aMasi is traditionally fermented milk that constitutes part of the South African heritage and is regarded as a supplementary staple food. Its inclusion into the South African Food Based Dietary Guidelines has led to the encouraged consumption of this product. Given the fact that aMasi is a rich source of lactic acid bacteria (LAB), such bacteria are of economic importance to the food, feed and pharmaceutical industries. The main concern regarding food safety is ability to acquire and disseminate antibiotic-resistant genes. Although LAB bility of resistance genes to human and animal opportunistic and pathogenic bacteria which could make treatment of bacterial infections more complex to treat in the future. Numerous reports globally, have documented antibiotic resistance among LAB isolated from commercial dairy and pharmaceutical products over the last decade. Therefore, the aim of this study was to determine if LAB isolated from commercial aMasi samples harbour antibiotic-resistant genes. To achieve this aim, the total bacterial population and LAB population of 10 aMasi samples were surveyed using culture-dependent techniques and the proportional prevalence of LAB to the total bacterial population were determined by using a 100% stacked-column. In all 10 samples, LAB was the predominating population ranging from 87.44% to 99.77%. A total of 30 LAB isolates were characterised after isolation and sequencing of 16S rDNA of these isolates showed that LAB were Leuconostoc pseudomesenteroides and Leuconostoc mesenteroides with two isolates being identified as Lactococcus lactis CP028160.1. The relationship between the growth of LAB and selected physicochemical properties (pH, titratable acidity, water activity (aw), moisture content, fat content and estimation of reducing sugars (lactose)) were determined using principal component analysis (PCA) and classification and regression tree (CART) to illustrate the likelihood of LAB present in aMasi samples based on LAB count and pH. From the PCA results, approximately 75.25% of variances in the data were retained by the first three principal components (PCs). The first principal component (PC1) had accounted for the highest total variance of 33.16%. PC1 increased with an increase in lactic acid % and aw, whilst it negatively correlated with LAB count, moisture % and lactose (mg/25ml lactose·H2O). The results showed an increase in LAB count with an increase in moisture % and lactose (mg/25ml lactose·H2O) whilst, LAB count had decreased with an increase in lactic acid % and aw. Moreover, pH and fat % had no effect on PC1, high LAB counts were observed for samples 6 and 7 whist low LAB counts were observed for samples 9 and 10. On the other hand, PC2 had accounted for approximately 27.53% of the total variance. PC2 increased with an increase in fat % and lactose (mg/25ml lactose·H2O), whilst it negatively correlated with LAB count and pH. It was observed that the growth of LAB had increased with an increase in pH, whilst it decreased with an increase in fat % and lactose (mg/25ml lactose·H2O). Moreover, lactic acid %, aw and moisture % had no effect on PC2. High LAB counts were observed for samples 7 and 8 and low LAB counts were observed for samples 2 and 4. Nine out of the 30 LAB isolates were selected due to these isolates having a different GenBank Accession number and were subjected to antibiotic susceptibility testing using the disc diffusion method against a total of 11 antibiotics. Most of the LAB isolates exhibited multiple resistance towards some of the most commonly used antibiotics as well as last-resort antibiotics. All the isolates showed high levels of resistance towards vancomycin, colistin sulphate, fosfomycin and pipemidic acid except for Lactococcus lactis CP028160.1 which was susceptible to vancomycin. All isolates were susceptible to tetracycline and erythromycin whilst eight out of nine isolates were susceptible to chloramphenicol with seven out of nine isolates being susceptible to ampicillin. Furthermore, the isolates had displayed intermediate resistance mainly towards kanamycin and streptomycin. The present study showed that multiple antibiotic resistance is prevalent in different species of starter culture strains, which may pose a food safety concern. LAB that exhibit phenotypic resistance to antibiotics should also be evaluated on a molecular level to monitor their resistance. The presence of such a variety of expressed AR genes in probiotic isolates is a worrying trend. The impact of the interactions of these bacteria with pathogenic strains and their transfer of these AR genes is yet to be assessed. Furthermore, antibiotic sensitivity is an important criterion in the safety assessment for the evaluation of food-grade and potential food-grade LAB.
Use of the alr Gene as a Food-Grade Selection Marker in Lactic Acid Bacteria