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Abdullahi, Binta Sambo (2019) Enumeration, isolation, identification and probiotic characterisation of lactic acid bacteria from Nigerian human breast milk. Doctoral thesis, London Metropolitan University.
The aims of the research were to investigate the population and diversity of lactic acid bacteria (LAB) in human breast milk, to assess the probiotic potential of the identified LAB, to examine the antimicrobial resistance profile of the LAB and to investigate the possible relationship between the identified bacterial population and diversity with stage of lactation, number of children and diet. Samples of breast milk were collected from 20 breast feeding mothers. Standard spread plating on MRS agar and MRS agar supplemented with L-cysteine HCl (MRS-cys) was used for the enumeration of the organisms. Phenotypic identification of 108 recovered isolates was carried out. All isolates were Gram positive and oxidase negative, some were catalase positive and others were catalase negative. Further identification was carried out by grouping the isolates using repetitive sequence based PCR (rep-PCR). A total of nineteen groups were generated from the rep-PCR DNA profiles. This was followed by genotypic identification using 16S rRNA gene sequencing. The 16S rRNA gene sequencing identified LAB and non-LAB. Most of the LAB isolates belonged to the Lactobacillus genus, the rest were Leuconostoc, Weissella, Streptococcus and Enterococcus spp. The non-LAB were Staphylococcus epidermidis and Staphylococcus hominis. To examine the potential probiotic characteristics of the isolates, at least one representative isolate from each species of LAB (11 in total) were selected and characterised through a series of experiments. Initially the acid and bile resistance of the selected isolates were studied. All eleven LAB showed good tolerance to low pH and high concentration of bile salt. The antimicrobial activity of the isolates was also assessed. Again all eleven LAB produced antibacterial metabolites that inhibited the growth of all the four indicator bacteria (Bacillus cereus, Escherichia coli, Salmonella Enteritidis and Staphylococcus aureus) in both unbuffered and buffered agar spot tests. The antimicrobial activity of the LAB using Agar well diffusion assay revealed antimicrobial properties of some of the studied LAB. The ability of the eleven selected LAB to produce exopolysaccharide, deconjugate bile salt and reduce cholesterol was investigated. The entire eleven LAB produced exopolysaccharide, deconjugate bile salt and reduce cholesterol. The phenotypic antimicrobial resistance profile of the eleven LAB was assessed. Furthermore, the genetic background of the phenotypic resistance was also investigated. All eleven LAB were sensitive to ciprofloxacin, gatifloxacin and quinupristin/dalfopristin and resistant to daptomycin. Most of the LAB were resistant to erythromycin, gentamicin tetracycline and vancomycin. Antimicrobial resistance genes for erythromycin and tetracycline were confirmed for three LAB.
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