Putative Amplification of 16S rRNA Markers of Escherichia coli and Salmonella spp Isolates From Raw Milk Sample of Cattle

Abstract

Milk derived from milk of dairy cows harbour a variety of microorganisms and can be sources of food borne pathogens. The presence of specific amplicons of stx, stx2, in timin genes, InvA, and stn genes are responsible for the virulence of the organisms. The aim of this study was to amplify the 16S rRNA marker genes of E.coli and Salmonella spp.A total number of ten raw milk samples were collected from Fulani dairy farm at Ago-Aare, Oke-Ogun area, Oyo State, Nigeria. Both total viable bacteria counts (TBC) and total coliform counts (TCC) analysis were performed to determine the microbial load of the raw milk samples. The antimicrobial susceptibility testing for both E.coli and Salmonella were carried out using the disk diffusion method. A more specific identification of the bacterial 16S rRNA genes was performed by PCR and Sanger sequencing technique by using specific primers which are specific for the amplification of E.coli and Salmonella genes. Total viable bacteria count of all the samples ranged from 1.23 x105 cfu/ml (R7) to 2.60 x105 cfu/ml (R1). Total coliform count ranged from 1.05 x105 cfu/ml (R7) to 1.93 x105 cfu/ml (R9). Both counts were significant at (P >0.05). The raw milk samples had 80% E.coli, 70% Salmonella, 20% Shigella spp, 60% Klebsiella spp, while 50% level of contamination was observed with Staphylococcus aureus, 20% Proteus spp and 20% Pseudomonas spp. Isolates were catalase (+), glucose and maltose (+) and oxidase (-). Antibiotic susceptibility test showed that Salmonella spp and E.coli were resistant to most commonly used antibiotics and most susceptible to Ofloxacin and ceftriaxone. Molecular characterization of both E.coli and Salmonella spp indicated the presence of specific amplicons of stx1, stx2, intimin genes, InvA and stn genes respectively. The presence of these genes could justify the degree of virulence in the organisms that harboured the genes. Conversely the need for continuous molecular surveillance and genetic monitoring of raw milk and dairy products in order to minimize epidemics with Salmonella and E.coli.