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The present study aimed at the molecular characterization of pathogenic and non pathogenic F. oxysporum f. sp. lycopersici strains isolated from tomato. The causal agent isolated from symptomatic plants and soil samples was identified based on morphological and molecular analyses. Pathogenicity testing of 69 strains on five susceptible tomato varieties showed 45% of the strains were highly virulent and 30% were moderately virulent. Molecular analysis based on the fingerprints obtained through ISSR indicated the presence of wide genetic diversity among the strains. Phylogenetic analysis based on ITS sequences showed the presence of at least four evolutionary lineages of the pathogen. The clustering of F. oxysporum with non pathogenic isolates and with the members of other formae speciales indicated polyphyletic origin of F. oxysporum f. sp. lycopersici. Further analysis revealed intraspecies variability and nucleotide insertions or deletions in the ITS region among the strains in the study and the observed variations were found to be clade specific. The high genetic diversity in the pathogen population demands for development of effective resistance breeding programs in tomato. Among the pathogenic strains tested, toxigenic strains harbored the Fum1 gene clearly indicating that the strains infecting tomato crops have the potential to produce Fumonisin.

Increasing evidence suggests that altered intestinal microbial composition and function result in an increased risk of Clostridium difficile -associated diarrhoea (CDAD); however, the specific changes of intestinal microbiota in children suffering from CDAD and their associations with C. difficile strain toxigenicity are poorly understood. High-throughput pyrosequencing showed that reduced faecal bacterial diversity and dramatic shifts of microbial composition were found in children with CDAD. The Firmicutes/Bacteroidetes ratio was increased significantly in patients with CDAD, which indicated that dysbiosis of faecal microbiota was closely associated with CDAD. C. difficile infection resulted in an increase in lactate-producing phylotypes, with a corresponding decrease in butyrate-producing bacteria. The decrease in butyrate and lactate buildup impaired intestinal colonisation resistance, which increased the susceptibility to C. difficile colonisation. Strains of C. difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive and were associated with unusually abundant Enterococcus , which implies that the C. difficile toxins have different impacts on the faecal microbiota of children. Greater understanding of the relationships between disruption of the normal faecal microbiota and colonisation with C. difficile that produces different toxins might lead to improved treatment.

Interactions between bacterial microbiota and epibenthic species of the dinoflagellate Prorocentrum may define the onset and persistence of benthic harmful algal blooms (bHABs). Chemical ecological interactions within the dinoflagellate phycosphere potentially involve a complex variety of organic molecules, metabolites, and toxins, including undefined bioactive compounds. In this study, the bacterial diversity and core members of the dinoflagellate-associated microbiota were defined from 11 strains of three epibenthic Prorocentrum species, representing three geographically disjunct locations within Mexican coastal waters. Microbiota profiles in stable monoclonal Prorocentrum cultures were obtained by sequencing amplicons of the V3-V4 region of the 16S rRNA gene. Thirteen classes of bacteria were identified among dinoflagellate clones, where Alphaproteobacteria, Gammaproteobacteria, and Bacteroidia were consistently dominant. The bacterial community structure exhibited significantly different grouping by the location of origin of dinoflagellate clones. No significant diversity difference was found among free-living or unattached bacteria in the dinoflagellate culture medium (M) compared with those in closer association with the dinoflagellate host cells (H). Twelve taxa were defined as core members of the bacterial assemblage, representing the genera Algiphilus , Cohaesibacter , Labrenzia , Mameliella , Marinobacter , Marivita , Massilia , Muricauda , Roseitalea , and an unclassified member of the Rhodobacteraceae. The core members are inferred to significantly contribute to primary and secondary metabolic functions, but no direct correlation with dinoflagellate toxigenicity was apparent. Overall the bacterial profile and implied gene functionality indicated a suite of positive interactions, suggesting either mutualism or commensalism with the dinoflagellate. The further characterization and interpretation of specific gene functions and interactions between bacteria and dinoflagellates, such as epibenthic members of genus Prorocentrum , are key to understanding their role in toxigenesis and bHAB development.

Trichothecenes are toxins produced by certain species from several fungal genera, including Aspergillus , Fusarium , Isaria , Paramyrothecium , Stachybotrys, Trichoderma , and Trichothecium . These toxins are of interest because they contribute to the toxigenicity, plant pathogenicity, and/or biological control activities of some fungi. All trichothecenes have the same core (12,13-epoxytrichothec-9-ene or EPT) structure but can differ from one another by the presence or absence of a macrocyclic ring formed from polyketide and isoprenoid substituents esterified to carbon atoms 4 and 15 of EPT, respectively. Genes required for formation and some modifications of EPT have been elucidated, but almost nothing is known about genes specific to the formation of the macrocyclic ring. Therefore, we used genomic, transcriptomic, metabolomic, and gene deletion analyses to identify genes that are required specifically for the formation of the macrocyclic ring. These analyses identified one gene, TRI24 , that is predicted to encode an acyltransferase and that is required for macrocyclic ring formation during biosynthesis of macrocyclic trichothecenes by the fungus Paramyrothecium roridum . In addition, a TRI24 deletion mutant of P. roridum caused less severe disease symptoms on common bean and had less antifungal activity than its wild-type progenitor strain. We propose that the reduced aggressiveness and antifungal activity of the mutant resulted from its inability to produce trichothecenes with a macrocyclic ring. To our knowledge, this is the first report of a gene required specifically for the formation of the macrocyclic ring of trichothecenes and that loss of the macrocyclic ring of trichothecenes can alter the biological activities of a fungus. Key points • TRI24 gene is found in all known macrocyclic trichothecene-producing fungi. • A tri24-deletion mutant exhibits a reduction in antifungal and plant disease activities. • TRI24 is the first described gene specific to macrocyclic trichothecene biosynthesis. Graphical abstract

Bacillus cereus has long been recognized as an important pathogen in foodborne poisoning worldwide. Fresh vegetables are often contaminated with enterotoxigenic B. cereus and have been implicated as a vehicle for the transmission of this bacterium. This study reports on the occurrence, virulence gene profile, and antibiotic resistance of B. cereus in fresh vegetables. Of 102 examined samples, 48 (47%) of the samples were contaminated with B. cereus (>1 log CFU/g) and 7 (6.8%) of the samples showed more than 3 log CFU/g. In total, 118 B. cereus isolates were examined for the virulence genes nheA, nheB, nheC, hblA, hblC, hblD, cytK, and entFM and for resistance to antibiotics. Of these B. cereus isolates, 70% harbored nheA, nheB, nheC, and cytK. Eighteen (80%) of 21 isolates from bell peppers possessed eight enterotoxin genes. B. cereus isolates were susceptible to imipenem, vancomycin, gentamicin, erythromycin, ciprofloxacin, and chloramphenicol, whereas 22.4% of isolates from garlic chives, 48.7% from perilla leaf, and 40.5% from romaine lettuce showed antibiotic resistance to rifampin and 6% of isolates from garlic chives exhibited resistance to tetracycline. Three isolates from garlic chives were resistant to both tetracycline and rifampin. Raw vegetables were revealed to be major sources of B. cereus containing multiple toxin genes and exhibiting antibiotic resistance. Therefore, the potential health risks of consuming these vegetables raw or undercooked should not be underestimated. This study provides basic information for monitoring the antibiotic resistance and toxigenicity of B. cereus in the food chain during vegetable distribution and for developing food safety management to reduce the contamination with and transmission of B. cereus.

PurposeDiphtheria, still present in many countries of the world, is caused by toxigenic strains of species of the Corynebacterium diphtheriae complex, mainly Corynebacterium diphtheriae and the emerging zoonotic pathogen C. ulcerans. The immunoprecipitation test according to Elek is the gold standard for detection of the major virulence factor diphtheria toxin (DT) in toxigenic corynebacteria. Due to its sophisticated methodological requirements, the classical Elek test is performed mainly by specialized reference laboratories. It was revealed that the current modification of the Elek test does not detect the toxin in weakly toxigenic isolates. Therefore, a more robust method for detecting free DT is urgently needed, especially for toxigenic C. ulcerans strains which are known to produce often much lower amounts of DT than C. diphtheriae.MethodsThirty-one tox-positive C. ulcerans isolates with a negative standard Elek test result previously determined as NTTB (non-toxigenic tox bearing) were re-analyzed in this study using a modified immunoprecipitation method optimized regarding different parameters including type and concentration of antitoxin, medium volume, inoculum distance from the antitoxin disk and position of controls.ResultsAll 31 C. ulcerans strains tested positive in the optimized Elek test.ConclusionOnly with a reliable and easy-to-handle method for detecting the toxigenicity of C. ulcerans, it is possible to assess the etiological role of this emerging zoonotic bacterium in human pathology.

Despite great advancement in genetic typing, phenotyping is still an indispensable tool for categorization of bacteria. Certain amino acids may be essential for bacterial survival, growth, pathogenicity or toxin production, which prompts the idea that the intrinsic ability to utilize single amino acid under live-or-die situation could be a basis for differentiation of bacteria species. In this study, we determined the single amino acid consumption profiles of 7 bacterial species, and demonstrated that most bacteria have species-specific pattern of amino acid consumption. We also discovered that bacterial strains from different hosts, toxigenicity, and antibiotic-resistance presented distinct preference for certain amino acids. Taken altogether, the amino acid consumption profiles showed potential to be a novel tool complementary to study not only bacterial categorization but also biochemical characteristics of the bacteria such that its phenotyping can be used to uncover strategies for nutritional, pharmaceutical, taxonomic, and evolutionary aspects of bacterial researches.

This study determined the prevalence, characteristics, and risk factors of Campylobacter species contamination of chicken carcasses sold at informal poultry outlets in Gauteng province, South Africa. Within six townships, 151 chicken carcasses were collected from 47 outlets. Carcass swab, cloacal swab, and carcass drip samples were collected from each chicken, along with a matched questionnaire on risk factors regarding Campylobacter contamination. Sample-inoculated Bolton broth (BB) was cultured to isolate Campylobacter species by bacteriological methods. Subsequent confirmation and characterization of Campylobacter were conducted using polymerase chain reaction (PCR). Isolated Campylobacter strains were evaluated for the presence of six virulence genes (ciaB, dnaj, pldA, racR, flaA, and flaB), three toxin genes (cdtA, cdtB, and cdtC), and one antimicrobial resistance gene (tetO). The overall prevalence of Campylobacter was 23.4% (106 of 453), with sample type–specific prevalence being 17.2% (26 of 151), 25.8% (39 of 151), and 27.2% (41 of 151) for the carcass swabs, cloacal swabs, and carcass drip, respectively, following bacteriological isolation and confirmation by PCR. The overall prevalence of Campylobacter species was 93.5% by PCR, which varied significantly (P = 0.000) by sample: 99.2, 98.4, and 82.8% for carcass swabs, cloacal swabs, and carcass drip, respectively, by using PCR to detect Campylobacter in BB. Important risk factors for carcass contamination by Campylobacter included the slaughter of culled breeders and spent chickens, the use of stagnant water, and poor sanitation. Virulence and toxin gene frequencies were higher in C. jejuni–positive (82.5%) than in C. coli–positive (71.4%) BB cultures, but tetracycline resistance gene (tetO) frequency was higher in C. coli (75.9%) than in C. jejuni (48.10%). The observed high frequencies in C. jejuni recovered from street-vended chickens may pose food safety and therapeutic concerns to consumers. •Prevalence of Campylobacter in chickens from informal markets was determined.•Prevalence of Campylobacter was 23.4% (bacteriology) and 93.5% (PCR).•Virulence and toxin genes were detected in C. jejuni(81.5%) and C. coli (74.1%).•Tetracycline resistance gene was found in C. coli (75.9%) and C. jejuni (48.1%).•Virulent strains of C. jejuni in chickens could pose a food safety risk.

Mycotoxins pose significant health and economic challenges, affecting humans, animals, and commodities. This review examines fungal toxigenicity, its spread, and its impact on crops, ecosystems, and human health. The economic implications of mycotoxins, particularly their detrimental effect on the global economy, are also explored. Global agro-economic industry losses have been reported, emanating from cross-border import/export denials, product destruction, and routine analysis. Mainly victims of these exclusions are goods from regions accounting for 70% of global nut and dried fruit imports. Annual costs of mycotoxin contamination have been estimated to reach USD 100 million. The current review highlights the situation in the Americas, where Bt corn saves US farmers approximately USD 17 million annually by reducing damage from fumonisin and deoxynivalenol. In Africa and Asia, mycotoxin contamination presents severe health and economic challenges. Control measures include early harvesting, rapid drying, seed separation, sanitation, agronomic practices, insect control, the use of botanicals and synthetics, biological control, and detoxification of contaminated commodities.

This study evaluated the impact of seasonal and geographical variations on the toxigenicity of Aspergillus and Fusarium strains previously isolated from smallholder dairy cattle feeds and feedstuffs sampled during summer and winter in the Free State and Limpopo provinces of South Africa (SA). In total, 112 potential toxigenic fungal species were obtained and determined for their capability to produce mycotoxins on solid Czapek Yeast Extract Agar (CYA); followed by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Our result revealed that 41.96% of the fungal species produced their respective mycotoxins, including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), and zearalenone (ZEN), with higher levels of AFB1 (0.22 to 1045.80 µg/kg) and AFB2 (0.11 to 3.44 µg/kg) produced by fungal species isolated from summer samples than those in winter [(0.69 to 14.44 µg/kg) and (0.21 to 2.26 µg/kg), respectively]. The same pattern was also observed for AFB1 and AFB2 in Limpopo (0.43 to 1045.80 µg/kg and 0.13 to 3.44 µg/kg) and Free State (0.22 to 576.14 µg/kg and 0.11 to 2.82 µg/kg), respectively. More so, ZEN concentrations in summer (7.75 to 97.18 µg/kg) were higher than in winter (5.20 to 15.90 µg/kg). A similar observation was also noted for ZEN in Limpopo (7.80 to 97.18 µg/kg) and Free State (5.20 to 15.90 µg/kg). These findings were confirmed via Welch and Brown-Forsythe tests with significantly (p ≤ 0.05) higher mycotoxin levels produced by fungal strains obtained in samples during summer than those in winter. In contrast, the concentrations of mycotoxins produced by the fungal species from both provinces were not significantly (p > 0.05) different.