. Because this effect is independent of toxin production as shown by infection with a non-toxigenic strain, we focused on changes in the gut microbiota. The microbiota altered by C.difficile, featured with reduced proportions of g_Prevotellaceae_UCG-001 and g_Muribaculaceae, were confirmed to contribute to disease severity in colitis mice via fecal microbiota transplantations. The inflamed colon showed neutrophil accumulation by flow cytometric analysis and myeloperoxidase immunochemical staining. There was enrichment of upregulated genes in leukocyte chemotaxis or migration as shown by RNA sequencing analysis. The isolated neutrophils from C. difficile-infected mice with colitis showed a robust migratory ability and had enhanced expression of cytokines and chemokines. We observed a detrimental role of neutrophils in the progress of disease by hindering neutrophil recruitment with the CXCR2 inhibitor SB225002. Furthermore, neutrophil recruitment appeared to be regulated by interleukin (IL)-1β, as inhibition of IL-1β production by MCC950 markedly ameliorated inflammation with decreased neutrophil accumulation and neutrophil-derived chemokine expression. In conclusion, our study provides information on the complicated interaction between microbiota and immune responses in C. difficile-induced inflammation in mice with colitis. Our findings could help determine potential therapeutic targets for patients with IBD concurrent with C. difficile infection.

Genome sequence and probiotic potential of newly isolatedEnterococcus duransstrain MN187066

Enterococci are commensals of the human intestinal tract. Their use as probiotics is supported by their ability to confer several health benefits and eliminate foodborne pathogens but is controversial due to the presence of virulence and antibiotic resistance traits. To use them as probiotics requires thorough research to establish their safety. Here, we sequenced the whole genome of a newly isolated Enterococcus durans MN187066 and used a suite of bioinformatics tools to analyze its beneficial probiotic traits as well as antimicrobial resistance and virulence genes. The whole genome had a length of 2 978 152bp, and an average G+C content of 37.88%. The bopABCD genes involved in biofilm formation were annotated in the genome. However, further analysis showed that these genes are mostly helpful in strengthening their colonization and establishment in the gastrointestinal tract. Also, we identified secondary metabolite gene clusters and the bacteriocins Enterolysin A and Enterocin P. We also identified repUS15 and rep1 replicons and genes that were associated with antimicrobial resistance and virulence. Nevertheless, vancomycin resistance genes were not detected. Our results show that the Ent. durans strain MN187066 can be considered a nontoxigenic strain and produces beneficial metabolites that are critical for their success as probiotics.

Genomic Analysis of Corynebacterium diphtheriae Strains Isolated in the Years 2007-2022 with a Report on the Identification of the First Non-Toxigenic tox Gene-Bearing Strain in Poland.

Infections caused by non-toxigenic Corynebacterium diphtheriae have been reported every year in Poland since 2004, with the ST8 biovar gravis strains being most commonly isolated. This study analyzed thirty strains isolated between 2017 and 2022 and six previously isolated strains. All the strains were characterized using classic methods in terms of species, biovar level, and diphtheria toxin production, as well as by means of whole genome sequencing. The phylogenetic relationship based on SNP analysis was determined. The number of C. diphtheriae infections has been rising in Poland every year with a maximum of 22 cases in the year 2019. Since 2022, only the non-toxigenic gravis ST8 (most common) and mitis ST439 (less common) strains have been isolated. An analysis of the genomes of the ST8 strains showed that they had many potential virulence factors, such as adhesins and iron-uptake systems. The situation rapidly changed in 2022 and strains from different STs were isolated (ST32, 40, and 819). The ST40 biovar mitis strain was found to be non-toxigenic tox gene-bearing (NTTB), with the tox gene inactivated due to a single nucleotide deletion. Such strains were previously isolated in Belarus. The sudden appearance of new C. diphtheriae strains with different STs and the isolation of the first NTTB strain in Poland indicate that C. diphtheriae should be classified as a pathogen of special public health concern.

Conservation of cholera vibrios in complex microcosm containing green microalgae

BACKGROUND: In the summer-autumn period, the peak of phytoplankton reproduction and flowering occurs and results in the deterioration of the quality of river water is noted. At the same time, cases of gastrointestinal diseases in humans are recorded, and epidemic complications of cholera may occur in endemic areas. V. cholerae form biofilm on the surface of chitinous hydrobionts and plastic components can lead to the spread of V. cholerae, possibly explaining the autochthonous mechanism of their existence in water bodies.
 AIM: This study determines the duration of Vibrio cholerae preservation on biotic (chitin) and abiotic (plastic) substrates in the presence of green unicellular algae with a change in cultivation temperature under experimental conditions.
 MATERIALS AND METHODS: This study used bacteriological and molecular genetic methods to achieve its goal.
 RESULTS: The preservation of toxigenic and non-toxigenic strains of V. cholerae O1 El Tor and V. cholerae O139 (ctxАВ+tcpА+csh1 and ctxАtcpАcsh1+) for six months, including three months at a low temperature simulating the autumn-winter period, as part of biofilms in microcosms, where one component is green microalgae. We noted an excess of the concentration of V. cholerae O1 and O139 serogroups by two orders of magnitude in samples where one of the components is chitin and the reproduction of green microalgae in the presence of a chitin substrate. This is probably one stage in the food chain in the ecology of water bodies and, accordingly, can be a reservoir for biofilm forms of cholera vibrios. Preservation of V. cholerae O1 El Tor non-toxigenic strain in a viable state in biofilm samples on plastic at decreased temperatures 82C for three months is possibly due to the presence of a cold shock gene in its genome.
 CONCLUSION: Without a substrate for adhesion, colonization, and biofilm formation, vibrios are incapable of long-term persistence at low temperatures.

A rapid multiplex real-time PCR detection of toxigenic Clostridioides difficile directly from fecal samples

This study developed a new single-tube multiplex real-time PCR method for detecting toxigenic C. difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets, which could be performed on kinds of polymerase chain reaction device including point-of-care testing (POCT), with improved detection efficiency. The specificity, sensitivity, and repeatability of each gene was evaluated using 69 C. difficile isolates and 74 fecal samples. Results were compared with established PCR, qPCR, and ELISA methods. Interspecies specificity was 100% based on six common intestinal pathogens (Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum). The lower detection limit (LDL) for tcdA, tcdB, and cdtB with pure C. difficile DNA was 101,100, and 100 copies/μL, respectively, the coefficients of variation among different experimental batches and within each experimental batch were both less than 3%, which shows that this method has strong repeatability. And the LDL of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively. In addition, the efficiency for detection of tcdA was compared with established PCR and real-time PCR methods, demonstrating high consistency (98.4%) and similar sensitivity. ELISA was used to confirm inconsistent results, which were identical with our method. The sensitivity and specificity for detecting toxigenic C. difficile in fecal samples were 96.49% and 94.12% compared with the toxigenic culture (TC). This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China.

Comparative analysis of the structure and expression of the <i>vasH</i> regulatory gene of type VI secretion system in toxigenic and non-toxigenic <i>Vibrio cholerae</i> strains

Objective. The comparative analysis of the structure of the regulatory gene vasH of the type VI secretion system and its expression in toxigenic and non-toxigenic V. cholerae O1, biovar El Tor strains.
 Materials and methods. We used 35 strains isolated from patients and from the environmental samples in the territory of Russia and Ukraine between 1970 and 2017. Analysis of the structure of the vasH gene and the amino acid sequence of the protein was carried out using Ugene 1.32, Mega X, and Bioedit v. 7.0.9.0. The relative level of vasH expression was studied by 2Ct.
 Results. The The structure of the vasH gene and the amino acid sequence of VasH protein in toxigenic typical strains and genovariants of V. cholerae O1, El Tor biovar (genotype ctxA+tcpA+) have been shown to be identical to the reference V. cholerae n16961 O1, El Tor biovar strain. The vasH sequence is variable in isolates lacking ctxA and tcpA genes (ctxAtcpA), and does not differ from the reference in ctxAtcpA+ (with the exception of one strain). The studied toxigenic typical strains and the genovariants have a similar relative level of expression of the vasH gene. In isolates that do not contain the ctxA and tcpA genes, the expression of this gene is comparable to toxigenic strains, and is 3.1 times higher in ctxAtcpA+ strains than that of ctxAtcpA and 2.142.6 times higher than that of toxigenic ones.
 Conclusion. The analysis of toxigenic and non-toxigenic V. cholerae O1, biovar El Tor strains isolated in Russia and Ukraine in different periods of the current cholera pandemic confirmed the data of foreign researchers on vasH gene being intact in toxigenic isolates and variable in isolates lacking ctxA and tcpA genes. Meanwhile, the structure of vasH gene has been shown to be identical to that of toxigenic ones in 99% of the studied ctxAtcpA+ strains. The expression of the vasH gene has been detected in all studied strains, being the highest in ctxATtcpA+ strains. Only two non-toxigenic strains presumably synthesizing the functionally inactive VasH protein have been identified.

Detection of hydrolases of different subclasses in cell-free fractions of Vibrio cholerae O1 and O139 serogroups using radial enzymatic diffusion in agarose gel

Cholera remains an actual infection worldwide, which dictates the need for a comprehensive study of its pathogens and, in particular, their hydrolytic enzymes that interact with the cells of the macroorganism. We determined the presence of hydrolases from different subclasses in preparations of cell-free fractions obtained from 58 strains of Vibrio cholerae O1 and O139 serogroups of different epidemic significance and origins using radial enzyme diffusion in agarose gels with various substrates. The analysis revealed statistically significant differences in enzyme activity depending on the origin and epidemiological significance of the original strain. We found that preparations obtained from non-toxigenic strains had increased activity of proteases and chitinolytic enzymes, while those from toxigenic strains showed high activity of mucinases, lipolytic enzymes, and nucleases. These data can be applied in the microbiological diagnosis of cholera as an additional biochemical characterization of Vibrio cholerae strains, as well as contribute to the understanding of the role of enzymes in the pathogenesis and adaptation of cholera pathogens.

A New Perspective on Metabolites and Bioactive Compounds from Fungi.

Fungi play an important role in the solution to important global problems. Making use of processes and goods that are based on fungi can help promote sustainability by making the most efficient use of natural resources. Fungi stand apart from other organisms due to their extraordinary capacity to generate organic compounds. They are necessary for the psychological and physiological well-being of people worldwide. They are excellent producers of vitamins, pigments, hydrolytic enzymes, biofuels, organic acids, polysaccharides, and secondary metabolites such as antibiotics, anticancer treatments, hypocholesterolemic pharmaceuticals, and immunosuppressants. Other secondary metabolites include biofuels. In addition, polysaccharides are produced by them. We provide a condensed explanation of the significance of secondary metabolites in a variety of industries, such as the pharmaceutical industry, the food industry, the textile industry, and the transportation industry. In addition to providing a better understanding of biosynthetic regulation and the possibilities of genetic engineering, improved laboratory processes for the selection of nontoxigenic fungal strains have permitted the manufacture of larger quantities of safe commercial items. The significance of fungi in industrial settings is the topic that will be investigated in this review.

Seroepidemiology and Carriage of Diphtheria in Epidemic-Prone Area and Implications for Vaccination Policy, Vietnam

In 2019, a community-based, cross-sectional carriage survey and a seroprevalence survey of 1,216 persons 1-55 years of age were conducted in rural Vietnam to investigate the mechanism of diphtheria outbreaks. Seroprevalence was further compared with that of an urban area that had no cases reported for the past decade. Carriage prevalence was 1.4%. The highest prevalence, 4.5%, was observed for children 1-5 years of age. Twenty-seven asymptomatic Coerynebacterium diphtheriae carriers were identified; 9 carriers had tox gene-bearing strains, and 3 had nontoxigenic tox gene-bearing strains. Child malnutrition was associated with low levels of diphtheria toxoid IgG, which might have subsequently increased child carriage prevalence. Different immunity patterns in the 2 populations suggested that the low immunity among children caused by low vaccination coverage increased transmission, resulting in symptomatic infections at school-going age, when vaccine-induced immunity waned most. A school-entry booster dose and improved infant vaccination coverage are recommended to control transmissions.

Effect of Aspergillus Flavus Fungi Infection and Aflatoxin Contamination on Single Corn Kernel Mechanical Strength

HighlightsKernels were artificially inoculated with toxigenic and non-toxigenic strains of Aspergillus flavus in the lab.Corn kernel mechanical strength was assessed as single kernel compression rupture force (CRF).Fungus-infected kernels had lower mechanical strength than the controls.Kernels with high aflatoxin contamination had lower mechanical strength than others.ABSTRACT. One persistent food safety issue associated with corn is aflatoxin contamination. Aflatoxins are secondary metabolites produced mainly by the fungi Aspergillus flavus (A. flavus) and A. parasiticus. Under environmental conditions suitable for fungal growth and aflatoxin production, these fungi are capable of infecting corn kernels in the field and in storage. When corn kernels are infected, the fungi use nutrients from the kernels to grow and propagate. In general, moldy and contaminated kernels are discolored and lighter in weight. These features could aid in the identification and removal of infected kernels. The objective of this research was to investigate the relationship between the mechanical strength of corn kernels and fungal infection, as well as aflatoxin contamination as a consequence of fungal infection. Corn kernels were infected with aflatoxin producing (AF13) and non-toxin-producing (AF36) A. flavus strains in lab inoculations. A total of 900 kernels were assigned to three groups, non-inoculated control, AF13-inoculated, and AF36-inoculated. One hundred kernels from each group were incubated for 3, 5, and 8 days. After incubation each kernel was cleaned and dried, then subjected to a destructive mechanical test. The compression rupture force (CRF) of each kernel was measured using a Mark-10 material test gauge to assess its kernel strength. The results show that kernel strength was significantly different between the control and treatment groups, with the control kernels being the strongest and the AF36-inoculated kernels the weakest. The results also indicated that kernels with aflatoxin contamination of 300 ppb and above were significantly weaker than control kernels and those less contaminated in the AF13-inoculated group. It is expected that the results of this research could benefit the corn industry by mitigating the aflatoxin contamination problem in post-harvest management. Keywords: Aflatoxin, Aspergillus flavus, Compression rupture force, Corn, Fungus-infected, Inoculation, Mechanical strength.

Genomic analysis of an outbreak of toxin gene bearing Corynebacterium diphtheriae in Northern Queensland, Australia reveals high level of genetic similarity.

Toxigenic diphtheria is rare in Australia with generally fewer than 10 cases reported annually; however, since 2020, there has been an increase in toxin gene-bearing isolates of Corynebacterium diphtheriae cases in North Queensland, with an approximately 300% escalation in cases in 2022. Genomic analysis on both toxin gene-bearing and non-toxin gene-bearing C. diphtheriae isolated from this region between 2017 and 2022 demonstrated that the surge in cases was largely due to one sequence type (ST), ST381, all of which carried the toxin gene. ST381 isolates collected between 2020 and 2022 were highly genetically related to each other, and less closely related to ST381 isolates collected prior to 2020. The most common ST in non-toxin gene-bearing isolates from North Queensland was ST39, an ST that has also been increasing in numbers since 2018. Phylogenetic analysis demonstrated that ST381 isolates were not closely related to any of the non-toxin gene-bearing isolates collected from this region, suggesting that the increase in toxigenic C. diphtheriae is likely due to the expansion of a toxin gene-bearing clone that has moved into the region rather than an already endemic non-toxigenic strain acquiring the toxin gene.

Impact of Volatile Organic Compounds on the Growth of Aspergillus flavus and Related Aflatoxin B1 Production: A Review.

Volatile organic compounds (VOCs) are secondary metabolites of varied chemical nature that are emitted by living beings and participate in their interactions. In addition, some VOCs called bioactive VOCs cause changes in the metabolism of other living species that share the same environment. In recent years, knowledge on VOCs emitted by Aspergillus flavus, the main species producing aflatoxin B1 (AFB1), a highly harmful mycotoxin, has increased. This review presents an overview of all VOCs identified as a result of A. flavus toxigenic (AFB1-producing) and non-toxigenic (non AFB1-producing) strains growth on different substrates, and the factors influencing their emissions. We also included all bioactive VOCs, mixes of VOCs or volatolomes of microbial species that impact A. flavus growth and/or related AFB1 production. The modes of action of VOCs impacting the fungus development are presented. Finally, the potential applications of VOCs as biocontrol agents in the context of mycotoxin control are discussed.

Comparative biofilm-forming ability between Clostridioides difficile strains isolated in Latin America and the epidemic NAP1/027 strain.

One of the challenges in treating Clostridioides difficile infection (CDI) is that the bacterium forms biofilms, a critical virulence mechanism known to promote antibiotic resistance and, as a result, consequently, a higher recurrence of the disease. The goal of this study was to compare the ability of three MLST Clade 2 strains to form a biofilm in vitro: ICC-45 (ribotype SLO231/UK[CE]821), a ST41 toxinotype IXb isolated in Brazil; and two epidemic NAP1/027/ST01 strains: NAP1/027/ST01 (LIBA5756), isolated during a 2010 outbreak in Costa Rica and the reference epidemic strain NAP1/027/ST01 (R20291); and ATCC700057, a non-toxigenic strain. The ability of strains to form biofilm was evaluated using crystal violet staining. In addition, samples were stained with the Film Tracer biofilm matrix (Invitrogen®) and the biofilm matrix thickness was measured using confocal microscopy. The matrix architecture was determined using Scanning electron microscop. Confocal microscopy was used to detect the presence of toxin A (tcdA) using an anti-Clostridioides difficile TcdA antibody. The expression of virulence genes (tcdA, tcdB, tcdC, cdtB, spo0A, slpA, cwp66 and cwp84) was examined, as well as the effect of antibiotics metronidazole (MTZ) and vancomycin (VAN) on biofilm growth. All of the strains tested formed a moderate biofilm with 1.1 <DO570nm>3.5. After 72h, biofilm biomass of the NAP1/027/ST01 epidemic strains (LIBA5756 and R20291) was significantly higher than ICC-45 and ATCC 700057 biofilms, as confirmed by electron and confocal microscopy. At 120h, the LIBA5756 biofilm biomass decreased compared to other strains. The toxigenic strains R20291 or LIBA 5756 had higher expression of genes tcdA, tcdB, tcdC, cdtA, slpA and spo0A than ICC-45, but there were no significant differences in the expression levels of cdtB, cwp66 and cwp84. In epidemic strains, VAN and MTZ inhibited biofilm formation; however, in the ICC-45 strain, MIC concentrations of VAN and MIC and 4MIC of MTZ did not inhibit biofilm formation. The three MLST Clade 2 isolated from different rybotipes, two of which were isolated from Latin America, are competent biofilm-forming bacteria, indicating their ability to induce C. difficile infection recurrence, making treatment difficult.

Toxicity of the disinfectant benzalkonium chloride (C14) towards cyanobacterium Microcystis results from its impact on the photosynthetic apparatus and cell metabolism

Quaternary ammonium compounds (QACs) are commonly used in a variety of consumer and commercial products, typically as a component of disinfectants. During the COVID-19 pandemic, QACs became one of the primary agents utilized to inactivate the SARS-CoV-2 virus on surfaces. However, the ecotoxicological effects of QACs upon aquatic organisms have not been fully assessed. In this study, we examined the effects of a widely used QAC (benzalkonium chloride-C14, BAC-14) on two toxigenic Microcystis strains and one non-toxigenic freshwater Microcystis strain and carried out an analysis focused on primary, adaptive and compensatory stress responses at apical (growth and photosynthesis) and metabolic levels. This analysis revealed that the two toxic Microcystis strains were more tolerant than the non-toxic strain, with 96 hr-EC50 values of 0.70, 0.76, and 0.38 mg/L BAC-14 for toxigenic M. aeruginosa FACHB-905, toxigenic M. aeruginosa FACHB-469, and non-toxigenic M. wesenbergii FACHB-908, respectively. The photosynthetic activities of the Microcystis, assessed via Fv/Fm values, were significantly suppressed under 0.4 mg/L BAC-14. Furthermore, this analysis revealed that BAC-14 altered 14, 12, and 8 metabolic pathways in M. aeruginosa FACHB-905, M. aeruginosa FACHB-469, and M. wesenbergii FACHB-908, respectively. It is noteworthy that BAC-14 enhanced the level of extracellular microcystin production in the toxigenic Microcystis strains, although cell growth was not significantly affected. Collectively, these data show that BAC-14 disrupted the physiological and metabolic status of Microcystis cells and stimulated the production and release of microcystin, which could result in damage to aquatic systems.

Surface layer protein A from hypervirulent Clostridioides difficile ribotypes induce significant changes in the gene expression of tight junctions and inflammatory response in human intestinal epithelial cells

BackgroundSurface layer protein A (SlpA), the primary outermost structure of Clostridioides difficile, plays an essential role in C. difficile pathogenesis, although its interaction with host intestinal cells are yet to be understood. The aim of this study was to investigate the effects of SlpA extracted from C. difficile on tight junction (TJ) proteins expression and induction of pro-inflammatory cytokines in human colon carcinoma cell line HT-29. SlpA was extracted from three toxigenic C. difficile clinical strains including RT126, RT001, RT084 as well as C. difficile ATCC 700057 as non-toxigenic strain. Cell viability was performed by MTT assay, and the mRNA expression of TJ proteins and inflammation-associated genes was determined using quantitative RT-PCR. Additionally, the secretion of IL-8, IL-1β and TNF-α cytokines was measured by ELISA.ResultsC. difficile SlpA from selected RTs variably downregulated the expression level of TJs-assassinated genes and increased the expression level of TLR-4 and pro-inflammatory cytokines in HT-29 treated cells. SlpA from RT126 significantly (padj<0.05) decreased the gene expression level of claudins family and JAM-A and increased the secretion of IL-8, TNF-α and IL1-β as compared to untreated cells. Moreover, only SlpA from RT001 could significantly induce the expression of IL-6 (padj<0.05).ConclusionThe results of the present study highlighted the importance of SlpA in the pathogenesis of CDI and C. difficile-induced inflammatory response in the gut. Further studies are required to unravel the significance of the observed results in promoting the intestinal inflammation and immune response induced by C. difficile SlpA from different RTs.

Skin colonization of Staphylococcus aureus harboring superantigen toxin genes and its correlation with serum IL-22 level in psoriasis patients

Psoriasis is a chronic debilitating skin disease with an estimated prevalence reaching 2% of the worldwide population. Psoriatic disease is driven by the interactions among innate and adaptive immune systems with structural components of the skin. Interleukin (IL)-22 mediates keratinocyte proliferation and epidermal hyperplasia, and changes in the structure of skin flora can play a role in the secretion of IL-22. The aim of this study was to correlate serum levels of IL-22 and Staphylococcus aureus toxins with disease activity in plaque psoriasis. The study group included 50 patients with mild, moderate, and severe psoriasis. The control group comprised 20 sex- and age-matched apparently healthy volunteers. IL-22 concentration was assessed in sera of patients and the control group by using the ELISA technique. The serum levels of IL-22 in patients were higher than in the control group, but the difference was statistically insignificant (P=0.413). Serum IL-22 levels were positively correlated with the Psoriasis Area and Severity Index (PASI) score of psoriasis patients (P=0.0003). The IL-22 serum levels in patients colonized with toxigenic strains of S. aureus were significantly higher than in patients colonized with non-toxigenic strains (P= 0.028). In conclusion, IL-22 plays a role in the pathogenesis of psoriasis, and its secretion can be triggered by the toxins produced by S. aureus colonizing the skin of patients.

Determination of Diphtheria Toxin in Bacterial Cultures by Enzyme Immunoassay.

Since diphtheria toxin (DT) is the main virulence factor of Corynebacterium diphtheriae and C. ulcerans, the detection of DT in corynebacterial cultures is of utmost importance in the laboratory diagnosis of diphtheria. The need to measure the level of DT production (LTP) arises when studying the virulence of a strain for the purpose of diphtheria agent monitoring. To determine the LTP of diphtheria agents, an immunoassay based on monoclonal antibodies (mAbs) has been developed. A pair of mAbs specific to the fragment B of DT was selected, which makes it possible to detect DT in a sandwich ELISA with a detection limit of DT less than 1 ng/mL. Sandwich ELISA was used to analyze 218 liquid culture supernatants of high-, low- and non-toxigenic strains of various corynebacteria. It was shown that the results of ELISA are in good agreement with the results of PCR and the Elek test for the tox gene and DT detection, respectively. The diagnostic sensitivity of the assay was approximately 99%, and specificity was 100%. It has been found that strains of C. ulcerans, on average, produce 10 times less DT than C. diphtheriae. The mAbs used in the ELISA proved to be quite discriminatory and could be further used for the design of the LFIA, a method that can reduce the labor and cost of laboratory diagnosis of diphtheria.

Current and future approaches in Clostridioides difficile management

Clostridioides difficile is considered one of the main causes of the healthcare associated infections worldwide and the main cause of antibiotic-associated diarrhea, with forms of clinical disease ranging from mild to deadly. Both 2021 IDSA and ESCMID guidelines recommend that for the first episode of CDI Fidaxomicin should be considered as the preferred regimen of treatment and Vancomycin becomes 2nd Standard of Care (SOC) line, an alternative to fidaxomicin. Monoclonal antibodies, fecal microbiota transplantation and surgery remain other recommendations in the guidelines. New means of management of Clostridioides difficile infection are under development, these including prevention measures (vaccination, population with non-toxigenic strains of the colon, standardized fecal microbiota products) and new antibiotics.

Absence of toxin gene transfer from Clostridioides difficile strain 630Δerm to nontoxigenic C. difficile strain NTCD-M3r in filter mating experiments.

Nontoxigenic Clostridioides difficile strain M3 (NTCD-M3) protects hamsters and humans against C. difficile infection. Transfer in vitro of the pathogenicity locus (PaLoc) to nontoxigenic strain CD37 has been reported. We repeated these conjugations using toxigenic strain 630Δerm as donor and NTCD-M3 and CD37 as recipients. In order to conduct these matings we induced rifampin resistance (50ug/ml) in NTCD-M3 by serial passage on rifampin-containing media to obtain strain NTCD-M3r. 630Δerm/CD37 matings produced 21 PaLoc transconjugants in 5.5 x 109 recipient CFUs; a frequency of 3.8 x 10-9. All transconjugants carried the tcdB gene and produced toxin. 630Δerm/NTCD-M3r matings produced no transconjugants in 5 assays with a total of 9.4 x 109 NTCD-M3r recipient cells. Toxin gene transfer to NTCD-M3r could not be demonstrated under conditions that demonstrated transfer to strain CD37.

One-day prevalence of asymptomatic carriage of toxigenic and non-toxigenic Clostridioides difficile in 10 French hospitals

Asymptomatic faecal carriage of Clostridioides difficile has been widely evaluated, but its prevalence across a wide range of clinical departments and related risk factors are not well described. The objectives of the PORTADIFF study were to evaluate the prevalence and identifying risk factors leading to asymptomatic carriage of both toxigenic and non-toxigenic C.difficile. The PORTADIFF study was a 1-day prevalence study carried out in 10 different French hospitals. Adult patients, who agreed to participate, were included in this study and provided a fresh stool sample. C. difficile strains isolated from carriage were characterized by polymerase chain reaction (PCR) detection of tcdA, tcdB, cdtA and cdtB, and PCR ribotyping. In total, 721 patients were included in this study. The median age was 73 years (range 18-101 years) and the male/female ratio was 1.06. C. difficile (either toxigenic or non-toxigenic strains) was isolated from 79 (11%) patients; 42 (5.8%) strains were toxigenic. The prevalence rates of asymptomatic carriage ranged from 5% on surgical wards to 19% on long-term care wards. The main risk factors associated with asymptomatic carriage were antibiotic treatment within the preceding 3 months (81.8% vs 53.7%; P<0.01), hospitalization within the preceding 2 months (55.8% vs 33%; P<0.01), cumulative duration of hospital stay before study inclusion (mean 50.1 vs 34.5 days; P<0.047), and hospitalization on a ward with high global incidence of C. difficile infection. Eleven percent of hospitalized patients were asymptomatic carriers of toxigenic or non-toxigenic C. difficile, and may constitute a potential reservoir of C. difficile strains.