Genome mining of Lactiplantibacillus plantarum PA21: insights into its antimicrobial potential.

BACKGROUND: The dramatic increase of antimicrobial resistance in the healthcare realm has become inexorably linked to the abuse of antibiotics over the years. Therefore, this study seeks to identify potential postbiotic metabolites derived from lactic acid bacteria such as Lactiplantibacillus plantarum that could exhibit antimicrobial properties against multi-drug resistant pathogens. RESULTS: In the present work, the genome sequence of Lactiplantibacillus plantarum PA21 consisting of three contigs was assembled to a size of 3,218,706 bp. Phylogenomic analysis and average nucleotide identity (ANI) revealed L. plantarum PA21 is closely related to genomes isolated from diverse niches such as dairy products, food, and animals. Genome mining through the BAGEL4 and antiSMASH database revealed four bacteriocins in a single cluster and four regions of biosynthetic gene clusters responsible for the production of bioactive compounds. The potential probiotic genes indirectly responsible for postbiotic metabolites production were also identified. Additionally, in vitro studies showed that the L. plantarum PA21 cell-free supernatant exhibited antimicrobial activity against all nine methicillin-resistant Staphylococcus aureus (MRSA) and three out of 13 Klebsiella pneumoniae clinical isolates tested. CONCLUSION: Results in this study demonstrates that L. plantarum PA21 postbiotic metabolites is a prolific source of antimicrobials against multi-drug resistant pathogens with potential antimicrobial properties.

Recombinant lactococcal-based oral vaccine for protection against Streptococcus agalactiae infections in tilapia (Oreochromis niloticus).

Streptococcosis outbreaks caused by Streptococcus agalactiae infection in tilapia aquaculture have been consistently reported and associated with high mortality and morbidity leading to significant economic losses. Existing vaccine candidates against Streptococcus spp. are designed for intraperitoneal injections that are not practical and labor-intensive which have prompted farmers to protect aquatic animals with antibiotics, thus encouraging the emergence of multidrug resistant bacteria. In this study, a live recombinant L. lactis vaccine expressing a 1403 bp surface immunogenic protein (SIP) and a 1100 bp truncated SIP (tSIP) gene was developed and evaluated against S. agalactiae infection in tilapia. Both SIP and tSIP sequences were cloned and transformed into L. lactis. The recombinant L.lactis vaccine was orally administered to juvenile tilapia for a month. Detection of SIP-specific serum IgM in vaccinated groups compared to control groups indicated that recombinant proteins expressed from L. lactis could elicit immunogenic reactions in tilapia. Fish immunized with the tSIP vaccine also showed the highest level of protection compared to other test groups, and the mortality rate was significantly reduced compared to both control groups. The relative percentage of survival (RPS) against S. agalactiae for both SIP and tSIP-vaccinated groups was 50 % and 89 %, respectively, at 14 days post-challenge. Significant up-regulation of IgM, IL-1ß, IL-10, TNF-α and IFN-γ were observed at day 34 between the vaccinated and control groups. These results indicated that the recombinant lactococcal tSIP vaccine can elicit both cell-mediated and humoral responses and is recommended as a potential oral vaccine against S. agalactiae infection. Future work will include further in vivo challenge assessments of this vaccine candidate fused with adjuvants to boost immunogenicity levels in tilapia.

Development of optimized functional clove fortified probiotic yoghurt.

We focused on assessing the antimicrobial effects of functional yoghurts supplemented with clove and probiotics. The formulation of aqueous clove extract (ACE) incorporated with probiotic yoghurt (Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactococcus lactis) was optimised in terms of aqueous clove extract concentrations (2.5-7.5% v/v), fermentation temperature (32-42 °C), and total culture concentration (1.5-4.5% v/v). pH, titratable acidity, syneresis, water holding capacity, viscosity, springiness, color difference, lactic acid bacteria viability, and the antibacterial property of 17 runs were determined as responses using Box-Behnken design. The results indicate that elevated ACE concentration leads to a significant (p < 0.05) increase in titratable acidity, antibacterial effectiveness (against K. pneumoniae and P. aeruginosa), springiness, and color profile. Conversely, an elevated fermentation temperature significantly (p < 0.05) reduces pH and L. bulgaricus viability (log CFU/mL). Additionally, there is a significant (p < 0.05) decline in S. thermophilus and L. lactis viability (log CFU/mL) as well as springiness with an increased culture concentration. The optimal conditions identified are 7.5% (v/v) ACE concentration, a fermentation temperature of 36.6 °C (37 °C), and a total culture concentration of 4.5% (v/v), resulting in a 79% desirability score. The spectra of components were mainly obtained at wavelength of 3258 cm-1, 1636 cm-1 and 1075 cm-1 in Fourier transform infrared (FTIR) spectroscopy of optimized functional yoghurt. Where, 3258 cm-1 corresponds to the stretching vibration of O-H (hydroxyl) groups, 1636 cm-1 corresponds to the C=O (carbonyl) stretching vibration, and 1075 cm-1 corresponds to the C-O (ether or alcohol) stretching vibration.

Effect of Secretion Efficiency of Mutant KRAS Neoantigen by Lactococcus lactis on the Immune Response of a Mucosal Vaccine Delivery Vehicle Targeting Colorectal Cancer.

Colorectal cancer (CRC) is often caused by mutations in the KRAS oncogene, making KRAS neoantigens a promising vaccine candidate for immunotherapy. Secreting KRAS antigens using live Generally Recognized as Safe (GRAS) vaccine delivery hosts such as Lactococcus lactis is deemed to be an effective strategy in inducing specific desired responses. Recently, through the engineering of a novel signal peptide SPK1 from Pediococcus pentosaceus, an optimized secretion system was developed in the L. lactis NZ9000 host. In this study, the potential of the L. lactis NZ9000 as a vaccine delivery host for the production of two KRAS oncopeptides (mutant 68V-DT and wild-type KRAS) through the use of the signal peptide SPK1 and its mutated derivative (SPKM19) was investigated. The expression and secretion efficiency analyses of KRAS peptides from L. lactis were performed in vitro and in vivo in BALB/c mice. Contradictory to our previous study using the reporter staphylococcal nuclease (NUC), the yield of secreted KRAS antigens mediated by the target mutant signal peptide SPKM19 was significantly lower (by ~1.3-folds) compared to the wild-type SPK1. Consistently, a superior elevation of IgA response against KRAS aided by SPK1 rather than mutant SPKM19 was observed. Despite the lower specific IgA response for SPKM19, a positive IgA immune response from mice intestinal washes was successfully triggered following immunization. Size and secondary conformation of the mature proteins are suggested to be the contributing factors for these discrepancies. This study proves the potential of L. lactis NZ9000 as a host for oral vaccine delivery due to its ability to evoke the desired mucosal immune response in the gastrointestinal tract of mice.

Optimization of Signal Peptide via Site-Directed Mutagenesis for Enhanced Secretion of Heterologous Proteins in Lactococcus lactis.

Secretion efficiency of heterologous proteins in the Generally Regarded As Safe (GRAS) Lactococcus lactis is often reported to be insufficiently low due to limitations such as poor targeting and translocation by the signal peptide or degradation by the host proteases. In this study, the secretion efficiency in the host was enhanced through the utilization of a heterologous signal peptide (SP) SPK1 of Pediococcus pentosaceus. The SPK1 was subjected to site-directed mutations targeting its tripartite N-, H-, and C-domains, and the effect on secretion efficiency as compared to the wild-type SPK1 and native lactococcal USP45 was determined on a reporter nuclease (NUC) of Staphylococcus aureus. A Fluorescence Resonance Energy Transfer (FRET) analysis indicated that four out of eight SPK1 variants successfully enhanced the secretion of NUC, with the best mutant, SPKM19, showing elevated secretion efficiency up to 88% (or by 1.4-fold) and an improved secretion activity yield of 0.292 ± 0.122 U/mL (or by 1.7-fold) compared to the wild-type SPK1. Modifications of the SPK1 at the cleavage site C-domain region had successfully augmented the secretion efficiency. Meanwhile, mutations in the H-domain region had resulted in a detrimental effect on the NUC secretion. The development of heterologous SPs with better efficacy than the USP45 has been demonstrated in this study for enhanced secretion of heterologous production and mucosal delivery applications in the lactococcal host.

Anti-cancer and anti-inflammatory effects elicited by short chain fatty acids produced by Escherichia coli isolated from healthy human gut microbiota.

BACKGROUND: Extracellular metabolites of short chain fatty acids (SCFA) excreted by gut microbiota have been reported to play an important role in the regulation of intestinal homeostasis. Apart from supplying energy, SCFA also elicit immune stimulation in animal and human cells. Therefore, an attempt was conducted to isolate SCFA producing bacteria from healthy human microbiota. The anti-cancer and anti-inflammatory effects of extracellular metabolites and individual SFCA were further investigated by using breast, colon cancer and macrophage cells. Toxin, inflammatory and anti-inflammatory cytokine gene expressions were investigated by RT-qPCR analyses in this study. RESULTS: Escherichia coli KUB-36 was selected in this study since it has the capability to produce seven SCFA extracellularly. It produced acetic acid as the main SCFA. It is a non-exotoxin producer and hence, it is a safe gut microbiota. The IC50 values indicated that the E. coli KUB-36 metabolites treatment elicited more potent cytotoxicity effect on MCF7 breast cancer cell as compared to colon cancer and leukemia cancer cells but exhibited little cytotoxic effects on normal breast cell. Furthermore, E. coli KUB-36 metabolites and individual SCFA could affect inflammatory responses in lipopolysaccharide-induced THP-1 macrophage cells since they suppressed inflammatory cytokines IL-1ß, IL-6, IL-8 and TNF-α well as compared to the control, whilst inducing anti-inflammatory cytokine IL-10 expression. CONCLUSION: SCFA producing E. coli KUB-36 possessed vast potential as a beneficial gut microbe since it is a non-exotoxin producer that exhibited beneficial cytotoxic effects on cancer cells and elicited anti-inflammatory activity simultaneously. However, the probiotic characteristic of E. coli KUB-36 should be further elucidated using in vivo animal models.

Secretion of recombinant xylanase in Lactococcus lactis using signal peptides Usp45 and Spk1.

OBJECTIVE: The effect of two signal peptides, namely Usp45 and Spk1 on the secretion of xylanase in Lactococcus lactis was analysed. RESULTS: Xylanase was successfully expressed in Lactococcus lactis. Recombinant xylanase fused to either signal peptide Usp45 or Spk1 showed halo zone on Remazol Brilliant Blue-Xylan plates. This indicated that the xylanase was successfully secreted from the cell. The culture supernatants of strains secreting the xylanase with help of the Spk1 and Usp45 signal peptides contained 49.7 U/ml and 34.4 U/ml of xylanase activity, respectively. CONCLUSION: Although Usp45 is the most commonly used signal peptide when secreting heterologous proteins in Lactococcus lactis, this study shows that Spk1 isolated from Pediococcus pentosaceus was superior to Usp45 in regard to xylanase protein secretion.

Engineering integrative vectors based on phage site-specific recombination mechanism for Lactococcus lactis.

BACKGROUND: Site-specific integration system allows foreign DNA to be integrated into the specific site of the host genome, enabling stable expression of heterologous protein. In this study, integrative vectors for secretion and surface display of proteins were constructed based on a lactococcal phage TP901-1 integrating system. RESULTS: The constructed integration system comprises of a lactococcal promoter (PnisA or P170), phage attachment site (attP) from bacteriophage TP901-1, a signal peptide (USP45 or SPK1) for translocation of the target protein, and a PrtP344 anchor domain in the case of the integrative vectors for surface display. There were eight successfully constructed integrative vectors with each having a different combination of promoter and signal peptide; pS1, pS2, pS3 and pS4 for secretion, and pSD1, pSD2, pSD3 and pSD4 for surface display of desired protein. The integration of the vectors into the host genome was assisted by a helper vector harbouring the integrase gene. A nuclease gene was used as a reporter and was successfully integrated into the L. lactis genome and Nuc was secreted or displayed as expected. The signal peptide SPK1 was observed to be superior to USP45-LEISSTCDA fusion in the secretion of Nuc. As for the surface display integrative vector, all systems developed were comparable with the exception of the combination of P170 promoter with USP45 signal peptide which gave very low signals in whole cell ELISA. CONCLUSION: The engineered synthetic integrative vectors have the potential to be used for secretion or surface display of heterologous protein production in lactococcal expression system for research or industrial purposes, especially in live vaccine delivery.

Recovery of recombinant Mycobacterium tuberculosis antigens fused with cell wall-anchoring motif (LysM) from inclusion bodies using non-denaturing reagent (N-laurylsarcosine).

BACKGROUND: The current limitations of conventional BCG vaccines highlights the importance in developing novel and effective vaccines against tuberculosis (TB). The utilization of probiotics such as Lactobacillus plantarum for the delivery of TB antigens through in-trans surface display provides an effective and safe vaccine approach against TB. Such non-recombinant probiotic surface display strategy involves the fusion of candidate proteins with cell wall binding domain such as LysM, which enables the fusion protein to anchor the L. plantarum cell wall externally, without the need for vector genetic modification. This approach requires sufficient production of these recombinant fusion proteins in cell factory such as Escherichia coli which has been shown to be effective in heterologous protein production for decades. However, overexpression in E. coli expression system resulted in limited amount of soluble heterologous TB-LysM fusion protein, since most of it are accumulated as insoluble aggregates in inclusion bodies (IBs). Conventional methods of denaturation and renaturation for solubilizing IBs are costly, time-consuming and tedious. Thus, in this study, an alternative method for TB antigen-LysM protein solubilization from IBs based on the use of non-denaturating reagent N-lauroylsarcosine (NLS) was investigated. RESULTS: Expression of TB antigen-LysM fusion genes was conducted in Escherichia coli, but this resulted in IBs deposition in contrast to the expression of TB antigens only. This suggested that LysM fusion significantly altered solubility of the TB antigens produced in E. coli. The non-denaturing NLS technique was used and optimized to successfully solubilize and purify ~ 55% of the recombinant cell wall-anchoring TB antigen from the IBs. Functionality of the recovered protein was analyzed via immunofluorescence microscopy and whole cell ELISA which showed successful and stable cell wall binding to L. plantarum (up to 5 days). CONCLUSION: The presented NLS purification strategy enables an efficient and rapid method for obtaining higher yields of soluble cell wall-anchoring Mycobacterium tuberculosis antigens-LysM fusion proteins from IBs in E. coli.

Terpene Derivatives as a Potential Agent against Antimicrobial Resistance (AMR) Pathogens.

The evolution of antimicrobial resistance (AMR) in pathogens has prompted extensive research to find alternative therapeutics. Plants rich with natural secondary metabolites are one of the go-to reservoirs for discovery of potential resources to alleviate this problem. Terpenes and their derivatives comprising of hydrocarbons, are usually found in essential oils (EOs). They have been reported to have potent antimicrobial activity, exhibiting bacteriostatic and bactericidal effects against tested pathogens. This brief review discusses the activity of terpenes and derivatives against pathogenic bacteria, describing the potential of the activity against AMR followed by the possible mechanism exerted by each terpene class. Finally, ongoing research and possible improvisation to the usage of terpenes and terpenoids in therapeutic practice against AMR are discussed.

Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice.

BACKGROUND: Tuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world's population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0-80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed. RESULTS: A novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice's spleen, lung and GIT. CONCLUSIONS: This study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.

A review on Lactococcus lactis: from food to factory.

Lactococcus lactis has progressed a long way since its discovery and initial use in dairy product fermentation, to its present biotechnological applications in genetic engineering for the production of various recombinant proteins and metabolites that transcends the heterologous species barrier. Key desirable features of this gram-positive lactic acid non-colonizing gut bacteria include its generally recognized as safe (GRAS) status, probiotic properties, the absence of inclusion bodies and endotoxins, surface display and extracellular secretion technology, and a diverse selection of cloning and inducible expression vectors. This have made L. lactis a desirable and promising host on par with other well established model bacterial or yeast systems such as Escherichia coli, Saccharomyces [corrected] cerevisiae and Bacillus subtilis. In this article, we review recent technological advancements, challenges, future prospects and current diversified examples on the use of L. lactis as a microbial cell factory. Additionally, we will also highlight latest medical-based applications involving whole-cell L. lactis as a live delivery vector for the administration of therapeutics against both communicable and non-communicable diseases.

Oral vaccine of Lactococcus lactis harbouring pandemic H1N1 2009 haemagglutinin1 and nisP anchor fusion protein elevates anti-HA1 sIgA levels in mice.

OBJECTIVE: An oral lactococcal-based vaccine which haboured the haemagglutinin1 (HA1) antigen fused to nisP anchor protein for the purpose of surface displaying the HA1 antigen was developed against H1N1 virus. RESULTS: Recombinant L. lactis strains expressed HA1-nisP fusion proteins when induced with nisin, as confirmed through western blotting. However, immunofluorescense did not detect any surface-displayed proteins, suggesting that the protein was either unsuccessfully translocated or improperly displayed. Despite this, oral administration of recombinant L. lactis strains to BALB/c mice revealed that significant levels of anti-HA1 sIgA antibodies were detected in mice fecal suspension samples of mice group NZ9000 (pNZ:HN) when compared to the negative control NZ9000 (pNZ8048) group. CONCLUSION: Specific anti-HA1 sIgA antibodies were locally produced and live recombinant lactococcal vaccine was able to elicit humoral response of BALB/c mice despite unsuccessful surface display of the HA1 epitope.

Surface display of glycosylated Tyrosinase related protein-2 (TRP-2) tumour antigen on Lactococcus lactis.

BACKGROUND: The exploitation of the surface display system of food and commensal lactic acid bacteria (LAB) for bacterial, viral, or protozoan antigen delivery has received strong interest recently. The Generally Regarded as Safe (GRAS) status of the Lactococcus lactis coupled with a non-recombinant strategy of in-trans surface display, provide a safe platform for therapeutic drug and vaccine development. However, production of therapeutic proteins fused with cell-wall anchoring motifs is predominantly limited to prokaryotic expression systems. This presents a major disadvantage in the surface display system particularly when glycosylation has been recently identified to significantly enhance epitope presentation. In this study, the glycosylated murine Tyrosinase related protein-2 (TRP-2) with the ability to anchor onto the L. lactis cell wall was produced in suspension adapted Chinese Hamster Ovary (CHO-S) cells by expressing TRP-2 fused with cell wall anchoring LysM motif (cA) at the C-terminus. RESULTS: A total amount of 33 µg of partially purified TRP-2-cA from ~6.0 g in wet weight of CHO-S cells was purified by His-tag affinity chromatography. The purified TRP-2-cA protein was shown to be N-glycosylated and successfully anchored to the L. lactis cell wall. CONCLUSIONS: Thus cell surface presentation of glycosylated mammalian antigens may now permit development of novel and inexpensive vaccine platforms.

Inhibition of pathogenic and spoilage bacteria by a novel biofilm-forming Lactobacillus isolate: a potential host for the expression of heterologous proteins.

BACKGROUND: Bacterial biofilms are a preferred mode of growth for many types of microorganisms in their natural environments. The ability of pathogens to integrate within a biofilm is pivotal to their survival. The possibility of biofilm formation in Lactobacillus communities is also important in various industrial and medical settings. Lactobacilli can eliminate the colonization of different pathogenic microorganisms. Alternatively, new opportunities are now arising with the rapidly expanding potential of lactic acid bacteria biofilms as bio-control agents against food-borne pathogens. RESULTS: A new isolate Lactobacillus plantarum PA21 could form a strong biofilm in pure culture and in combination with several pathogenic and food-spoilage bacteria such as Salmonella enterica, Bacillus cereus, Pseudomonas fluorescens, and Aeromonas hydrophila. Exposure to Lb. plantarum PA21 significantly reduced the number of P. fluorescens, A. hydrophila and B. cereus cells in the biofilm over 2-, 4- and 6-day time periods. However, despite the reduction in S. enterica cells, this pathogen showed greater resistance in the presence of PA21 developed biofilm, either in the planktonic or biofilm phase. Lb. plantarum PA21 was also found to be able to constitutively express GFP when transformed with the expression vector pMG36e which harbors the gfp gene as a reporter demonstrating that the newly isolated strain can be used as host for genetic engineering. CONCLUSION: In this study, we evaluate the ability of a new Lactobacillus isolate to form strong biofilm, which would provide the inhibitory effect against several spoilage and pathogenic bacteria. This new isolate has the potential to serve as a safe and effective cell factory for recombinant proteins.

IRES-incorporated lactococcal bicistronic vector for target gene expression in a eukaryotic system.

Plasmid DNAs isolated from lactic acid bacteria (LAB) such as Lactococcus lactis (L. lactis) has been gaining more interests for its positive prospects in genetic engineering-related applications. In this study, the lactococcal plasmid, pNZ8048 was modified so as to be able to express multiple genes in the eukaryotic system. Therefore, a cassette containing an internal ribosome entry site (IRES) was cloned between VP2 gene of a very virulent infectious bursal disease (vvIBDV) UPM 04190 of Malaysian local isolates and the reporter gene, green fluorescent protein (GFP) into pNZ:CA, a newly constructed derivative of pNZ8048 harboring the cytomegalovirus promoter (Pcmv) and polyadenylation signal. The new bicistronic vector, denoted as pNZ:vig was subjected to in vitro transcription/translation system followed by SDS-PAGE and Western blot analysis to rapidly verify its functionality. Immunoblotting profiles showed the presence of 49 and 29kDa bands that corresponds to the sizes of the VP2 and GFP proteins respectively. This preliminary result shows that the newly constructed lactococcal bicistronic vector can co-express multiple genes in a eukaryotic system via the IRES element thus suggesting its feasibility to be used for transfection of in vitro cell cultures and vaccine delivery.

Construction of a novel inducible expression vector for Lactococcus lactis M4 and Lactobacillus plantarum Pa21.

A vector that drives the expression of the reporter gusA gene in both Lactobacillus plantarum and Lactococcus lactis was constructed in this study. This vector contained a newly characterized heat shock promoter (Phsp), amplified from an Enterococcus faecium plasmid, pAR6. Functionality and characterization of this promoter was initially performed by cloning Phsp into pNZ8008, a commercial lactococcal plasmid used for screening of putative promoters which utilizes gusA as a reporter. It was observed that Phsp was induced under heat, salinity and alkaline stresses or a combination of all three stresses. The newly characterized Phsp promoter was then used to construct a novel Lactobacillus vector, pAR1801 and its ability to express the gusA under stress-induced conditions was reproducible in both Lb. plantarum Pa21 and L. lactis M4 hosts.

Effect of Domain Manipulation in the Staphylococcal Phage Endolysin, Endo88, on Lytic Efficiency and Host Range.

The bacteriophage endolysin Endo88 targeting Staphylococcus aureus PS88 consists of the CHAP and Amidase-2 enzymatic domains and one SH3b targeting domain. In this study, the effects of domain manipulations on Endo88 functionality were determined. Three truncated mutants of Endo88 (CHAP, CHAPAmidase and CHAPSH3) and two chimeras (CHAPAmidase-Cpl7Cpl7 and Endo88-Cpl7Cpl7) containing the Cpl7Cpl7 targeting domains of the streptococcal LambdaSa2-ECC endolysin were cloned in E. coli (pET28a), expressed, and then purified. Lytic efficiency and host range were assessed through plate lysis assays and turbidity reduction assays. Endo88 required all domains for maximum functionality, with activity detected against Staphylococcus aureus PS88 (host strain), S. aureus Mu50 (VISA), CoNS (Staphylococcus epidermidis and Staphylococcus hominis), and Enterococcus faecalis. The truncated constructs maintained the original host range but with reduced lytic efficiency. The Amidase-2 and SH3b domains are interdependent in maximizing functionality. The chimera constructs demonstrated reduced functionality, without activity against Streptococcus agalactiae in both assays. This study provides insights into domain function in a staphylococcal endolysin, which could enable the development of prospective engineered antimicrobials against multidrug-resistant pathogens.

Effects of Individual and Combined Fermentation Factors on Antimicrobial Activity of Nisin by Lactococcus lactis ATCC 11454.

There is an increasing demand for natural food preservatives due to consumers' concern on the negative effects of chemical preservatives in food products. Nisin (bacteriocin) is an effective food biopreservative that has been approved globally. However, its low yield proves to be a limiting factor and must be addressed to meet the increasingly high demand from the food industry. The present work thus investigated the effects of individual and combined fermentation factors on Lactococcus lactis ATCC 11454 growth and nisin activity using the one-factor-at-a-time (OFAT) method. The level of each factor that gave the highest nisin production was then selected and combined to further improve its activity. The best combined conditions for highest cell growth and nisin activity were 30 °C, pH 6.0, and mild agitation with the addition of 1.0% w/v glucose, 1.0% w/v skim milk, and 0.5% v/v Tween 20. This increased nisin production by 22.7% as compared to control (basic condition). The present work provided critical information on the relationship between fermentation conditions, growth, and nisin activity of L. lactis ATCC 11454 that could be explored to understand the potential and limitation of the strain. This fermentation strategy can also serve as a benchmark to further enhance the production of bacteriocin or other biopreservative compounds.

Comprehensive Characterization of a Streptococcus agalactiae Phage Isolated from a Tilapia Farm in Selangor, Malaysia, and Its Potential for Phage Therapy.

The Streptococcus agalactiae outbreak in tilapia has caused huge losses in the aquaculture industry worldwide. In Malaysia, several studies have reported the isolation of S. agalactiae, but no study has reported the isolation of S. agalactiae phages from tilapia or from the culture pond. Here, the isolation of the S. agalactiae phage from infected tilapia is reported and it is named as vB_Sags-UPM1. Transmission electron micrograph (TEM) revealed that this phage showed characteristics of a Siphoviridae and it was able to kill two local S. agalactiae isolates, which were S. agalactiae smyh01 and smyh02. Whole genome sequencing (WGS) of the phage DNA showed that it contained 42,999 base pairs with 36.80% GC content. Bioinformatics analysis predicted that this phage shared an identity with the S. agalactiae S73 chromosome as well as several other strains of S. agalactiae, presumably due to prophages carried by these hosts, and it encodes integrase, which suggests that it was a temperate phage. The endolysin of vB_Sags-UPM1 termed Lys60 showed killing activity on both S. agalactiae strains with varying efficacy. The discovery of the S. agalactiae temperate phage and its antimicrobial genes could open a new window for the development of antimicrobials to treat S. agalactiae infection.