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    Profiling and antibiotic resistance of lactic acid bacteria isolated from commercial aMasi samples
    (2020-03) Pillay, Yovani; Ijabadeniyi, Oluwatosin Ademola; Swalaha, Feroz Mahomed
    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.
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    Antimicrobial activity of ciprofloxacin-coated gold nanoparticles on selected pathogens
    (2014-08-08) Moodley, Nivrithi; Odhav, Bharti
    Antibiotic resistance amongst bacterial pathogens is a crisis that has been worsening over recent decades, resulting in serious and often fatal infections that cannot be treated by conventional means. Diseases caused by these drug resistant agents result in protracted illnesses, greater mortality rates and increases in treatment costs. Improvements to existing therapies and the development of novel treatments are urgently required to deal with this escalating threat to human health. One of the more promising strategies to combat antibiotic resistance is the use of metallic nanoparticles. Research into this area has shown that the binding of antibiotics to nanoparticles enhances their antimicrobial effects, reduces side-effects due to requirement of lower dosages of the drug, concentrates the drug at the interaction site with bacterial cells and in certain cases, has re-introduced susceptibility into bacterial strains that have developed drug resistance. Furthermore, these nanoparticles can be used in cancer treatment in similar drug delivery roles. Based on the promising data that demonstrated the synergistic effects of antimicrobial agents with nanoparticles, the aim of our research is to determine the effect of ciprofloxacin-conjugated gold nanoparticles as antimicrobial agents. To achieve this aim our objectives were: (i) to synthesize citrate-capped and ciprofloxacin-conjugated gold nanoparticles; (ii) to determine the physical and chemical characteristics of the ciprofloxacin-nanoparticle hybrid molecule; (iii) to investigate the antimicrobial activity of the conjugated nanoparticles against various species of common pathogens and (iv) to investigate the anti-cancer potential of the citrate-capped nanoparticles against a Caco-2 cell line. In this study, citrate-capped gold nanoparticles were conjugated to the antibiotic, ciprofloxacin, and their antibacterial and anti-cancer activity was evaluated. Initial experiments involved the synthesis and characterization of gold nanoparticles and ciprofloxacin conjugated nanoparticles. The gold nanoparticles were synthesized using the Turkevich citrate reduction technique which has been extensively used in studies thus far. The synthesized nanoparticles were characterized for specific absorbance using a UV-Spectrophotometer. The bond between the nanoparticles and ciprofloxacin was characterized by FTIR. Ultra structural details of the gold nanoparticles were established by TEM. The colloidal stability of the nanoparticles was determined by spectroscopic analysis. The antibacterial activity of the ciprofloxacin-conjugated gold nanoparticles was studied by exposure to pathogenic bacteria (Staphyloccocus aureus, E. coli, Klebsiella pneumoniae, Enterocococcus spp., Enterobacter spp., and Psuedomonas spp.). MIC values were measured to give indication of antimicrobial effect. These bactericidal properties of the conjugate nanoparticles were further investigated by electron microscopy. To evaluate the action of the citrate capped gold nanoparticles on cancer cells, we exposed Caco-2 cells to various concentrations of the nanoparticles and its effect was evaluated by measuring the viability of the cells. The results showed that 0.5 mM trisodium citrate reduced gold chloride to yield gold nanoparticles, which were spherical and 15 to 30 nm (by TEM characterization) and had an absorption maxima of 530 nm. The ciprofloxacin conjugated nanoparticles had an absorption maxima of 667nm. The colloidal stability, which is used to assess whether the synthesized particles will retain their integrity in solution showed that citrate-capped GNPs were most stable at 37°C over a 14 day storage period while ciprofloxacin-conjugated GNPs were found to be most stable at 4°C over a 14 day period. The FTIR results showed that chemical bonding in the conjugated nanoparticles occurs between the pyridone moiety of ciprofloxacin and the nanoparticle surface. The antimicrobial results of ciprofloxacin-conjugated GNPs had a significantly improved killing response compared to ciprofloxacin on both Gram positive and Gram negative bacteria. The citrate-capped GNPs are shown to exert a similar cytotoxic effect to gemcitabine on the Caco-2 cell line at a concentration of 0.5 mM. These results indicate that combining gold nanoparticles and ciprofloxacin enhances the antimicrobial effect of the antibiotic. The conjugate nanoparticles increase the concentration of antibiotics at the site of bacterium-antibiotic interaction, and thus enhance the binding and entry of antibiotics into bacteria. This has great implications for treatment of infection, as these antibiotic-conjugated nanoparticles can be incorporated into wound dressings, be administered intravenously as drug delivery agents, be engineered to possess multiple functionalities in addition to antibacterial activity and act as dual infection tracking and antimicrobial agents. Likewise, in this study, gemcitabine, an anticancer drug and gold nanoparticles were shown to kill cancer cells. In addition to their use in photothermal therapy and as drug delivery agents, the nanoparticles themselves possess anti-cancer activity against the Caco-2 cells. Thus, they have potential to act alone as a form of cancer treatment if functionalized with certain targeting agents that are specific to cancer cells, reducing the side-effects that come with regular chemotherapeutic drugs. It can be concluded that ciprofloxacin-conjugated gold nanoparticles enhance antibacterial effects of the antibiotic ciprofloxacin against bacterial cells and citrate-capped gold nanoparticles have anti-cancer activity against the Caco-2 cell line.
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    Antimicrobial activity of plant phenols from Chlorophora excelsa and Virgilia oroboides
    (Academic Journals, 2013-04-24) Padayachee, Thiriloshani; Odhav, Bharti
    The anti-bacterial and anti-fungal activity of four aqueous plant extracts (1 x 104 µg/ml) of 2,3'4,5'-tetra hydroxy-4'-geranylstilbene (chlorophorin) and 3',4, 5' - trihydroxy - 4' - geranylstilbene (Iroko) from the tree Chlorophora excelsa and (6aR,11aR)-3-hydroxy-8,9-methylenedioxypterocarpan (Maackiain) and 7-hydroxy-4'-methoxyisoflavone (formononetin) from Virgilia oroboides were evaluated by the seeded agar overlay well diffusion method. The test organisms and bioautography used included: Bacillus coagulans, Streptococcus pneumoniae, Klebsiella pneumoniae, Escherichia coli, Mycobacteria tuberculosis, Aspergillus flavus and Fusarium verticilloides. Vancomycin, the drug of choice for these organisms was used as the control at 30 µg/ml. The extracts showed that chlorophorin at 1.95 µg/ml and Iroko at 3.125 and 6.25 µg/ml respectively were active in inhibiting the growth of S. pneumoniae and B. coagulans and not active against K. pneumoniae and E. coli. Maackiain; formononetin and formononetin acetate showed little activity against S. pneumonia, B. coagulans, K. pneumoniae and E. coli. None of the extracts showed activity against M. tuberculosis. Maackiain, formononetin, chlorophorin and Iroko inhibited F. vertiicilloides, maackiain being the most active compound. Formononetin, chlorophorin and Iroko inhibited A. flavus. A. flavus was most sensitive to chlorophorin and Iroko. The bioautography method confirmed these results and was attributed to the phenolic nature of the compounds.