Categorization of Listeria monocytogenes from food, environmental, and clinical origin in the Western Cape (South Africa)

Thesis (MScFoodSc)--Stellenbosch University, 2020.ENGLISH ABSTRACT: Listeria monocytogenes is a ubiquitous food pathogen responsible for the often-fatal infection, listeriosis. Foods that act as vectors of L. monocytogenes include meat products, seafood and fish products, dairy products, and ready-to-eat (RTE) foods. South Africa recently experienced the largest ever listeriosis outbreak during 2017-18, and despite this, information on L.monocytogenes is still lacking. Internationally, there is a high incidence of L. monocytogenesresistance towards antibiotics currently used as a treatment for listeriosis. However, in South Africa there is little information available on the resistance patterns of L. monocytogenes to these antibiotics. Additionally, due to the difficulty in controlling L. monocytogenes in the food processing environment, new methods such as bacteriophage treatment are being investigated to reduce L. monocytogenes numbers in these environments. Worldwide, studies have demonstrated the efficacy of bacteriophages against L. monocytogenes, but in South Africa, this technology is still unfamiliar and untested. Seeing as South Africa has a high burden of serious diseases, such as tuberculosis and HIV/AIDS, which often amplify the impacts of listeriosis, data on the efficacy of phage treatment, as well as currently used antibiotics, against L. monocytogenes, is greatly needed. Using L. monocytogenes isolates from various origins (Clinical, Environmental, Raw meats, Raw seafood, and RTE) in the Western Cape, South Africa, the first objective was to classify L.monocytogenes isolates into lineage groups by using a recently developed PCR-RFLP method(based on SNPs within the hlyA gene). The results showed an overrepresentation of Lineage I in Clinical environments and Raw seafood, while in the Environmental, Raw meats, and RTE categories, Lineages I and II were somewhat equally distributed. The prevalence of a high number of Lineage I isolates in categories other than Clinical contrasts with previous evidence that Lineage I is mostly associated with human listeriosis, while Lineage II is mostly associated with foods. The second objective was to determine the susceptibility of L. monocytogenes isolates to a commercial bacteriophage (ListexTM P100), using spot tests. Additionally, the lineage group data was used to determine whether or not lineage classifications influenced bacteriophage susceptibility. The L. monocytogenes isolates from the Clinical, Environmental, Raw meats, and Raw Seafood categories were significantly susceptible to phage activity. However, a large fraction of isolates in the RTE category were tolerant to the phage, which disagrees with the findings of others. Additionally, both lineage groups were significantly susceptible to phage activity when considering all categories combined and lineage grouping did not significantly influence phage susceptibility. The final objective was to determine the antibiotic resistance of L. monocytogenes isolates to five antibiotics, namely ampicillin, chloramphenicol, erythromycin, gentamicin, and tetracycline, using the disc diffusion method (EUCAST). The results indicated that all isolates were significantly susceptible to ampicillin, and many isolates were resistant to chloramphenicol, erythromycin, and tetracycline. Clinical and Raw seafood isolates were significantly susceptible to all antibiotics, while Raw meats had the highest number of resistant strains.AFRIKAANSE OPSOMMING: Listeria monocytogenes is 'n alomteenwoordige voedselpatogeen wat verantwoordelik is vir die dikwels noodlottige infeksie, listeriose. Voedselsoorte wat dien as vektore van L. monocytogenes sluit in vleisprodukte, seekos en visprodukte, suiwelprodukte, en gereed-om-te eet (RTE) voedsel. Suid-Afrika het onlangs die grootste listeriose-uitbraak gedurende 2017-18 ervaar, en ten spyte hiervan, ontbreek inligting oor L. monocytogenes nogsteeds in Suid-Afrika. Internasionale studies het hoë weerstandigheid gerapporteer teen antibiotika wat tans gebruik word as 'n behandeling vir listeriose. In Suid-Afrika is daar egter min inligting beskikbaar oor die weerstandigheid van L. monocytogenes aan hierdie antibiotika. Daarbenewens, as gevolg van die probleme in die beheer van L. monocytogenes in die voedselverwerkings-omgewing (voedselfabrieke), word nuwe metodes soos bakteriofaag-behandeling ondersoek om die teenwoordigheid van L. monocytogenes in hierdie omgewings te verminder. Wêreldwyd het studies die doeltreffendheid van bakteriofage teen L. monocytogenes gedemonstreer, maar in Suid-Afrika is hierdie tegnologie steeds onbekend en ongetoets. Aangesien Suid-Afrika 'n hoë las het van ernstige siektes soos tuberkulose en MIV/VIGS, wat dikwels die impak van listeriose vererger, is data oor die doeltreffendheid van bakteriofage, asook antibiotikum weerstandigheid, baie nodig. Die eerste doel was om L. monocytogenes isolate van verskeie bronne (Kliniese, Omgewing, Rou vleis, Rou seekos, en RTE) in die Wes-Kaap, Suid-Afrika, te klassifiseer in een van drie linie groepe, deur die gebruik van 'n onlangs-ontwikkelde metode vir PCR-RFLP (gebaseer op enkel-nukleotied polimorfisme binne die hlyA-geen). Die resultate het getoon dat daar ‘n oorverteenwoordigheid van Linie I was in isolate komende van listeriosis pasiënte (Klinies) en rou seekos, terwyl Linie I en Linie II iewat ewe versprei was in isolate komende van die Omgewing, Rou vleis, en RTE. Die voorkoms van 'n hoë aantal Linie I isolate in kategorieë anders as Kliniese, is in teenstelling met baie ander studies wat Linie I gevind het meestal van listeriose gevalle (d.w.s. kliniese), terwyl Linie II meer dikwels met voedsel geassosieer word. Die tweede doel was om die vatbaarheid van L. monocytogenes isolate te bepaal vir 'n kommersiële bakteriofaag (ListexTM P100), deur middel van kol-toetse. Verder is die linie groepering gebruik om te bepaal of vatbaarheid vir die bakteriofaag daardeur beinvloed word. Die L. monocytogenes isolate van die Kliniese, Omgewings-, Rou vleis, en Rou seekos-kategorieë was aansienlik vatbaar vir die bakteriofaag. 'n Groot fraksie van isolate in die RTE-kategorie was egter onvatbaar vir die bakteriofaag, in teenstelling met ander se bevindings. Daarbenewens was beide linie groepe beduidend vatbaar vir bakteriofaag aktiwiteit by die oorweging van alle kategorieë gekombineer en linie groepering het nie beduidend hierdie vatbaarheid beïnvloed nie. Die finale doel was om die antibiotikum-weerstand van L. monocytogenes te bepaal teen vyf antibiotika, naamlik ampisillien, chloramphenicol, eritromisien, gentamisien en tetrasikliene, met behulp van die skyfdiffusie-metode (EUCAST). Die resultate het aangedui dat alle isolate beduidend vatbaar was vir ampisillien, en baie isolate was weerstandig teen chloramphenicol, eritromisien en tetrasikliene. Kliniese en Rou seekos-isolate was aansienlik vatbaar vir alle antibiotika, terwyl die Rou vleis kategorie die meeste weerstandige isolate bevat het.Master