Antimicrobial Effects of Plant Compounds against Virulent <i>Escherichia coli</i>O157:H7 Strains Containing Shiga Toxin Genes in Laboratory Media and on Romaine Lettuce and Spinach

Escherichia coli strains produce Shiga-toxins Stx-1 and Stx-2 that contribute to their virulence. The objective was to evaluate antimicrobial activities of plant essential oils (oregano, cinnamon, lemongrass), their active components (carvacrol, cinnamaldehyde, citral) and plant-extracts (green tea polyphenols, apple skin, black tea, decaffeinated black tea, grapeseed and pomace extracts) against E. coli O157:H7 strains containing Stx-1 and Stx-2 genes, as determined by Multiplex Polymerase Chain Reaction, in vitro and on leafy greens. Antimicrobials at various concentrations in sterile PBS were added to bacterial cultures (~3 - 4 logs CFU/ml), mixed thoroughly, and incubated at 37°C. Surviving bacteria were enumerated at 0, 1, 3, 5 and 24 h. The most effective essential oil (oregano oil;0.5%) and plant extract (green tea;3%) were evaluated against E. coli O157:H7 on romaine lettuce and spinach stored at 4°C for 7 days. Microbial survival was a function of the concentration of antimicrobials and incubation times. All antimicrobials reduced bacterial population to below detection levels in vitro;however, essential oils and active components exhibited greater activity than plant extracts. Oregano oil and green tea reduced E. coli O157:H7 on lettuce and spinach to below detection. Plant-based antimicrobials have the potential to protect foods against E. coli O157:

Microbial Quality and Molecular Identification of Enterotoxigenic Staphylococcus Strains Isolated from Dried, Smoked, and Braised Fish Sold in Ouagadougou Markets

Background: The investigation of toxin genes in strains involved in staphylococcal food poisoning contributes to food safety. The aim of this study was to isolate and identify enterotoxigenic Staphylococcus strains from dried, smoked, and braised fish sold in Ouagadougou markets. Methodology: Staphylococci were isolated using standard microbiology methods. Staphylococcus strains were identified using API Staph kit (Reference # 20500, BioMerieux S.A., Marcy l'Etoile, France). The molecular identification of isolated Staphylococcus aureus strains was specifically confirmed by PCR using the Staur4 and Staur6 primers. The genes encoding enterotoxins, enterotoxin-like toxins, exfoliative toxins, and TSST-1 toxin were detected by multiplex PCR using specific primers from Inquaba Biotec West Africa Ltd, Africa's Genomics Company. Results: The results of the microbiological quality assessment indicated that most of the samples analyzed were found to be of unsatisfactory microbiological quality according to the Staphylococcus aureus microbiological criteria (m = 102). Overall, only 12.55% of samples were satisfactory, while 97.45% were unsatisfactory. The STAPH API gallery allowed the identification of the following species: Staphylococcus aureus, Staphylococcus xylosus, Staphylococcus lugdunensis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus lentus, Staphylococcus sciuri and Staphylococcus capitis. Of the 108 Staphylococcus isolates, 81 (75%) showed at least one (1) toxin gene. Among the 21 toxin genes tested in this study, 20 genes were detected in all strains analyzed. The staphylococcal toxin genes detected were present in both Staphylococcus aureus and the other coagulase-negative strains isolated in this study. In addition, these genes are found individually or in association in certain strains. The most frequent genes detected in toxin gene-positive strains were: the tsst-1 gene in 45 isolated strains (41.7%), sei (16/14.8%), seg (13/12%), ser (7/6.5%) sec (6/5.5%), and sea (5/4.6%) for staphylococcal enterotoxins, seln (14/12.9%), selq (8/7.4%), for enterotoxin-like toxin gene and eta (3/2.7%) for exfoliative toxin genes. Conclusion: This study highlighted the pathogenicity of Staphylococcus strains isolated from dried, smoked, and braised fish sold in Ouagadougou markets. Monitoring toxin-producing strains of Staphylococcus is invaluable for better prevention of food poisoning.

Establishment and Application of a Real-time PCR Method for Detecting stx2 Gene in Shiga Toxin-producing Escherichia coli(STEC)

[Objective] This study aimed to establish a real-time PCR method for de- tecting stx2 gene in Shiga toxin-producing E. coli （STEC）. [Method] According to the known STEC stx2 gene sequences published in GenBank, PCR primers and probes were designed based on the conserved region to construct recombinant plasmid as a positive template, thus optimizing the reaction conditions and establishing the real- time PCR method. [Result] A standard curve was established based on the opti- mized real-time PCR system, indicting a good linear correlation between the initial template concentration and Ct value, with the correlation coefficient F^e of above 0.995. The established method had a good specificity, without non-specific amplifica- tion for 10 non-STEC intestinal bacterial strains; the detection limit of initial template was 1.0x102 copies/μI, indicating a high sensitivity; furthermore, the coefficients of variation within and among batches were lower than 1% and 5% respectively, sug- gesting a good repeatability. [Conclusion] In this study, a real-time PCR method was successfully established for detecting STEC stx2 gene, which provided technical means for rapid detection of STEC in samples.

Mycobacterium bovis BCG as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin

Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.

Clinical and molecular characteristics of Staphylococcus aureus isolated from Chinese children:association among the agr groups and genotypes,virulence genes and disease types

Background This study was aimed to investigate the clinical and molecular epidemiology of Staphylococcus aureus(S.aureus)isolated from Chinese children and determine the possible relationship among the accessory gene regulator(agr)groups and genotypes,as well as among the virulence genes and disease types.Methods S.aureus strains were isolated from Beijing Children's Hospital between October 2017 and October 2019.The isolates and 19 virulence genes were characterized using multi-locus sequence typing,staphylococcal protein A(spa),staphylococcal cassette chromosome mec,and agr typing.Results A total of 191 non-repetitive S.aureus clinical isolates were divided into 33 sequence types(STs),18 clonal com-plexes(CCs),and 59 spa types.ST59(39.8%),t437(37.7%),and agrⅠ(84.8%)were the predominant types.CC59,CC25,CC22,CC951,CC8,and CC398 belonged to agrⅠ.CC5 and CC15 were assigned to agrⅡ,and CC30 was characterized as agrⅢ.CC121 was classified under agrⅣ.The eta,etb,and bbp genes were more prevalent in agrⅣ(P<0.001 for each),while tst was more prevalent in agr groupⅢcompared to the other groups(P<0.001).Nearly all isolates that harbored lukS/F-PV belonged to agrⅠ(P=0.005).However,the correlation between disease types and agr groups was not significant(P>0.05).Conclusions An association among the agr groups and genotypes,as well as specific toxin genes,was observed among the S.aureus strains isolated from Chinese children.However,a statistical correlation was not found among the agr groups and disease types.

Lack of correlation of vacA genotype, cagA gene of Helicobacter pylori and their expression products with various gastroduodenal diseases

Abstract:Objective To investigate the correlation among vacA genotypes, cagA gene, VacA, serum CagA antibodies of Helicobacter pylori (H. pylori) and gastroduodenal diseases. Methods vacA genotypes and cagA gene of 62 H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were tested by polymerase chain reaction, and Hela cell assay for VacA activity in vitro. Serum CagA antibodies were measured by EIA method in the same patients.Results All 62 H. pylori strains possessed the vacA gene and vacA genotypes of all strains were type s1a/m2. Total positive rate of cagA gene was 56.45%; the positive rates of cagA gene of H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were 55.56%, 54.17% and 63.64%, respectively (P>0.05). The total positive rate of VacA was 37.10%; the positive rates of VacA produced by H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were 33.33%, 29.17% and 63.64%, respectively (P>0.05). The positive rates of CagA antibodies in patients with chronic gastritis, peptic ulcer and gastric cancer were 70.37%, 79.17% and 40.00%, respectively (P>0.05). The total positive rate of CagA antibodies was 68.85%.Conclusion There was no correlation among cagA gene and vacA genotypes of H. pylori, VacA, serum CagA antibodies and various gastroduodenal diseases.