Milk hygiene assessment and characterization of staphylococcus aureus associated with the dairy value of chain in Western, Lusaka and Southern provinces of Zambia.

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Phiri, Bruno Stephen July
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The University of Zambia
Raw milk provides a good environment for the growth of a wide variety of microorganisms including Staphylococcus aureus (S. aureus) and can be a vehicle to transmit such pathogens to humans. The study aimed to investigate milk hygiene practices and characterize S. aureus/MRSA associated with the Zambian dairy chain in the selected Provinces and reduce the risk to consumers. A total of 1939 samples (of different matrices) were collected from 288 facilities (farms, milk collection centres (MCCs), traders, processing plants, traditional markets, and supermarkets/shops). Microbiological methods were used to analyse the samples with regard to S. aureus, including methicillin-resistant S. aureus (MRSA). A total of 348 presumptive S. aureus isolates (including one presumptive MRSA) were isolated. Molecular characterisation was used to type and characterise isolates of which 295 were confirmed by MALDI-TOF MS and PCR. No MRSA was detected. Raw milk from farms was widely contaminated with S. aureus in all the three provinces (Western: 46 percent, Lusaka: 43 percent and Southern: 33 percent) including that from MCCs and traders in Western and Southern provinces. Raw milk and/or sour milk samples from traditional markets and raw milk samples from milk processing plants were contaminated with S. aureus in all three provinces. S. aureus was less frequently detected from hands and nasal swabs from milkers, milk buckets and mastitis cows. No S. aureus was detected from water and commercially processed (heat-treated) dairy products from supermarkets/shops. A total of 36 known spa types were detected including seven novel types: t18396, t18397, t18398, t18399, t18400, t18402 and t18416. Spa type t355, t267 and t084 were common in all the three provinces. Many isolates from Lusaka province belonged to t189 and t267, while in Southern province t521 was dominant. Diversity and differences in the distribution and pattern of virulence, enterotoxin and antibiotic resistance genes were detected. A total of 29 S. aureus strains (10 percent) carried genes that codes for the virulence factor Panton-Valentine leucocidin (PVL). In addition, many of the 77 isolates selected for detailed characterisation by microarray or whole genome sequencing carried the virulence genes hlgA, hlgB, hlgC, lukD, lukE, lukY, lukX, and/or hlb; some isolates carried at least SplA, SplB, sak, chp and/or scn while other strains harboured eta and tst1. Genes for staphylococcal enterotoxins (SEs) were found in only 16 out of 77 isolates. Of the classical SE genes, sea was found in two isolates, seb and sec in three each, and sed in one. The predominant antimicrobial resistance in isolates included penicillin (41.7 percent), tetracycline (33.3 percent) and trimethoprim (27.8 percent) and 19.4 percent were multi drug resistant (MDR). Enterotoxigenic, virulent and antibiotic resistant S. aureus strains are circulating in the Zambian dairy value chain and represent a public health threat. The possible transmission of S. aureus to humans through cow milk is indicated. Therefore, there is a need for improved milk handling and hygienic practices at different stages/levels along the entire Zambian dairy value chain.
Thesis of Doctor of philosophy in Microbiology