Toxin-producing Genes Research Articles

Prevalence and antimicrobial resistance of porcine O157 and non-O157 Shiga toxin-producing Escherichia coli from India.

The aims of this study were to determine the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains in pigs as a possible STEC reservoir in India as well as to characterize the STEC strains and to determine the antimicrobial resistance pattern of the strains. A total of 782 E. coli isolates from clinically healthy (n = 473) and diarrhoeic piglets (309) belonging to major pig-producing states of India were screened by the polymerase chain reaction (PCR) assay for the presence of virulence genes characteristic for STEC, that is, Shiga toxin-producing gene(s) (stx1, stx2), intimin (eae), enterohemolysin (hlyA) and STEC autoagglutinating adhesin (Saa). Overall STEC were detected in 113 (14.4%) piglets, and the prevalence of E. coli O157 and non-O157 STEC were 4 (0.5%) and 109 (13.9%), respectively. None of the O157 STEC isolates carried gene encoding for H7 antigen (fliCh7). The various combinations of virulence genes present in the strains studied were stx1 in 4.6%, stx1 in combination with stx2 gene in 5.1%, stx1 in combination with stx2 and ehxA in 0.6%, stx1 in combination with stx2 and eae in 0.2% and stx2 alone in 3.7%. All STEC isolates were found negative for STEC autoagglutinating adhesin (Saa). The number of STEC isolates which showed resistance to antimicrobials such as ampicillin, tetracycline, streptomycin, lincomycin, nalidixic acid, sulfadiazine, penicillin, gentamicin, kanamycin and ceftriaxone were 100, 99, 98, 97, 95, 94, 92, 88, 85 and 85, respectively. Ninety-seven isolates showed resistance to more than 2 antimicrobials, and 8 resistance groups (R1 to R8) were observed. This study demonstrates that pigs in India harbour both O157 and non-O157 STEC, and this may pose serious public health problems in future.

Differentiation of Pseudomonas syringae pathovars originating from stone fruits

Due to an overlapping host range, similar symptomatology and many common characteristics, Pseudomonas syringae pathovars originating from stone fruits can easily be misidentified. In order to select tests for rapid and efficient differentiation of P. s. pvs. syringae, morsprunorum and persicae, we studied the suitability and differentiating potential of some standard bacteriological and molecular methods. Differentiation of the strains was performed using LOPAT, GATTa and ice nucleation tests, nutrient sucrose broth growth and utilization of various carbon sources. PCR method enabled the detection of toxin-producing genes: syrB and syrD in P. s. pv. syringae, and cfl gene in P. s. pv. morsprunorum race 1. Syringomycin production by pv. syringae was confirmed in bioassay using Geotrichum candidum, Saccharomyces cerevisiae and Rhodotorula pilimanae as indicator organisms. Pathogenicity test on lemon and immature nectarine fruits, as well as on string bean pods, showed different intensity of reaction of the inoculated material which could separate pv. syringae from the other two pathovars. PCR-based repetitive sequences, Rep-PCR with REP, ERIC and BOX primers revealed different genetic profiles within P. syringae pathovars.

Growth Profile and Toxigenicity of Bacillus cereus in Ready-to-eat Food Products of Animal Origin

The growth profile of Bacillus cereus in ready-to-eat (RTE) food products of animal origin was examined under different temperature and incubation conditions. In sandwiches and Kimbab, B. cereus did not grow or exhibited only minimal growth at 4 and <TEX>$10^{\circ}C$</TEX>, but it grew rapidly at ambient temperature. In sandwiches, B. cereus did not grow efficiently at <TEX>$25^{\circ}C$</TEX>, however, in ham, the main ingredient of sandwiches, B. cereus growth was observed at the same temperature, with bacterial levels reaching 7.94 Log CFU/g after incubation for 24 h at <TEX>$25^{\circ}C$</TEX>. Toxigenicity of B. cereus was observed only at temperatures above <TEX>$25^{\circ}C$</TEX>. In Kimbab, B. cereus produced toxin after 9 h at <TEX>$30^{\circ}C$</TEX> and after 12 h at <TEX>$25^{\circ}C$</TEX>. Ingredients of sandwiches and Kimbab were collected from 3 different Korean food-processing companies to investigate the source of contamination by B. cereus. Among the 13 tested food items, 6 items including ham were found to be contaminated with B. cereus. Of these ingredients, B. cereus isolates from 3 items produced enterotoxins. None of these isolates harbored the emetic toxin-producing gene. The findings of the present study can be used for risk assessments of food products, including ham and cheese, contaminated with B. cereus.

Oligonucleotide microarray for the identification of potential mycotoxigenic fungi

BackgroundMycotoxins are secondary metabolites which are produced by numerous fungi and pose a continuous challenge to the safety and quality of food commodities in South Africa. These toxins have toxicologically relevant effects on humans and animals that eat contaminated foods. In this study, a diagnostic DNA microarray was developed for the identification of the most common food-borne fungi, as well as the genes leading to toxin production.ResultsA total of 40 potentially mycotoxigenic fungi isolated from different food commodities, as well as the genes that are involved in the mycotoxin synthetic pathways, were analyzed. For fungal identification, oligonucleotide probes were designed by exploiting the sequence variations of the elongation factor 1-alpha (EF-1 α) coding regions and the internal transcribed spacer (ITS) regions of the rRNA gene cassette. For the detection of fungi able to produce mycotoxins, oligonucleotide probes directed towards genes leading to toxin production from different fungal strains were identified in data available in the public domain. The probes selected for fungal identification and the probes specific for toxin producing genes were spotted onto microarray slides.ConclusionsThe diagnostic microarray developed can be used to identify single pure strains or cultures of potentially mycotoxigenic fungi as well as genes leading to toxin production in both laboratory samples and maize-derived foods offering an interesting potential for microbiological laboratories.

Protein kinase A regulates production of virulence determinants by the entomopathogenic fungus, Metarhizium anisopliae

Metarhizium anisopliae is a model system for studying insect fungal pathogenesis. The role of cAMP signal transduction in virulence was studied by disrupting the class I PKA catalytic subunit gene ( MaPKA1). The PKA mutant ( ΔMaPKA1) showed reduced growth and greatly reduced virulence. PKA was dispensable for differentiation of infection structures (appressoria), but differentiation was delayed and the appressoria were defective because of reduced turgor pressure. ΔMaPKA1 germinated at similar rates as the wild type in glucose and glycerol, but germination was delayed on alanine. Conidial adhesion and appressorium formation by ΔMaPKA1 against a plastic surface was fully inhibited with glucose as sole nutrient source. Adhesion to plastic was not inhibited with glycerol or alanine, but appressorium formation was delayed. ΔMaPKA1 showed reduced tolerance to the oxidative agent diamide, but not to H 2O 2 and methyl-viologen. Comparative transcriptome analysis of ΔMaPKA1 and the wild type strain showed that PKA is responsible for up-regulating approximately one-third of the genes induced by insect cuticle, including subsets of those responsible for differentiation of appressoria and penetration pegs, cuticle degradation, nutrient acquisition, pH regulation, lipid synthesis, cell cycle control and the cytoskeleton. PKA was not however required for expression of toxin-producing genes. We conclude therefore that MaPKA1 is required for sensing host-related stimuli and transduction of these signals to regulate many infection processes.

Genomics‐based food‐borne pathogen testing and diagnostics: Possibilities for the U.S. department of agriculture's food safety and inspection service

The use of genomic technologies at the U.S. Department of Agriculture could enhance inspection, monitoring, and risk assessment capabilities within its Food Safety and Inspection Service (FSIS). Molecular assays capable of detecting hundreds of microbial DNA sequences within a single food sample that identify food-borne pathogens of concern and characterize their traits most relevant to human health risk are of great interest for FSIS. For example, a high-density assay, or combination of assays, could screen FSIS inspected food for pathogens relevant to public health (e.g., Salmonella, Listeria, and toxic E. coli) as well as their associated virulence factors and antibiotic resistance genes. Because most genotype assays can be completed in one working day with a minimum of reagents, use of such assays could potentially save FSIS a significant amount of cost/time for analyses. Further, a genotype assay can detect specific microbial traits relevant to human health risk based on the DNA sequence of toxin producing genes, antibiotic resistance alleles, and more. By combining rapid analysis with specific data on human health risks, information from such high-density genotype assays could provide expanded support for test and hold situations, recalls, outbreak management, and microbial risk assessments (e.g., provide data needed for food-borne illness source attribution). Environ. Mol. Mutagen.

Transduction of the Plasmid Encoding Antiseptic Resistance Gene qacB in Staphylococcus aureus

The plasmid-borne qacA and qacB genes encode a multidrug efflux protein. The proteins encoded by qacA and qacB mediate efflux of cationic antiseptic agents such as quaternary ammonium compounds. In methicillin-resistant Staphylococcus aureus (MRSA), qacA and qacB are widely prevalent and decrease antiseptic susceptibility. However, it is difficult to find the plasmids encoding qacA or qacB in community-associated MRSA (C-MRSA) isolated from patients with impetigo. Most MRSA, the strains causative of impetigo, carry the plasmid-borne exfoliative toxin-producing gene etb. To find the reason for the paucity of qacA or qacB in MRSA isolated from patients with impetigo, we performed transfer experiments of the plasmid pTZ2162qacB encoding qacB. The pTZ2162qacB was transferred to S. aureus strain RN4220 by transduction, although no pTZ2162qacB was transferred by conjugation. Additionally, pTZ2162qacB was transduced to MRSA carrying etb, and was coexistence with the plasmid encoding etb. Our results showed that pTZ2162qacB was horizontally transferred by transduction and was compatible with the plasmid encoding etb. Consequently, there will be risk of the emergence of C-MRSA with decreased antiseptic susceptibility among patients with impetigo.

Changes in drug susceptibility and toxin genes in Staphylococcus aureus isolated from blood cultures at a university hospital.

We studied changes in toxin-producing genes and drug susceptibility in Staphylococcus aureus isolated from blood cultures at the University of Tokyo Hospital between 1980-1984 (six mecA gene-positive methicillin resistant S. aureus [MRSA] strains and 20 mecA gene-negative methicillin-susceptible S. aureus [MSSA] strains) and 1999 (11 MRSA and 20 MSSA strains). The prevalence of strains with toxin-producing genes increased from 66.7% to 90.9% in MRSA, and from 30.0% to 55.0% in MSSA during the interval. Among toxin-producing gene-positive S. aureus, the dominant strains shifted from those with the enterotoxin (ET) - A gene in 1980-1984 to those with both the toxic shock syndrome toxin-1 and the ET-C genes in 1999. All strains were susceptible to vancomycin and teicoplanin. Mupirocin and arbekacin inhibited all strains at concentrations of less than or equal to 0.5 micro g/ml and 4 micro g/ml, respectively. More than half of the MRSA strains in 1999 were considered to be nonsusceptible to flomoxef. Because almost all MRSA and more than half of MSSA among recently isolated strains possessed the toxin-producing genes, we should pay attention to whether toxin-related diseases caused by MRSA and MSSA are increasing.

Detection of bacterial pathogens by phage antibody display.

Vibrio parahaemolyticus is a halophilic Gramnegative facultative anaerobe commonly found in estuarine waters and in seafood like shellfish. It has been implicated in outbreaks of gastro-enteritis in several countries [1]. Food borne illness caused by V. parahaemolyticus results chiefly from the consumption of insufficiently heated or raw seafood, especially in the summer months. The standard method for detection of V. parahaemolyticus (Bacteriological Analytical Manual procedure) is a culture-based procedure which can take up to four days for positive identification. Recent outbreaks of V. parahaemolyticus related illnesses have heightened the need to develop a rapid and reliable method to detect this pathogen in shellfish. PCR methods, based on the amplification of the toxin producing genes, unfortunately give false postive results with other Vibrio species [2]. In our studies we have used phage antibody display to differentiate between pathogenic V. parahaemolyticus and other pathogenic and non-pathogenic Vibrio sp.