The efficacy of bacteriophages FO1a and S16 in the reduction of Salmonella on chicken carcasses in a South African poultry processing environment

Thesis (PhD)--Stellenbosch University, 2022.

ENGLISH ABSTRACT: Much of the research surrounding bacteriophages (phages) as a processing aid for the control of Salmonella on chicken meat has been conducted in vitro in the laboratory. Information about the efficacy and application of bacteriophages as part of a hurdle concept in the chicken processing environment is limited. In South Africa, the use of certain antibiotics in live broilers and the use of chlorine-containing antimicrobials in the processing environment, are still permitted as Salmonella control methods. The incidence of Salmonella in chicken meat in South Africa is unclear, but previous research has repeatedly shown that the use of antibiotics and/or chlorine selects for resistance in Salmonella. The aim of this study was to determine the efficacy of a commercial phage cocktail PhageGuard S™ (PGS) (FO1a and S16 phages) in the reduction of Salmonella on chicken carcasses through a validated spraying system in a South African chicken processing plant. This study also investigated the incidence and antibiotic susceptibility profiles of Salmonella isolated from chicken carcasses in the plant. The PGS was applied at a 1% (v/v) concentration onto chicken carcasses via a spraying system (validated specifications: 530 µm nozzle diameter, 200 mesh strainer and 3 Bar pump pressure) after the chlorine spin chilling step. Neck skins samples were collected before the inside- outside wash step (N= 80) and after the PGS application step (N= 160). The neck skin samples were tested for Salmonella presence/absence (EN ISO 6579/A1 (02/2006)) and confirmed using Vitek®. Confirmed Salmonella isolates were screened for antibiotic susceptibility using the Kirby-Bauer disk diffusion method according to M100 from the Clinical and Laboratory Standards Institute (CLSI, 2020). Confirmed Salmonella isolates from neck skin samples collected after PGS application were re-exposed to PGS in the laboratory via a killing assay (Micreos Food Fafety, NL) to determine if the isolates were resistant to the PGS. Before the inside-outside step the Salmonella incidence was 60% with a large portion of these isolates showing resistance to tetracycline (56.3%) and sulfonamide (43.8%). After the combination of the inside-outside wash step, chlorine spin chilling and PGS application, the Salmonella incidence decreased to 23.75%, where more than half of these isolates showed resistance to tetracycline (63.2%) and sulfonamide (55.3%). For the killing assay, all isolates which survived PGS in the processing environment were reduced by 100% in the laboratory, highlighting that the phages were unable to reach the Salmonella via the spray application, and not that the Salmonella was resistant to the phages. The results in this study showed that the multi-drug resistant Salmonella in the chicken neck skins survived a complete immersion in chlorine but were successfully reduced by PGS, making phages a potential solution to many persistent microbial problems. This study also provides valuable insight into implementing phages into the large-scale hurdle concept of a processing environment and highlights the importance of the application method to ensure safe delivery of the phages to the target bacteria for a high efficacy.

AFRIKAANSE OPSOMMING: Navorsing wat fokus om bakteriofaag as ‘n hulpmiddel te gebruik in die beheer van Salmonella in hoenders, word hoofsaaklik in vitro in ‘n laboratorium uitgevoer. Inligting rakende die sukses en aanwending van bakteriofaag in hierdie omgewing is egter beperk. In Suid-Afrika word the gebruik van sekere antibiotikas en antimikrobiese middels what chloor bevat, nog toegelaat om Salmonella in lewendige braaihoenders te beheer. Die omvang van Salmonella in hoendervleis in Suid-Afrika is nog onseker, alhoewel vorige navorsing bewys dat salmonella weerstandigheid ontwikkel het teen die gebruik van antibiotikas en/of chloor. Die doel van hierdie studie is om die effektiwiteit te bepaal van die ‘n kommersiële bakteriofaag middel PhageGuard S™ (PGS) (FO1a en S16) in die vermindering van Salmonella in hoenderkarkasse. Die middel word toegedien deur ‘n sproeistelsel op a Suid-Afrikaanse hoenderplaas. Die studie ondersoek ook die antibiotika vatbaarheidsprofiele van Salmonella geisoleer van hoenderkarkasse op die plaas. Die PSG is toegedien op die hoenderkarkasse direk na die chloor spin-verkoelde stap, met ‘n sproeistelsel van ‘n 1% (v/v) konsentrasie (spesifikasies van ‘n 530 diameter pyp, 200 digte sif en ‘n 3 Bar pompdruk). Monsters van die hoendervel is geneem na die binne- en buite wasproses (N= 80) en na die PGS toegepas is (N= 160). Die velmonsters is getoets vir Salmonella (EN ISO 6579/A1 (02/2006)) en Vitek® is gebruik om die resultaat te bevestig. Die positiewe Salmonella gevalle is getoets vir antibiotika met die Kirby-Bauer skyf diffusie metode in ooreenstemming met M100 van die Clinical and Laboratory Standards Institute (CLSI, 2020). Die geisoleerde Salmonella gevalle van die velmonsters wat versamel is na die PSG toediening, is weer ‘n keer in die laboratorium blootgestel aan PSG deur middel van die dodertoets (Micreos Food Fafety, NL). Dit is om te bepaal of die geisoleerde gevalle PGS weerstandig is. Voor die binne- buiteproses was die Salmonella gevalle 60% waar ‘n groot gedeelte van die geisoleerde gevalle weerstandigheid getoon het teen tetracycline (56.3%) en sulfonamide (43.8%). Na die kombinasie van die binne- en buite wasproses, chloor spin-verkoelde stap, en die PSG toediening, het die Salmonella verminder na 23.75%, terwyl meer as die helfe van die geisoleerde gevalle weerstand getoon het teen tetracycline (63.2%) en sulfonamide (55.3%). Die geisoleerde gevalle wat PSG oorleef het op die slagplaas,met die dodertoets, is verminder met 100% in die laboratorium. Dit bevestig dat die bakteriofaag nie die Salmonella kon bereik met die sproeimetode nie, en nie dat die Salmonella weerstandig is teen die bakteriofaag is nie. Die studie se resultate bewys dat die multi-medikasie weerstandige Salmonella in die velmonsters ‘n volledige onderdompeling in chloor oorleef het, maar dat dit wel suksesvol verminder is met PSG. Dit maak bakteriofaag ‘n moontlike oplossing vir baie weerstandige mikrobiese probleme. Die studie gee ook waardevolle insig in die implementering van bakteriofaag in ‘n slagplaas omgewing en die belangrike toepassing van hierdie metode om die bakteriofaag op ‘n veilige wyse op spesifieke bakterië toe te pas vir die hoogste effektiwiteit.

Doctoral