Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

none13sìListeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.openGiuditta Fiorella Schiavano * , Collins Njie Ateba , Annalisa Petruzzelli , Veronica Mele , Giulia Amagliani , Fabrizia Guidi , Mauro De Santi , Francesco Pomilio , Giuliana Blasi , Antonietta Gattuso , Stefania Di Lullo , Elena Rocchegiani, Giorgio BrandiSchiavano, GIUDITTA FIORELLA; Njie Ateba, Collins; Petruzzelli, Annalisa; Mele, Veronica; Amagliani, Giulia; Guidi, Fabrizia; DE SANTI, Mauro; Pomilio, Francesco; Blasi, Giuliana; Gattuso, Antonietta; Di Lullo, Stefania; Rocchegiani, Elena; Brandi, Giorgi

The detection and molecular characterisation of Shiga Toxigenic Escheria coli (STEC) O157 strains from humans, cattle and pigs in the North–West Province, South Africa

MSc. (Agric.) North-West University, Mafikeng Campus, 2006The prevalence and antibiotic resistant profiles of shiga-toxin producing Escherichia coli 0157 strains isolated from faeces samples of cattle, pigs and human stool samples were determined. The strains were further characterised by molecular methods for the presence of shiga-toxin virulence genes and antibiotic resistant genes. Seventy-six Escherichia coli 0157 strains were isolated and the prevalence was higher among E. coli isolated from faeces from pigs (44.2% to 50%) than those from cattle faeces (5.4% to 20.0%) or human stool samples (7 .5%). On testing E. coli 0157 isolates for their resistance to 9 antimicrobial agents, multiple antibiotic resistance (MAR) was observed in all of the isolates arising from resistance to three or more antibiotics. Seventy (92.1 %) of the E. coli 0157 isolated from humans, cattle and pigs were resistant to tetracycline. 73 (96.1 %) were resistant to sulphamethoxazole, 63 (82.9%) were resistant to erythromycin. 40 (52.6%) were resistant to streptomycin and 26 (34.2%) were resistant to ampicillin. The highest frequency of resistance was observed among the human isolates (n=3 ), where 3 (I 00%) of the isolates were resistant to tetracycline, sulphamethoxazole, erythromycin and ampicillin. Furthermore, among the pig isolates (n=60), 58 (96. 7%) were resistant to tetracycline, 57 (95%) were resistant to sulphamethoxazole, 47 (78.3%) were resistant to erythromycin. 38 (63.3%) were resistant to streptomycin and 22 (36. 7%) were resistant to ampicillin. The MAR phenotypes S-Smx-T-E, Smx-T-Ap and Smx-T-E were the dorminant phenotypes among the E. coli 0157 isolated from the faeces samples of communal pigs in 30.4%, 21 .7% and 17.4% of these isolates, respectively. However, phenotypes Smx-T -E and S-Smx-T-E-Ne were identified at I6.2% and 10.8%, respectively within the isolates obtained from commercial pig faeces. The phenotype Smx-T-E was the only MAR phenotype identified among the E. coli 0157 isolated from the faecal samples of commercial cattle at Lichtenburg. Furthermore, MAR phenotypes Smx-T-E-C, K-S-Smx-T-E, S-Smx-T-E and Smx-T-E-Ap were obtained at 25%, respectively for the isolates obtained from communal cattle at Mogosane while Smx-T-E-Ap was the dorminant (66.7%) phenotype among the isolates of human origin. The phenotype Smx-T fom1ed the basis of all the MAR phenotypes obtained and this was similar to the percentage antibiotic resistance data. The distribution of the resistant determinants for tetracycline was determined by PCR analysis in resistant isolates. A tetB gene was detected in E. coli 0157 of pig origin. Based on the characterisation of 30 isolates for the presence of STEC virulence genes by PCR, 18 (60%) possessed the hlyA gene, 7 (23.7%) possessed the eae gene and 5 ( 16. 7%,) harboured both genes. The average MAR indices for pig, cattle and human E. coli 0157 isolates were 0.4n2, 0.3419 and 0.4814, respectively. Among the cattle isolates, the group MAR index was highest for the communal (Mogosane) population while the values for the commercial populations at Lichtenburg and Rustenburg were 0.33 and 0.22, respectively. £. coli 0157 isolated from pigs revealed MAR index results that were 0.508 and 0.415 for the commercial and communal populations respectively and 0.1851 for the E. coli control strains. Characterisation by cluster analysis to determine the commonness and resolve differences between the E. coli 0157 isolated from the Various sources revealed a close association between pig (Tlapeng and Mareetsane), cattle (Mogosane) and human isolates. Interestingly, E. coli 0157 isolated from pigs occurred at the highest frequency in all the clusters. which suggested their role in the dissemination of resistant determinants.Master

Genetic diversity, host range and molecular analysis of the virulence determinants of escherichia coli O157:H7 isolated from different sources

Thesis (PhD (Agric) North-West University, Mafikeng Campus, 2011Doctora

Genotypic Characterization of Escherichia coli O157:H7 Isolates from Different Sources in the North-West Province, South Africa, Using Enterobacterial Repetitive Intergenic Consensus PCR Analysis

In many developing countries, proper hygiene is not strictly implemented when animals are slaughtered and meat products become contaminated. Contaminated meat may contain Escherichia coli (E. coli) O157:H7 that could cause diseases in humans if these food products are consumed undercooked. In the present study, a total of 94 confirmed E. coli O157:H7 isolates were subjected to the enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) typing to generate genetic fingerprints. The ERIC fragments were resolved by electrophoresis on 2% (w/v) agarose gels. The presence, absence and intensity of band data were obtained, exported to Microsoft Excel (Microsoft Office 2003) and used to generate a data matrix. The unweighted pair group method with arithmetic mean (UPGMA) and complete linkage algorithms were used to analyze the percentage of similarity and matrix data. Relationships between the various profiles and/or lanes were expressed as dendrograms. Data from groups of related lanes were compiled and reported on cluster tables. ERIC fragments ranged from one to 15 per isolate, and their sizes varied from 0.25 to 0.771 kb. A large proportion of the isolates produced an ERIC banding pattern with three duplets ranging in sizes from 0.408 to 0.628 kb. Eight major clusters (I–VIII) were identified. Overall, the remarkable similarities (72% to 91%) between the ERIC profiles for the isolate from animal species and their corresponding food products indicated some form of contamination, which may not exclude those at the level of the abattoirs. These results reveal that ERIC PCR analysis can be reliable in comparing the genetic profiles of E. coli O157:H7 from different sources in the North-West Province of South Africa

Untapped Potentials of Endophytic Fungi: A Review of Novel Bioactive Compounds with Biological Applications

Over the last century, endophytic fungi have gained tremendous attention due to their ability to produce novel bioactive compounds exhibiting varied biological properties and are, therefore, utilized for medicinal, pharmaceutical, and agricultural applications. Endophytic fungi reside within the plant tissues without showing any disease symptoms, thus supporting the physiological and ecological attributes of the host plant. Ground breaking lead compounds, such as paclitaxel and penicillin, produced by endophytic fungi have paved the way for exploring novel bioactive compounds for commercial usage. Despite this, limited research has been conducted in this valuable and unique niche area. These bioactive compounds belong to various structural groups, including alkaloids, peptides, steroids, terpenoids, phenols, quinones, phenols, and flavonoids. The current review focuses on the significance of endophytic fungi in producing novel bioactive compounds possessing a variety of biological properties that include antibacterial, antiviral, antifungal, antiprotozoal, antiparasitic, antioxidant, immunosuppressant, and anticancer functions. Taking into consideration the portal of this publication, special emphasis is placed on the antimicrobial and antiviral activities of metabolites produced by endophytes against human pathogens. It also highlights the importance of utilization of these compounds as potential treatment agents for serious life-threatening infectious diseases. This is supported by the fact that several findings have indicated that these bioactive compounds may significantly contribute towards the fight against resistant human and plant pathogens, thus motivating the need enhance the search for new, more efficacious and cost-effective antimicrobial drugs

Characterisation of Escherichia coli O157 strains from humans, cattle and pigs in the North-West Province, South Africa

http://dx.doi.org/10.1016/j.ijfoodmicro.2008.08.01

Genotypic and Phenotypic Evaluation of Biofilm Production and Antimicrobial Resistance in Staphylococcus aureus Isolated from Milk, North West Province, South Africa

Background: Biofilm formation in S. aureus may reduce the rate of penetration of antibiotics, thereby complicating treatment of infections caused by these bacteria. The aim of this study was to correlate biofilm-forming potentials, antimicrobial resistance, and genes in S. aureus isolates. Methods: A total of 64 milk samples were analysed, and 77 S. aureus were isolated. Results: Seventy (90.9%) isolates were biofilm producers. The ica biofilm-forming genes were detected among 75.3% of the isolates, with icaA being the most prevalent (49, 63.6%). The icaB gene was significantly (P = 0.027) higher in isolates with strong biofilm formation potentials. High resistance (60%–90%) of the isolates was observed against ceftriaxone, vancomycin, and penicillin, and 25 (32.5%) of S. aureus showed multidrug resistance (MDR) to at least three antibiotics. Five resistance genes, namely blaZ (29, 37.7%), vanC (29, 37.7%), tetK (24, 31.2%), tetL (21, 27.3%), and msrA/B (16, 20.8%) were detected. Most MDR phenotypes possessed at least one resistance gene alongside the biofilm genes. However, no distinct pattern was identified among the resistance and biofilm phenotypes. Conclusions: The high frequency of potentially pathogenic MDR S. aureus in milk samples intended for human consumption, demonstrates the public health relevance of this pathogen in the region

Genetic characterization of antibiotic-resistant Staphylococcus aureus from milk in the North-West Province, South Africa

Food borne diseases are a major public health concern worldwide. Staphylococcus aureus is one of the potential food borne pathogens which causes nosocomial and community acquired infections. In the present study, 74 representative strains of S. aureus isolated and characterized in previous study from different milk samples were subjected to random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus (ERIC)-PCR to generate fingerprints to determine the genetic relationships of the isolated strains. A total of 20 RAPD patterns were generated and the number of amplified fragments obtained ranged from 0 to 8 with molecular weight ranging from 250 to 2000 bp. A dendrogram based on fingerprinting pattern grouped isolates into twelve major clusters (I–XII). In the case of ERIC-PCR 9 banding patterns were obtained with amplicons ranging from 1 to 8 and band sizes ranging from 250 to 2000 bp. A total of four major clusters (I–IV) were observed in the dendrogram based on ERIC fingerprints. The discrete banding patterns obtained both from ERIC-PCR and RAPD-PCR showed remarkably the genetic diversity of S. aureus. The findings of this study indicate that raw, bulk and pasteurized milk in the North-West Province was contaminated with toxigenic and multi-drug resistant S. aureus strains. This emphasizes the need to implement appropriate control measures to reduce contamination as well as the spread of virulent S. aureus strains to reduce the burden of disease in humans. Keywords: ERIC-PCR, Genetic fingerprinting, Milk, RAPD-PCR, Staphylococcus aureu

Polyphasic Assessment of Aflatoxin Production Potential in Selected Aspergilli

This study investigated the aflatoxin production potentials of selected fungi using a polyphasic approach. Internally transcribed spacer region of the fungi was amplified using the polymerase chain reaction. Forty-five Aspergillus strains were further assessed for aflatoxin production using the conventional methods such as growth on yeast extract sucrose, β-cyclodextrin neutral red desiccated coconut agar (β-CNRDCA); expression of the aflatoxin regulatory genes and the use of both thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A large proportion (82.22%) of the isolates harbored the Nor-1 gene while 55.56%, 68.89%, and 80% possessed the ver-1, omt-A, and aflR genes, respectively. All 100% the isolates harbored the aflJ gene. Twenty-three isolates were positive for aflatoxin production based on the yeast extract sucrose medium (YES) test; ammonium vapor test (51%), yellow pigment production (75.5%), and β-CNRDCA tests; and blue/green fluorescence (57.7%). Based on TLC detection 42.2% produced aflatoxins while in the HPLC, total aflatoxin (AFTOT) production concentrations ranged from 6.77–71,453 µg/g. Detectable aflatoxin B1 (AFB1) concentrations obtained from the HPLC ranged between 3.76 and 70,288 µg/g; 6.77 and 242.50 µg/g for aflatoxin B2 (AFB2); 1.87 and 745.30 µg/g for aflatoxin G1 (AFG1); and 1.67 and 768.52 µg/g for aflatoxin G2 (AFG2). AFTOT contamination levels were higher than European Union tolerable limits (4 µg/kg). The regression coefficient was one (R2 = 1) while significant differences exist in the aflatoxin concentrations of Aspergillus (p ≤ 0.05). This study reports the potentials of Aspergillus oryzae previously known as a non-aflatoxin producer to produce AFG1, AFG2, AFB1, and AFB2 toxins. Aspergillus species in feedlots of animals reared for food are capable of producing aflatoxins which could pose hazards to health