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Aims@#Lactococcus lactis is a non-colonizing, generally-regarded as safe (GRAS) lactic acid bacteria that has been frequently studied as a potential vector for bactofection. To mediate bactofection, a series of interaction between the bacteria and the host cell needs to occur. This study aims to investigate the in vitro bacterial-cell interaction between a locally-isolated L. lactis M4 strain with human colorectal cancer line, Caco-2.@*Methodology and results@#Bacterial interaction was evaluated via adherence and internalisation assays. A 250:1 ratio of bacteria to cancer cell was selected as the optimum multiplicity of infection for all assays. After 2 h, L. lactis M4 was able to adhere to and internalise into Caco-2 cells at comparable rates to commercial strains L. lactis NZ9000 and MG1363. @*Conclusion, significance and impact of study@#Findings from this study showed that this strain has similar interaction properties with the commercial strains and would make a promising candidate for future bactofection studies and development of bacteria-mediated DNA vaccination against various diseases.
Subject(s)
Lactococcus lactis , Colorectal Neoplasms , Caco-2 CellsABSTRACT
@#Introduction: Multidrug resistance bacteria is alarming worldwide. A lot of research were done and are ongoing to search for the best, convenient and economically affordable ways to fight them. With the latest genome editing tool; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, this research was performed to develop a novel strategy to genetically modify the genome and inhibit the growth of Klebsiella pneumoniae (UPM ESBLKP1), an Extended Spectrum Beta Lactamases (ESBL) organism. Methods: A CRISPR-Cas9 vector was constructed together with guide RNAs designed specifically for the targeted uppP gene, a gene responsible for bacterial cell growth and protection. Results: The growth and cell wall integrity of the modified Klebsiella pneumoniae (ΔUPM ESBLKP1) were significantly inhibited and reduced, respectively. Interestingly, wild type Klebsiella pneumoniae showed a normal growth curve while modified strains showed a faster doubling rate when supplemented with Luria-Bertani media. In contrast, slower growth rate of modified strain was observed in the M9 minimal media. This explained the higher doubling rate of mutants on nutrient rich medium earlier is being related to gene recovery. They grew slowly in the minimal media as they were adapting to a new environment while recovering the uppP gene and surviving, proving the success of its gene modification. Conclusion: The developed CRISPR-gRNA system was able to modify the targeted Klebsiella pneumoniae gene hence providing an opportunity to develop a new drug for Klebsiella pneumoniae infection as an alternative to antibiotics.
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Objective To investigate the antibacterial effect of selected lactic acid bacteria (LAB) biofilms on the planktonic and biofilm population of methicillin-resistant Staphylococcus aureus (MRSA) (S547). Methods In this study, biofilm-forming LAB were isolated from tairu and kefir. Isolate Y1 and isolate KF were selected based on their prominent inhibition against test pathogens (using spot-on-agar method and agar-well-diffusion assay) and efficient biofilm production (using tissue culture plate method). They were then identified as Lactobacillus casei (L. casei) Y1 and Lactobacillus plantarum (L. plantarum) KF, respectively using 16S rDNA gene sequencing. The influence of incubation time, temperature and aeration on the biofilm production of L. casei Y1 and L. plantarum KF was also investigated using tissue culture plate method. The inhibitory activity of both the selected LAB biofilms was evaluated against MRSA (Institute for Medical Research code: S547) using L. plantarum ATCC 8014 as the reference strain. Results L. casei Y1 showed the highest reduction of MRSA biofilms, by 3.53 log at 48 h while L. plantarum KF records the highest reduction of 2.64 log at 36 h. In inhibiting planktonic population of MRSA (S547), both L. casei Y1 and L. plantarum KF biofilms recorded their maximum reduction of 4.13 log and 3.41 log at 24 h, respectively. Despite their inhibitory effects being time-dependent, both LAB biofilms exhibited good potential in controlling the biofilm and planktonic population of MRSA (S547). Conclusions The results from this study could highlight the importance of analysing biofilms of LAB to enhance their antibacterial efficacy. Preferably, these protective biofilms of LAB could also be a better alternative to control the formation of biofilms by pathogens such as MRSA.
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Aims: Lactobacillus sp. has capability of producing an array of bioactive metabolites that exhibit probiotic effects. Therefore, the objective of this study was to determine the cytotoxicity effect of proteinaceous postbiotic metabolites (PPM) produced by Lactobacillus plantarum I-UL4 cultivated in different media composition on MCF-7 breast cancer cell. Methodology and results: L. plantarum I-UL4 was cultivated in yeast extract and modified de Man, Rogosa and Sharpe broth containing Tween 80 (CRMRS+T80) or without Tween-80 (CRMRS-T80). Human breast adenocarcinoma cell (MCF-7) was employed as cancer cell in this study. Cytotoxicity and antiproliferative effects of PPM were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide assay and Trypan Blue Dye Exclusion assay, whereas Acridine Orange/Propidium Iodide staining was employed to determine the cytotoxicity mechanism. PPM produced in CRMRS+T80 exerted cytotoxicity in a time and dose dependent manner that was selective towards MCF-7 cancer cell. Profound cytotoxicity with the lowest IC50 concentration of 10.83 µg was detected at 72 h of incubation, whereas the most potent antiproliferative effect revealed by the lowest viable cell population was observed at 24 h of incubation. PPM cultivated in CRMRS+T80 induced 80.87% of apoptotic MCF-7 cells at 48 h of incubation. Conclusion, significance and impact of study: PPM of L. plantarum I-UL4 cultivated in different media composition induced different levels of MCF-7 cancer cell death. The percentage of apoptotic MCF-7 cells treated with PPM cultivated in CRMRS+T80 increased significantly (p < 0.05) from 24 to 48 h of incubation. The results obtained in this study have revealed the potential of PPM produced by L. plantarum I-UL4 as human health supplement and as anticancer preventive agent. Keywords: Lactobacillus plantarum I-UL4; cytotoxic effect; proteinaceous postbiotic metabolites; media composition; breast cancer
Subject(s)
Lactobacillus , ProbioticsABSTRACT
Aims: Postbiotic metabolites are metabolic compounds produced by probiotic lactic acid bacteria. These compounds produced by Lactobacillus sp. have been shown to be effective substitutes for in-feed antibiotic in livestock due to their broad inhibitory activity. Therefore, the aim of this study was to determine the effects of various carbon and nitrogen sources on the bacteriocin-inhibitory activity of postbiotic metabolites produced by Lactobacillus plantarum I-UL4. Methodology and results: The effects of various combinations of carbon and nitrogen sources on the bacteriocininhibitory activity (expressed as modified bacteriocin activity, MAU/mL) of postbiotic metabolites produced by L. plantarum I-UL4 were determined in basal media without micronutrients. The combination of glucose (20 g/L) and yeast extract (22 g/L) gave the best bacteriocin-inhibitory activity as compared to other combinations. Maximum bacteriocininhibitory activity of 1440 MAU/mL was achieved when 36.20 g/L of yeast extract was added as the sole nitrogen source in modified de Man, Rogosa and Sharpe (MRS) medium. The glucose concentration was further optimised to enhance the bacteriocin-inhibitory activity of the postbiotic metabolites. Lower bacteriocin-inhibitory activity was observed at 5, 10, 15 and 40 g/L in comparison to 20 g/L of glucose. Conclusion, significance and impact of study: Maximum bacteriocin-inhibitory activity of postbiotic metabolites was achieved at 1440 MAU/mL when 20 g/L of glucose and 36.20 g/L of yeast extract were added as the sole carbon and nitrogen sources respectively in the modified MRS medium. Optimisation of other micronutrients present in MRS media is necessary to further enhance the bacteriocin-inhibitory activity of postbiotic metabolites produced by L. plantarum IUL4.