Toxigenic Strains Research Articles

Prevalence and antimicrobial susceptibility pattern of toxigenic Clostridium difficilestrains isolated in Iran

Background/aimClostridium difficile is a frequent cause of nosocomial infections and has become a major public health concern in developed nations. In the present study, the prevalence and antimicrobial susceptibility pattern of toxigenic C. difficile strains isolated in Iran were investigated.Materials and methods Between June 2016 and May 2017, 2947 inpatient fecal samples were taken from symptomatic adult hospitalized patients in different units of 32 care facilities in Tehran, Iran. C. difficile strains were identified by microbiological/biochemical methods. Susceptibility to 20 antimicrobials was measured by E-test method. Toxin-specific immunoassays and cytotoxicity assays were used to determine in vitro toxin production.Results Out of 2947 fecal samples, 538 (18.25%) C. difficile isolates were obtained among those with suspected CDI. In E-test method, all C. difficile isolates were susceptible to fidaxomicin, vancomycin, amoxicillin/clavulanate, and meropenem and were resistant to penicillin G. The prevalence of multidrug resistant C. difficile was 69.33% (373/538). Among 538 C. difficile, 147 (27.32%), 169 (31.41%), and 222 (41.26%) isolates were TcdA+/TcdB+, TcdA-/TcdB+, and TcdA-/TcdB-, respectively.Conclusion The results evidently support the hypothesis of a probable role of toxigenic strains of C. difficile in developing gastrointestinal complaints in patients with diarrhea.

The role of extrolites secreted by nonaflatoxigenic Aspergillus flavus in biocontrol efficacy.

Field control of aflatoxin contamination is thought to occur through competitive exclusion of native aflatoxigenic fungi by introduced nonaflatoxigenic Aspergillus flavus biocontrol strains. In this study, we explored another possible mechanism that could increase the efficacy of biocontrol strains-the production of secreted compounds termed extrolites. Using four Aspergillus strains (one nonaflatoxigenic and three aflatoxigenic) from the same geographic region (Louisiana), we devised experiments whereby each aflatoxigenic strain was grown on media that had first been colonized by the nonaflatoxigenic strain. We observed noticeable reduction in growth and reduced production of aflatoxin and cyclopiazonic acid for all three aflatoxigenic strains when grown in the presence of extrolite secretions from the nonaflatoxigenic strain. We provide evidence that biocontrol strain extrolites may improve the efficacy of pre and postharvest aflatoxin reduction. Our finding, that extrolites secreted by nonaflatoxigenic A. flavus strains potentially abate growth and toxin levels of aflatoxin-producing strains, should allow for us to elucidate the mechanism of how the reduction in toxigenic strains occurs, and potentially identify better biocontrol strains. Identification and isolation of the active extrolites may afford a supplemental method to mitigate aflatoxin production.

Alternative Biosynthetic Starter Units Enhance the Structural Diversity of Cyanobacterial Lipopeptides.

Puwainaphycins (PUWs) and minutissamides (MINs) are structurally analogous cyclic lipopeptides possessing cytotoxic activity. Both types of compound exhibit high structural variability, particularly in the fatty acid (FA) moiety. Although a biosynthetic gene cluster responsible for synthesis of several PUW variants has been proposed in a cyanobacterial strain, the genetic background for MINs remains unexplored. Herein, we report PUW/MIN biosynthetic gene clusters and structural variants from six cyanobacterial strains. Comparison of biosynthetic gene clusters indicates a common origin of the PUW/MIN hybrid nonribosomal peptide synthetase and polyketide synthase. Surprisingly, the biosynthetic gene clusters encode two alternative biosynthetic starter modules, and analysis of structural variants suggests that initiation by each of the starter modules results in lipopeptides of differing lengths and FA substitutions. Among additional modifications of the FA chain, chlorination of minutissamide D was explained by the presence of a putative halogenase gene in the PUW/MIN gene cluster of Anabaena minutissima strain UTEX B 1613. We detected PUW variants bearing an acetyl substitution in Symplocastrum muelleri strain NIVA-CYA 644, consistent with an O-acetyltransferase gene in its biosynthetic gene cluster. The major lipopeptide variants did not exhibit any significant antibacterial activity, and only the PUW F variant was moderately active against yeast, consistent with previously published data suggesting that PUWs/MINs interact preferentially with eukaryotic plasma membranes.IMPORTANCE Herein, we deciphered the most important biosynthetic traits of a prominent group of bioactive lipopeptides. We reveal evidence for initiation of biosynthesis by two alternative starter units hardwired directly in the same gene cluster, eventually resulting in the production of a remarkable range of lipopeptide variants. We identified several unusual tailoring genes potentially involved in modifying the fatty acid chain. Careful characterization of these biosynthetic gene clusters and their diverse products could provide important insight into lipopeptide biosynthesis in prokaryotes. Some of the variants identified exhibit cytotoxic and antifungal properties, and some are associated with a toxigenic biofilm-forming strain. The findings may prove valuable to researchers in the fields of natural product discovery and toxicology.

Deciphering the effects of nitrogen, phosphorus, and temperature on cyanobacterial bloom intensification, diversity, and toxicity in western Lake Erie

AbstractAlthough cyanobacterial harmful algal blooms (CHABs) are promoted by nutrient loading and elevated temperatures, the effects of these processes on bloom diversity are unclear. This study used traditional and next‐generation sequencing approaches to assess shifts in phytoplankton, cyanobacterial (16S rRNA), and microcystin‐producing (mcyE) communities during CHABs in western Lake Erie (Maumee and Sandusky Bays) in response to natural and experimental gradients of nitrogen (N), phosphorus (P), and temperature. CHABs were most intense near the Maumee and Sandusky Rivers and were dominated by Microcystis and Planktothrix, respectively. Sequencing of 16S amplicons revealed cryptic cyanobacterial diversity (47 genera) including high abundances of two distinct clades of Synechococcus in both bays and significant differences in community structure between nutrient‐rich nearshore sites and less eutrophic offshore sites. Sequencing of mcyE genes revealed low taxonomic (n = 3) but high genetic diversity (n = 807), with toxigenic strains of Planktothrix being more abundant than Microcystis and more closely paralleling microcystin concentrations. Cyanobacterial abundance significantly increased in response to elevated N, with the greatest increases in combined high N, P, and temperature treatments that concurrently suppressed green and brown algae. N significantly increased microcystin concentrations and the relative abundance of nondiazotrophic genera such as Planktothrix, while diazotrophic genera such as Dolichospermum and Aphanizomenon were, in some cases, enhanced by high P and temperature. While nutrients and elevated temperatures promote CHABs, differing combinations selectively promote individual cyanobacterial genera and strains, indicating management of both N and P will be required to control all cyanobacteria in Lake Erie, particularly as lake temperatures rise.

Phenotypic Characterization of Non-toxigenic Clostridioides difficile Strains Isolated From Patients in Mexico.

Clostridioides difficile is a Gram positive, sporulated, rod-shape, anaerobic pathogen responsible for nosocomial diarrhea and colitis, mainly in antibiotic treated patients. C. difficile produce two toxins responsible for disease, toxin A (TcdA) and toxin B (TcdB), although not all strains produce them. Non-toxigenic C. difficile (NTCD) strains are able to colonize the intestinal mucosa and are often isolated from asymptomatic individuals. NTCD are poorly studied, their evolutionary history has not been elucidated, and their relationship with illness remains controversial. The aim of this work was to analyze the phenotype of NTCD strains isolated from clinical cases in hospitals of México, and whether NTCD strains present characteristics that differentiate them from the toxigenic strains. Seventy-four C. difficile strains isolated from patients were tested for cytotoxicity and 14 were identified as NTCD strains. We analyzed phenotypical characteristics that are important for the biology of C. difficile like colony morphology, antibiotic resistance, motility, sporulation, and adherence. Strains were also genotyped to determine the presence of genes coding for TcdA, TcdB and binary toxin and ribotyped for 027 type. When compared with toxigenic strains, NTCD strains presented an enlarged branched colony morphology, higher resistance to metronidazole, and increased sporulation efficiency. This phenotype has been reported associated with mutations that regulates phenotypic characteristics like swimming, sporulation or adhesion. Our results show that phenotype of NTCD strains is heterogeneous but still present characteristics that differentiate them from toxigenic strains.

Clostridium difficile infection in cancer patients with hospital acquired diarrhea at the teaching hospitals in Iran: Multilocus sequence typing analysis (MLST) and Antimicrobial resistance pattern.

This study aimed to investigate the phenotype and genotype characterization of Clostridium difficile isolates among cancer patients with hospital-acquired diarrhea in 4 teaching hospitals in Isfahan, Iran. This was a cross sectional study conducted on adult (>18 years old) between April 2015 and May 2017. Over two years, 67 diarrheic fecal samples were collected. C. difficile isolates were characterized according to the presence of toxin genes and antibiotic resistance. Multilocus sequence typing (MLST) was performed to evaluate the genetic relationships between different lineages of toxigenic strains. Seven toxigenic and 12 non- toxigenic strains were detected among stool samples. Patients with a history of previous surgery during hospitalization were more than 7 times likely to develop Clostridium difficile infection (CDI). All isolates were susceptible to metronidazole, vancomycin and fusidic acid. Toxigenic C. difficile strains were divided into 3 different sequence types. The detected types were ST-54, ST-2 and ST-37, while none of the isolates was identified as ST-1 or ST-11. This is the first description of the MLST analysis of C. difficile strains isolated from cancer patients in Iran. All of the studied population were exposed to multiple antibiotics and chemotherapeutic agents. Further research and clinical studies are recommended in the treatment through good antimicrobial stewardship and prevention of C. difficile infection in all healthcare settings.

NGS-based phylogeny of diphtheria-related pathogenicity factors in different Corynebacterium spp. implies species-specific virulence transmission

BackgroundDiphtheria toxin (DT) is produced by toxigenic strains of the human pathogen Corynebacterium diphtheriae as well as zoonotic C. ulcerans and C. pseudotuberculosis. Toxigenic strains may cause severe respiratory diphtheria, myocarditis, neurological damage or cutaneous diphtheria. The DT encoding tox gene is located in a mobile genomic region and tox variability between C. diphtheriae and C. ulcerans has been postulated based on sequences of a few isolates. In contrast, species-specific sequence analysis of the diphtheria toxin repressor gene (dtxR), occurring both in toxigenic and non-toxigenic Corynebacterium species, has not been done yet. We used whole genome sequencing data from 91 toxigenic and 46 non-toxigenic isolates of different pathogenic Corynebacterium species of animal or human origin to elucidate differences in extracted DT, DtxR and tox-surrounding genetic elements by a phylogenetic analysis in a large sample set.ResultsSequences of both DT and DtxR, extracted from whole genome sequencing data, could be classified in four distinct, nearly species-specific clades, corresponding to C. diphtheriae, C. pseudotuberculosis, C. ulcerans and atypical C. ulcerans from a non-toxigenic toxin gene-bearing wildlife cluster. Average amino acid similarities were above 99% for DT and DtxR within the four groups, but lower between them. For DT, subgroups below species level could be identified, correlating with different tox-comprising mobile genetic elements. In most C. diphtheriae, tox genes were located within known prophages. In contrast, in C. ulcerans diverse tox-including mobile elements could be identified: either prophages differing from C. diphtheriae prophages or an alternative pathogenicity island (PAI) described previously. One isolate showed a different, shorter tox-comprising putative PAI. Beyond the tox-overlapping elements, most isolates harbored a variety of additional prophages.ConclusionOur NGS data from 137 isolates indicate the existence of different genetic backgrounds of DT-mediated pathogenicity in different Corynebacterium species and evolution of once acquired pathogenicity features with the strains. Different groups of pathogenicity-related elements within C. ulcerans imply that tox transmission pathways between isolates may differ in the zoonotic species and contribute to their emerging pathogenic potential.

Clostridium difficile. A review on an emerging infection.

Clostridium difficile causes antibiotic-associated diarrhoea and pseudomembranous colitis. The main virulence factors of C. difficile are the toxins A (TcdA) and B (TcdB). A third toxin, binary toxin (CDT), which pathogenetic role had been remained largely overlooked until few years ago, nowadays have been detected in 5%-23% of strains. C. difficile has spread around world. Clostridium difficile infection (CDI) is one of the most common health-care associated infections and a significant cause of morbidity and mortality among older adult hospitalized patients. Diagnosis of CDI is often difficult and has a substantial impact on the management of patients with disease. It is usually based on a clinical history of recent antimicrobial usage and diarrhoea in combination with laboratory tests. Although the conventional methods are crucial for the diagnosis and the subsequent treatment of CDI, a timely laboratory diagnosis is essential for the detection of toxigenic strains providing either to an effective and immediately treatment or to the prevention of further disease transmission. In this review we provide general recommendations for testing of samples collected from patients with suspected CDI.

Biodiversity of the bacteria closely associated with the toxigenic dinoflagellate, Alexandrium minutum, and screening of its toxigenic bacteria strains

Biodiversity of the bacteria closely associated with the toxigenic dinoflagellate, Alexandrium minutum, and screening of its toxigenic bacteria strains

Pediatric oncology and stem cell transplant patients with healthcare-associated Clostridium difficile infection were already colonized on admission.

A prospective cohort study was conducted over a three-year period in an inpatient pediatric oncology and stem cell transplant setting. Baseline stool samples were collected within three days of admission and were genotypically compared with clinically indicated samples submitted after three days of admission. A total of 175 patients were recruited with a total of 536 admissions. The adjusted prevalence of baseline toxigenic C. difficile colonization among admissions was 32.8%. Seventy-eight percent of positive admissions did not have history of CDI. Colonization with a toxigenic strain on admission was predictive of CDI (OR=28.6; 95% CI, 6.58-124.39; P<0.001). Nearly all clinical isolates (8/9) shared identical pulsed-field gel electrophoresis patterns with baseline isolates or were closely related (1/9). Only one of the 11 cases that were considered HA-CDI was potentially nosocomially acquired. The prevalence of colonization with toxigenic C. difficile in our cohort is high. Unfortunately, the current CDI surveillance definitions overestimate the incidence of HA-CDI in pediatric oncology and stem cell transplantation settings.

Of mice and men: Interaction of Corynebacterium diphtheriae strains with murine and human phagocytes

ABSTRACTSeven non-toxigenic C. diphtheriae strains and one toxigenic strain were analyzed with regard to their interaction with murine macrophages (BMM) and human THP-1 macrophage-like cells. Proliferation assays with BMM and THP-1 revealed similar intracellular CFUs for C. diphtheriae strains independent of the host cell. Strain ISS4060 showed highest intracellular CFUs, while the toxigenic DSM43989 was almost not detectable. This result was confirmed by TLR 9 reporter assays, showing a low signal for DSM43989, indicating that the bacteria are not endocytosed. In contrast, the non-pathogenic C. glutamicum showed almost no intracellular CFUs independent of the host cell, but was recognized by TLR9, indicating that the bacteria were degraded immediately after endocytosis. In terms of G-CSF and IL-6 production, no significant differences between BMM and THP-1 were observed. G-CSF production was considerably higher than IL-6 for all C. diphtheriae strains and the C. glutamicum did not induce high cytokine secretion in general. Furthermore, all corynebacteria investigated in this study were able to induce NFκB signaling but only viable C. diphtheriae strains were able to cause host cell damage, whereas C. glutamicum did not. The absence of Mincle resulted in reduced G-CSF production, while no influence on the uptake of the bacteria was observed. In contrast, when MyD88 was absent, both the uptake of the bacteria and cytokine production were blocked. Consequently, phagocytosis only occurs when the TLR/MyD88 pathway is functional, which was also supported by showing that all corynebacteria used in this study interact with human TLR2.

Biosynthesis of Silver Nanoparticles from &amp;lt;i&amp;gt;Allium ampeloprasum&amp;lt;/i&amp;gt; Leaves Extract and Its Antifungal Activity

Development of natural based nanomaterial immense interest of researchers involved in eco-friendly biosynthesis of nanoparticles. This research set out to investigate the potential of Allium ampeloprasum leaves extract to synthesize silver nanoparticles (AgNPs), and evaluate their antifungal activity against some toxigenic strains of Aspergillus isolates. In order to achieve this, AgNPs were prepared using plant extracts at room temperature and the formation of AgNPs was visually detected after the color changed to dark brown within few minutes. The biosynthesized AgNPs were characterized using the UV-vis spectroscopy, which confirmed the formation of AgNPs with a maximum peak at 437 nm due to the Plasmon resonance. The main active reduction agents were detected by Fourier Transmission infrared spectroscopy (FTIR). Also, the nanoparticles were characterized using Scanning electron microscope (SEM). The antifungal activity of AgNPs was investigated by agar well diffusion method, which revealed inhibition zones of 8 mm, 10 mm, 11 mm, 11 mm and 14 mm against Aspergillus flavus 1, A. parasiticus, A. flavus 2, A. ochraceus, and A. niger. The minimum inhibition concentration (MIC) was determined using micro broth dilution method. MIC values of AgNPs ranged from 652 - 2500 μg/ml. The fungi treated with AgNPs, were examined with (SEM), it was observed that the treated fungi were damaged. These results suggest that AgNPs have the potential to be used as an ideal eco-friendly approach to control toxigenic fungi.

An efficient and affordable laboratory method to produce and sustain high concentrations of microcystins by Microcystis aeruginosa

Microcystis aeruginosa is a cosmopolitan cyanobacteria that continues to jeopardize freshwater ecosystem services by releasing the hepatotoxin microcystin, which can, in some cases, cause death to aquatic fauna and even humans. Currently, our abilities to understand the mechanisms of microcystin toxicology are limited by the lack of a method for producing high concentrations, which are central to large-scale and long-term research in natural systems. Here we present an efficient and affordable laboratory method to produce high concentrations of microcystins by a toxigenic strain of M. aeruginosa. Through batch culture studies, we yielded microcystins at concentrations that are environmentally relevant to freshwaters around the world (1–300 μg L−1), maintained these concentrations without resupplying fresh medium (further reducing costs), and utilized rate equations to model the relationship between the environmental conditions in the cultures and changes occurring within the M. aeruginosa cells. Our assessment suggests that steady production of microcystins depends on the availability of carbon throughout the experiment. Hence, we recommend the use of tissue culture treated flasks with a vented cap to ensure the production of microcystins is uninterrupted. This method demonstrates that microcystins can be produced in the laboratory at concentrations relevant to freshwater ecosystems.•The method demonstrates M. aeruginosa CPCC 300 is a reliable strain of freshwater cyanobacteria that can yield microcystins at environmentally relevant concentrations.•Validation showed M. aeruginosa CPCC 300 is resilient in carbon-limited situations and may respond to stress by shifting the ratio of microcystin congeners.•Cell culture flasks with vented caps —filled no more than 50 % of the flask volume to allow for sufficient air exchange— are an excellent and cost-effective approach to maintaining cell growth and producing microcystins at a range between 300 to 1200 μg L−1.

A study on clinical application of a novel reagent detecting toxigenic strains of Clostridium difficile

Objective: To evaluate the performances of a "ready-to-use" liquid chromogenic medium for detecting toxigenic strains of C. difficile from stool specimens, by comparing with the reference method of combined GeneXpert detection with MALDI-TOF MS identification. Methods: This study was a clinical application research. A total of 322 stool specimens from patients with diarrhea in Henan Provincial People's Hospital were detected by using this liquid chromogenic mediumor by combination of GeneXpert and MALDI-TOF MS identification after CCFA culture from March to November 2017. The reference method was combined GeneXpert detection with MALDI-TOF MS identification. Data were analyzed with kappa analysis by use of SPSS 13.0. In this study, the limit of detection and anti-interference capability of chromogenic medium were determined with standard strains. Results: This assay had a sensitivity of 92.86% (52/56), a specificity of 96.99% (258/266), a positive predictive value of 86.67% (52/60), and a negative predictive value of 98.47% (258/262). Furthermore, this assay was well consistent with the reference method (kappa=0.874, P<0.01). The limit of detection of this assay was 10(3) CFU/ml for toxigenic strain of ATCC9689, and there was no interference by non-toxigenic strain of ATCC43593 or Clostridium perfringens (ATCC13124) even.When the concentration of ATCC9689 was as low as 0.5 McFarland. Conclusions: It is worth expanding clinical research for this "ready-to-use" chromogenic medium and preparing for its popularization in primary medical institutions, due to its similar performance to the reference method for detecting toxigenic strains of C. difficile, simple operation and no need of anaerobic condition.

The role of ear environment in postharvest susceptibility of maize to toxigenic Aspergillus flavus

AbstractA kernel screening assay (KSA) was used to assess the genetic and environmental effects on the vulnerability of maize to aflatoxin accumulation. Kernels of 26 inbred lines that had been grown in seven environments, and 190 lines of the Intermated B73xMo17 (IBM) population grown in one location in the United States, were inoculated with a toxigenic strain of A. flavus and incubated in the dark at 30°C for 6 days. Percent kernel colonization (PKC), sporulation and aflatoxin were influenced by the maize genotypes (G), the location (“ear environment” or E) and the GxE interactions. Overall, low broad‐sense heritabilities were observed for PKC, sporulation and aflatoxin. PKC was significantly correlated with sporulation in all environments. Aflatoxin was positively correlated with colonization for two and with sporulation for all ear environments. Higher grain sulphur or magnesium in IBM was associated with less colonization or aflatoxin. Postharvest susceptibility of maize to aflatoxin is thus influenced by factors that are modulated by the ear environment. In a KSA, sporulation could be a proxy test for aflatoxin accumulation.

Molecular genetic basis of biofilm formation as a component of Vibrio Cholerae persistence in water reservoirs of Russian Federation

Background. The problem of cholera remains acute for world health service and risks of importation of Vibrio cholerae strains from endemic countries to Russia do exist. Toxigenic strains (carrying cholera toxin genes ctxAB) can cause epidemic outbreaks of cholera and non-toxigenic (ctxAB-) single or multiple cases of cholera-like diarrhea. Investigation of their ability to survive in water reservoirs in climatic conditions of middle latitudes by means of forming biofilms is essential for potential threat evaluation.&#x0D; Materials and methods. Biofilm formation by 15 V. cholerae strains on abiotic surfaces was studied in microcosms with tap water and cover glasses. Identification of responsible genetic determinants in whole genome sequences and bioinformatics analysis were performed using BioEdit 7.2.5, BLASTN 2.2.29, Blastp and Vector NTI Advance 11 software.&#x0D; Results. The strains investigated differed in terms of biofilm formation which correlated with structural features of genes for MSHA pili (msh), matrix polysaccharides (vps) and proteins (rbm) as well as for certain regulatory factors. Strains with none or few genetic deviations from prototypes formed mature biofilms in 5-7 days while those containing truncated genes mshL, mshN, rbmC only in 13 days. One strain with truncated gene for positive regulator vpsR formed an immature biofilm. Acceleration of the process in some strains up to 2-3 days correlated with either truncated gene hapR (negative regulator) or altered structure of both msh and vps-rbm gene clusters.&#x0D; Conclusion. Analysis of genetic determinants responsible for biofilm formation may be used for prediction of V. cholerae ability to survive in environmental objects of Russia and thus the potential danger of the latters as sources of infection.

Tuna Condensate Waste with Molasses as a Renewable Substrate for Antifungal Compounds by Streptomyces philanthi RL-1-178 Against Aflatoxingenic B1 (AFB1) Aspergillus flavus

This study aims to utilize tuna condensate as a renewable resource for production of antifungal compounds by Streptomyces philanthi RL-1-178 against aflatoxingenic B1 (AFB1) Aspergillus flavus. Among ten isolates of A. flavus tested, the strain PSRDC-4 was the most toxigenic strain (1432 ppb of AFB1) and aggressive to S. philanthi (85.9% inhibition). The effective dose (100% inhibition) of the culture filtrate RL-1-178 was at 10.0% (v/v) with 1 h exposure time. The antifungal compounds, identified by GC–MS analysis, were consisted of 105 components with 2,4-imidazolidinedione (31.2%) followed by acetic acid (25.27%) was the most abundant. The optimum condition for growth and production of antifungal compounds from S. philanthi RL-1-178 was as following; tuna condensate of 15,000 mg L−1 COD, the initial pH at 7.0, incubation temperature at 30 °C and supplemented with 8 g L−1 of molasses. Therefore, tuna condensate exhibited a high potential to be utilized as an alternative medium for antifungal production by S. philanthi RL-1-178.

Development of a technique for DNA detection and identification of toxigenic strains of Clostridium botulinum types A, B, E by the Real-Time PCR method

Botulism is dangerous toxic infection caused by a toxin produced by the bacterium Clostridium botulinum. The mortality rate from botulism can reach 70% of all cases of illness in case of untimely initiation of treatment. The pathogenesis of botulism involves the damage to the central nervous system by a toxin produced by C. botulinum. Currently there are seven recognized antigenic types of this toxin. Botulinum toxin is included into the group of biological agents and it is one of the most likely agents to be used in a biological attack. Since botulinum neurotoxin is a complex nucleoprotein complex and the traces of DNA can be detected even in purified toxin preparations, we have elaborated a technique for detecting and identifying DNA of toxigenic strains of Clostridium botulinum types A, B, E, that cause human botulism in most cases. This technique is based on the the detection of residual amounts of this DNA in botulinum toxin using multiplex real-time polymerase chain reaction (PCR) assay with fluorescent hybridization detection. The main obstacle to development of a technique for the detection and identification of DNA of toxigenic strains is the high variability of the genes responsible for the synthesis of botulinum toxin. We have established a region of the gene with the lowest homology in all strains. This requirement is met by a fragment of the bont gene that encodes a light chain of a neurotoxin and is highly conserved in the strains of C. botulinum producing one type of toxin. The paper represents the results of the definition of analytical sensitivity and specific activity of the developed method. The specificity of the determination is 100%, the analytical sensitivity – 1×10 2 mc./ml. The method can be used to analyze food, samples of clinical materials and environmental samples suspected of being contaminated with toxigenic strains of C. botulinum

A Method for the Simultaneous Detection of Vibrio cholerae Strains and Drug-Resistant Genes in their Genome by Real-Time PCR

A method was developed for the simultaneous detection of V. cholerae strains and the presence of drug-resistant genes in their genomes by real-time PCR. Resistance to four antibiotics used for cholera treatment (tetracycline, trimethoprim, chloramphenicol and ciprofloxacin) was analyzed. The sensitivity of the panel was 1 × 103 CFU/mL for pure cultures, simulated clinical material, and environmental samples. PCR efficiency was confirmed by the analysis of 60 natural V. cholerae strains isolated from patients and the environment in different years. It was established that all of the studied V. cholerae strains of the O1 serogroup El Tor biovar isolated before 1993 did not contain the tested drug-resistant genes. At the same time, 18 toxigenic clinical strains (90%) imported within 1993–2010 were characterized by multiple drug resistance; their genomes contained trimethoprim resistance genes (dfrA1) and chloramphenicol resistance genes (floR), and 11 strains additionally contained a tetracycline-resistant gene tetR. In addition, nontoxigenic clinical and aqueous V. cholerae strains carrying a ciprofloxacin-resistant gene, the qnrVC (qnrVC1) gene, have been detected in the last few years (Kalmykia, 2011–2013).

Genome-wide comparison of Corynebacterium diphtheriae isolates from Australia identifies differences in the Pan-genomes between respiratory and cutaneous strains

BackgroundCorynebacterium diphtheriae is the main etiological agent of diphtheria, a global disease causing life-threatening infections, particularly in infants and children. Vaccination with diphtheria toxoid protects against infection with potent toxin producing strains. However a growing number of apparently non-toxigenic but potentially invasive C. diphtheriae strains are identified in countries with low prevalence of diphtheria, raising key questions about genomic structures and population dynamics of the species. This study examined genomic diversity among 48 C. diphtheriae isolates collected in Australia over a 12-year period using whole genome sequencing. Phylogeny was determined using SNP-based mapping and genome wide analysis.ResultsC. diphtheriae sequence type (ST) 32, a non-toxigenic clone with evidence of enhanced virulence that has been also circulating in Europe, appears to be endemic in Australia. Isolates from temporospatially related patients displayed the same ST and similarity in their core genomes. The genome-wide analysis highlighted a role of pilins, adhesion factors and iron utilization in infections caused by non-toxigenic strains.ConclusionsThe genomic diversity of toxigenic and non-toxigenic strains of C. diphtheriae in Australia suggests multiple sources of infection and colonisation. Genomic surveillance of co-circulating toxigenic and non-toxigenic C. diphtheriae offer new insights into the evolution and virulence of pathogenic clones and can inform targeted public health actions and policy. The genomes presented in this investigation will contribute to the global surveillance of C. diphtheriae both for the monitoring of antibiotic resistance genes and virulent strains such as those belonging to ST32.