Influence of biofilm-forming lactic acid bacteria against methicillin-resistant Staphylococcus aureus (MRSA S547)

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.

Effects of carbon and nitrogen sources on bacteriocin-inhibitory activity of postbiotic metabolites produced by Lactobacillus plantarum I-UL4

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.

Nutrients and culture conditions requirements for the degradation of phenol by Rhodococcus UKMP-5M.

The capability of Rhodococcus UKMP-5M, isolated from petroleum contaminated soil, in the degradation of phenol was studied using shake flask culture. The effect of nutrients and cultivation conditions on growth of this bacterium and phenol degradation was investigated. Among the different types of medium tested (M1, M2, M3 and M4), M1 was found to be the preferred medium for growth of this bacterium and phenol degradation. The optimized cultivation conditions for growth of Rhodococcus UKMP-5M and phenol degradation were; 30 degrees C, initial pH 7.5 and buffer concentration ranged from 5 to 50 mM. Improvement of growth and phenol degradation was achieved in medium supplemented with 2 g l(-1) glucose. In addition, NaCl at a concentration of 0.1 g l(-1) was required to enhance growth and phenol degradation. The addition of 0.4 g l(-1) (NH4)2SO4 into the culture medium greatly enhanced phenol degradation. At optimal medium composition and cultivation condition, Rhodococcus UKMP-5M was able to utilize phenol at concentration up to 900 mg l(-1). Results of this study showed that Rhodococcus UKMP-5M has potential to be used in the degradation of phenol.

Isolation and identification of lactic acid bacteria from fermented red dragon fruit juices.

UNLABELLED: Red dragon fruit or red pitaya is rich in potassium, fiber, and antioxidants. Its nutritional properties and unique flesh color have made it an attractive raw material of various types of food products and beverages including fermented beverages or enzyme drinks. In this study, phenotypic and genotypic methods were used to confirm the identity of lactic acid bacteria (LAB) appeared in fermented red dragon fruit (Hylocereus polyrhizus) beverages. A total of 21 isolates of LAB were isolated and characterized. They belonged to the genus of Enterococcus based on their biochemical characteristics. The isolates can be clustered into two groups by using the randomly amplified polymorphic DNA method. Nucleotide sequencing and restriction fragment length polymorphism of the 16S rRNA region suggested that they were either Enterococcus faecalis or Enterococcus durans. PRACTICAL APPLICATION: Current research revealed the use of biochemical analyses and molecular approaches to identify the microbial population particularly lactic acid bacteria from fermented red dragon fruit juices.

Effect of signal peptides on the secretion of ß-cyclodextrin glucanotransferase in Lactococcus lactis NZ9000.

Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme which catalyzes the formation of cyclodextrin from starch. The production of CGTase using lactic acid bacterium is an attractive alternative and safer strategy to produce CGTase. In this study, we report the construction of genetically modified Lactococcus lactis strains harboring plasmids that secrete the Bacillus sp. G1 ß-CGTase, with the aid of the signal peptides (SPs) SPK1, USP45 and native SP (NSP). Three constructed vectors, pNZ:NSP:CGT, pNZ:USP:CGT and pNZ:SPK1:CGT, were developed in this study. Each vector harbored a different SP fused to the CGTase. The formation of halo zones on starch plates indicated the production and secretion of ß-CGTase by the recombinants. The expression of this enzyme is shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. A band size of ∼75 kDa corresponding to ß-CGTase is identified in the intracellular and the extracellular environments of the host after medium modification. The replacement of glucose by starch in the medium was shown to induce ß-CGTase production in L. lactis. Although ß-CGTase production is comparatively low in NZ:SPK1:CGT, the SP SPK1 was shown to have higher secretion efficiency compared to the other SPs used in this study.

Lactococcus lactis M4, a potential host for the expression of heterologous proteins.

BACKGROUND: Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins. RESULTS: Several bacterial strains were isolated from cow's milk and eight of those were identified as Lactococcus lactis by 16S rRNA sequence analysis. Antibiotic susceptibility tests that were carried out showed that 50% of the isolates had almost identical antibiotic resistance patterns compared to the control strains MG1363 and ATCC 11454. Plasmid profiling results indicated the lack of low molecular weight plasmids for strain M4. Competent L. lactis M4 and MG1363 were prepared and electrotransformed with several lactococcal plasmids such as pMG36e, pAR1411, pAJ01 and pMG36e-GFP. Plasmid isolation and RE analyses showed the presence of these plasmids in both M4 and the control strain after several generations, indicating the ability of M4 to maintain heterologous plasmids. SDS-PAGE and Western blot analyses also confirmed the presence of GFP, demonstrating the potential of heterologous protein expression in M4. CONCLUSIONS: Based on the 16S rRNA gene molecular analysis, eight Gram-positive cocci milk isolates were identified as L. lactis subsp. lactis. One of the strains, L. lactis M4 was able to maintain transformed low molecular weight plasmid vectors and expressed the GFP gene. This strain has the potential to be developed into a new lactococcal host for the expression of heterologous proteins.