Bacteriocin-like Substance Research Articles

Bacteriocin-Like Inhibitory Substances in Staphylococci of Different Origins and Species With Activity Against Relevant Pathogens.

Bacteriocins are antimicrobial peptides with relevance in the modulation of human and animal microbiota that have gained interest in biomedical and biotechnological applications. In this study, the production of bacteriocin-like inhibitory substances (BLIS) was tested among a collection of 890 staphylococci of different origins (humans, animals, food, and the environment) and species, both coagulase-positive (CoPS, 238 isolates of 3 species) and coagulase-negative staphylococci (CoNS, 652 isolates of 26 species). Of the 890 staphylococci, 60 (6.7%) showed antimicrobial activity by the spot-on-lawn method against at least one of the 25 indicator bacteria tested. BLIS-producer (BLIS+) isolates were detected in 8.8% of CoPS and 6.0% of CoNS. The staphylococcal species with the highest percentages of BLIS+ isolates were S. chromogenes (38.5%), S. pseudintermedius (26.7%), and S. warneri (23.1%). The production of BLIS was more frequently detected among isolates of pets, wild animals, and food. Moreover, 13 BLIS+ isolates showed wide antimicrobial activiy spectrum, and 7 of these isolates (of species S. aureus, S. pseudintermedius, S. sciuri, and S. hominis) demonstrated antimicrobial activity against more than 70% of the indicator bacteria tested. The genetic characterization (by PCR and sequencing) of the 60 BLIS+ isolates revealed the detection of (a) 11 CoNS and CoPS isolates carrying putative lantibiotic-like genes; (b) 3 S. pseudintermedius isolates harboring the genes of BacSp222 bacteriocin; and (c) 2 S. chromogenes isolates that presented the gene of a putative cyclic bacteriocin (uberolysin-like), being the first report in this CoNS species. Antimicrobial susceptibility testing was performed in BLIS+ isolates and one-third of the CoNS isolates showed susceptibility to all antibiotics tested, which also lacked the virulence genes studied. These BLIS+ CoNS are good candidates for further characterization studies.

Bacteriocin-like inhibitory substances (BLIS) synthesized by Lactococcus lactis LLH20: Antilisterial activity and application for biopreservation of minimally processed lettuce (Lactuca sativa L.)

This work aimed to evaluate the ability of the isolate L. lactis LLH20 to synthesize bacteriocin-like inhibitory substances (BLIS) with antilisterial activity and its potential application for the biopreservation of minimally processed lettuce (Lactuca sativa L.). The BLIS showed antilisterial activity, sensitivity to proteolytic enzymes and stability at 100 °C, pH 2 to 10, chemical agents and temperatures of −20 °C for 105 days. The isolate LLH20 did not present positive results for lantibiotics related genes (lanB, lanC, lanM) and nisin, lacticin 481, lacticin 3147, and lactococcin 972. In 14 h of fermentation (MRS broth), the highest BLIS produced by LLH20 was identified, and the best condition found was a medium containing 1.25% glucose, pH 7 at 32 °C. The LLH20 has been shown to be safe for use in food by phenotypic tests. Lettuce was prepared, inoculated with Listeria monocytogenes (4.0 log CFU/g) and antimicrobial agents LLH20 (8.0 log CFU/g), nisin (2.5 ppm), BLIS of LLH20 (1600 AU/mL), or association of BLIS and nisin (1600 AU/mL and 2.5 ppm), and stored at 4 °C for 7 days. The L. monocytogenes counts in lettuce decreased by 3.14 and 1.63 log CFU/g, when inoculated with LLH20 or with BLIS from LLH20, respectively, being significantly different from the positive control (p < 0.05). The association of BLIS and nisin eliminated L. monocytogenes in minimally processed lettuce. These results indicate that L. lactis LLH20 synthesized a BLIS capable of controlling the growth of L. monocytogenes in lettuce stored at 4 °C, demonstrating its potential as a biopreservative.

Probiotic Characteristics and Antimicrobial Potential of a Native Bacillus subtilis Strain Fa17.2 Rescued from Wild Bromelia sp. Flowers.

In the present study, we identified the Bacillus subtilis strain annotated Fa17.2 isolated from Bromelia flower inflorescences collected from the subtropical humid mesothermal region, Santo Domingo de Los Tsachilas Province, Ecuador. The probiotic capacity and antimicrobial potential against four foodborne pathogens were assessed. The cell culture of Fa17.2 is highly resistant to synthetic gastric acid (pH 2.5, 3.0, and 3.5), bile salts (0.3%), tolerating different sodium chloride concentrations (1, 3, and 5%), and growth conditions (15 °C and 45 °C), suggesting its potential probiotic features. The isolate showed no antibiotic resistance and was considered safe as no hemolysis was detected on sheep blood agar. The optimum medium for bacterial growth and the release of antimicrobial compounds was MRS with 10% glucose. The active components released in the neutralized crude extract (NCE) were insensitive to organic solvents, surfactants, and nonproteolytic enzymes and sensitive to proteolytic enzymes suggesting their proteinaceous nature. The antimicrobial activity was enhanced by heat and maintained active over a wide range of pH (2.0–8.0). Moreover, the crude extract (CE) showed inhibitory activity against several Gram-negative and Gram-positive bacteria. The molecular weight of partially purified precipitated bacteriocin-like substances (BLISs) was about 14 kDa in 20% Tricine-SDS-PAGE. The CE obtained from Fa17.2 inhibits the growth of four foodborne pathogens, Staphylococcus aureus, Escherichia coli, Kosaconia cowanii, and Shigella dysenteriae, which implies its potential as an antimicrobial producer strain.

Bacteriocinogenic probiotic bacteria isolated from an aquatic environment inhibit the growth of food and fish pathogens

The conditions of aquatic environments have a great influence on the microbiota of several animals, many of which are a potential source of microorganisms of biotechnological interest. In this study, bacterial strains isolated from aquatic environments were bioprospected to determine their probiotic profile and antimicrobial effect against fish and food pathogens. Two isolates, identified via 16S rRNA sequencing as Lactococcus lactis (L1 and L2) and one as Enterococcus faecium 135 (EF), produced a bacteriocin-like antimicrobial substance (BLIS), active against Listeria monocytogenes, Salmonella Choleraesuis and Salmonella Typhimurium. Antimicrobial activity of BLIS was reduced when exposed to high temperatures and proteolytic enzymes (trypsin, pepsin, papain and pancreatin). All strains were sensitive to 7 types of antibiotics (vancomycin, clindamycin, streptomycin, gentamicin, chloramphenicol, rifampicin and ampicillin), exhibited a high rate of adherence to Caco-2 cells and expressed no hemolysin and gelatinase virulence factors. EF showed some resistance at pH 2.5 and 3.0, and L2/EF showed higher resistance to the action of bile salts. Finally, the presence of bacteriocin genes encoding for proteins, including Nisin (L1 and L2), Enterocin A, B, P, and Mundticin KS (EF) was detected. The molecular and physiological evidence suggests that the bacterial isolates in this study could be used as natural antimicrobial agents and may be considered safe for probiotic application.

Evaluation of inhibitory and probiotic properties of lactic acid bacteria isolated from vaginal microflora.

Lactic acid bacteria (LABs) are known to secrete species-specific secondary metabolites that could be utilized as novel therapeutics against multi-drug resistant pathogens. This study aimed to investigate the antagonistic and probiotic properties of LABs isolated from the vaginal ecosystem of healthy women and to assess the stability of their antagonistic metabolites. Among 43 strains isolated from healthy women, eight LAB strains exhibited detectable BLISs (bacteriocin-like substances) producing ability against E. faecalis (JH-86), S. aureus (JH-68), Streptococcus sp. (JH-80), and E. coli (JH-101), with zone of inhibition (ZI) ranging from 9.00 to 20.33mm and minimum inhibitory concentrations (MICs) from 62.5 to 500 μL/mL, respectively. The partially purified compounds extracted from cell free supernatant (CFS) displayed an increase in antagonistic activity based on ZI, 9.67-30.17mm and MICs, 3.91-15.63mg/mL, respectively. In a time-kill study, both crude and partially purified compounds of Limosilactobacillus reuteri (MT180537), Pediococcus pentosaceus (MT176555), Limosilactobacillus pontis (MW362838), and Levilactobacillus brevis (MW362790) exhibited significant bactericidal action against E. faecalis (MW051601), the most frequent etiological agent of aerobic vaginitis (AV). The active secondary metabolites from L. reuteri (MT180537), P. pentosaceus (MT176555), and L. pontis (MW362838) were protein in nature and remained stable under different physicochemical conditions. Regarding probiotic properties, the strains presented probiotic characteristics, i.e., good acid, bile salt tolerance, aggregation properties, and biofilm formation. The strains were susceptible to most of the commonly used antibiotics and had no hemolytic activity. In conclusion, antagonistic compounds or BLIS produced by L. reuteri (MT180537) could be investigated further for preparation of ointments to treat AV.

Antimicrobial Activity of Bacteriocin-Like Inhibitory Substances Produced by Bacillus subtilis Isolated from “Iru”

Antimicrobial Activity of Bacteriocin-Like Inhibitory Substances Produced by Bacillus subtilis Isolated from “Iru”

Bacteriocin-like inhibitory substances production by Enterococcus faecium 135 in co-culture with Ligilactobacillus salivarius and Limosilactobacillus reuteri.

The use of lactic acid bacteria (LAB) and probiotic cultures in the breeding of animals such as poultry and swine are quite common. It is known that those strains can produce bacteriocins when grown in pure culture. However, the production of bacteriocin using co-culture of microorganisms has not been much studied so far. The present study contributes with innovation in this area by embracing the production of bacteriocin-like inhibitory substances (BLIS) by a newly isolated strain of Enterococcus faecium 135. Additionally, the co-cultivation of this strain with Ligilactobacillus salivarius and Limosilactobacillus reuteri was also investigated. The antimicrobial activity of the produced BLIS was evaluated against Listeria monocytogenes, Listeria innocua, Salmonella enterica, and Salmonella enterica serovar Typhimurium using two methods: turbidimetric and agar diffusion. In addition, the presence of enterocin genes was also evaluated. The BLIS produced showed a bacteriostatic effect against the bio-indicator strains, and the highest antimicrobial activities expressed by arbitrary units per mL (AU/mL) were obtained against L. monocytogenes in monoculture (12,800 AU/mL), followed by the co-culture of E. faecium with Limosilactobacillus reuteri (400 AU/mL). After concentration with ammonium sulfate, the antimicrobial activity raised to 25,600 AU/mL. Assays to determine the proteinaceous nature of the BLIS showed susceptibility to trypsin and antimicrobial activity until 90°C. Finally, analysis of the presence of structural genes of enterocins revealed that four enterocin genes were present in E. faecium 135. These results suggest that BLIS produced by E. faecium 135 has potential to be a bacteriocin and, after purification, could potentially be used as an antimicrobial agent in animal breeding.

Study on the applicability of potentially probiotic strain Lactobacillus plantarum L4 to be included in the development of strategies for bio-preservation of food products

The antimicrobial activity of potentially probiotic strains is important for their possible application in the development of successful strategies for bio-preservation of different foods. The antimicrobial activity of the potentially probiotic strain Lactobacillus plantarum L4 against the pathogenic bacteria Salmonella abony; Staphylococcus aureus; Escherichia coli and the phytopathogenic fungi Aspergillus niger and Aspergillus flavus was determined using agar-diffusion assay with wells. Lb. plantarum L4 exhibited high antagonistic activity against all test microorganisms included in the study, which was due not only to the production and accumulation of lactic acid and other organic acids, but also to the production of other bioactive substances. The bioactive substances were then submitted to the action of different enzymes. This allowed to observe that they have protein nature, suggesting that they are likely bacteriocins or bacteriocin-like substances. The substances produced by Lb. plantarum L4 did not lose their antimicrobial activity during heat treatment at temperatures above 100 °С for 15 min and they exhibited high inhibitory activity against the test-microorganisms when pH ranged between 4 and 7. After further examination of the properties of the produced antimicrobial compounds, Lb. plantarum L4 can be used in the development of strategies for bio-preservation of different food products.

Pan-genomic and comparative analysis of Pediococcus pentosaceus focused on the in silico assessment of pediocin-like bacteriocins

Bacteriocins are antimicrobial peptides produced by different species of bacteria, especially the Gram-positive lactic acid bacteria (LAB). Pediococcus pentosaceus is widely applied in the industry and stands out as Bacteriocin-Like Inhibitory Substances (BLIS) producer known to inhibit pathogens commonly considered a concern in the food industries. This study aimed to perform in silico comparisons of P. pentosaceus genomes available in the public GenBank database focusing on their pediocin-like bacteriocins repertoire. The pan-genome analysis evidenced a temporal signal in the pattern of gene gain and loss, supporting the hypothesis that the complete genetic repertoire of this group of bacteria is still uncovered. Thirteen bacteriocin genes from Class II and III were predicted in the accessory genome. Four pediocin-like bacteriocins (54% of the detected bacteriocin repertoire) and their accompanying immunity genes are highlighted; penocin A, coagulin A, pediocin PA-1, and plantaricin 423. Additionally, in silico, modeling of the pediocin-like bacteriocins revealed different configurations of the helix motif compared to other physically determined pediocin-like structures. Comparative and phylogenomic analyses support the hypothesis that a dynamic mechanism of bacteriocin acquisition and purging is not dependent on the bacterial isolation source origin. Synteny analysis revealed that while coagulin A, pediocin PA-1, and Plantaricin 423 loci are associated with insertion sequences mainly from the IS30 family and are likely of plasmid origin, penocin A lies in a conserved chromosomal locus. The results presented here provide insights into the unique pediocin-like bacteriocin peptide fold, genomic diversity, and the evolution of the bacteriocin genetic repertoire of P. pentosaceus, shedding new insights into the role of these biomolecules for application in inhibiting bacterial pathogens, and suggesting that prospecting and sequencing new strains is still an alternative to mining for new probiotic compounds.

Genomic analysis of Enterococcus durans NT21, a putative bacteriocin-producing isolate.

Enterococcus species are a long-standing and non-pathogenic commensal bacterium, representing an important part of the normal. Enterococcus durans is a rarely isolated species from animals and humans, and it was a tiny constituent of human oral cavity and animal intestinal flora, as well as animal-derived foods, particularly dairy products. This study evaluated the security of our strain E. durans NT21 by using whole-genome sequencing (WGS), physicochemical features, and antimicrobial activity. The complete genomic of our strain Enterococcus durans NT21was sequenced and analyzed by using several bioinformatics tools to identify bacteriocin genes, virulence genes, antibiotic resistance genes, Crispr-Cas and pathogenicity islands. The results showed that our strain NT21 lacks the presence of virulence genes, pathogenicity islands, plasmids and has only two antibiotic resistance genes. On the other hand, it produces three bacteriocin-like inhibitory substances (Enterolysin A, P and L50a). It has six gene-encoded Crisper-Cas and one cluster Crispr-Cas gene. According to our findings, E. durans NT21 is a possible probiotic strain that is safe for both human and animal use.

Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from the Gut of Chanos chanos.

Bacteriocin-like inhibitory substances (BLIS) have sparked great interest because of their promising use in food as natural antimicrobial agents. In this work, six Streptomyces isolates obtained from the gut of Chanos chanos demonstrated their ability to produce extracellular metabolites with inhibitory activity against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. Exposure of the extracellular metabolites to proteolytic enzymes (i.e., proteinase-K, trypsin, and pepsin) revealed high sensitivity and confirmed their proteinaceous nature. The metabolites were stable at high temperatures (up to 100°C for 30 min) and a wide range of pH (pH 2.0–7.0). Fractionation of the crude BLIS by filtration yielded three fractions based on molecular weight: <3 kDa, 3–10 kDa, and >10 kDa. Analysis of the antibacterial activity of these fractions showed increased specific activity, especially in the fraction with a molecular weight (MW) of <3 kDa, relative to the crude sample. The fraction with MW < 3 kDa had minimum inhibitory and bactericidal concentrations in ranges 0.04–0.62 mg·mL−1 and 0.08–1.25 mg·mL−1, respectively. This fraction also showed better temperature and pH stability compared with crude BLIS. Brine shrimp toxicity assay revealed that this fraction has moderate toxicity with a 50% lethal concentration of 226.975 μg·mL−1 (i.e., moderate toxicity) to Artemia salina. Identification of the peptide sequences of this fraction by liquid chromatography–tandem mass spectrometry yielded 130 proteins with retention times of 15.21–19.57 min. Eleven proteins with MWs of 1345.66–2908.35 Da and composed of less than 30 amino acid residues with high hydrophobicity (15.34–26.22 kcal·mol−1) appeared to be responsible for the antibacterial activity of the fraction. This study revealed the potential application of BLIS from Streptomyces, especially BLIS SCA-8, as antibacterial agents.

Extractive Fermentation for Recovery of Bacteriocin-Like Inhibitory Substances Derived from Lactococcus lactis Gh1 Using PEG2000/Dextran T500 Aqueous Two-Phase System

This work aimed to optimize the parameters affecting partitioning of a bacteriocin-like inhibitory substances (BLIS) from Lactococcus lactis Gh1 in extractive fermentation using polyethylene glycol (PEG)/dextran aqueous two-phase system (ATPS). This system was developed for the simultaneous cell cultivation and downstream processing of BLIS. Results showed that the molecular weight of PEG, PEG concentration, and dextran T500 affect the partition coefficient (K), purification factor (PF), and yield of BLIS partitioning. ATPS composed of 10% (w/w) PEG2000 and 8% (w/w) dextran T500, provided the greatest conditions for the extractive BLIS production. The K (1.00 ± 0.16), PF (2.92 ± 0.37) and yield (77.24 ± 2.81%) were increased at selected orbital speed (200 rpm) and pH (pH 7). Sustainable growth of the cells in the bioreactor and repeated fermentation up to the eighth extractive batch were observed during the scale up process, ensuring a continuous production and purification of BLIS. Hence, the simplicity and effectiveness of ATPS in the purification of BLIS were proven in this study.

Insight into the pilot‐scale fed-batch fermentation for production of Enterococcus faecium CW3801 using molasses-based medium

Enterococcus sp. has been used as starters in food fermentation due to their probiotic and antimicrobial properties in food biopreservation. The antimicrobial properties were mainly contributed by the bacteriocin called enterocin. Hence, the availability of a cost-effective pilot-scale cultivation conditions is a necessity for the production of probiotic bacteria. This study aims to investigate optimization of medium composition using sugarcane molasses as a carbon source using response surface methodology and the potential use of fed-batch cultivation for improvement of the cell viability of Enterococcus faecium CW3801 for the use as a probiotic starter culture. Two feeding strategies (ramp and constant) were applied in fed-batch cultivation for enhancement of the production of E. faecium in a 2-L stirred tank bioreactor using the optimized medium and scaled up to a 15-L bioreactor. Optimized fermentation medium which comprised of 10% (v/v) of molasses and 10 g/L of yeast extract at pH 7 yielded maximum cell viability of 29.4 × 1011 CFU/mL with 3900 AU/mL of bacteriocin-like inhibitory substances (BLIS) activity. In the fed-batch, the cell viability (8.4 × 1013) and dry cell weight (6.34 g/L) reached the highest in optimized medium when the ramp (stepwise) feeding was applied. In scaling up to 15-L bioreactor, the growth of E. faecium was achieved at 2.3 × 1013 CFU/mL with the dry cell weight of 5.28 g/L under the same condition. The BLIS in 15-L bioreactor was 6% higher than the 2-L bioreactor. This study demonstrated that molasses and yeast extract are good feedstock for the growth of E. faecium. The E. faecium, a non-vancomycin resistant enterococcus (VRE) was successfully produced by a fed-batch cultivation approach and scaled up to a 15-L bioreactor using a ramp feeding strategy. Results from this study revealed that the fed-batch cultivation using molasses-based medium has industrial potential for the production of probiotics.

Bacteriocin producing microbes with bactericidal activity against multidrug resistant pathogens

BackgroundBacteriocins are proteins or peptides synthesized by bacteria that show inhibitory or killing activities against various bacteria. Bacteriocins are mainly considered for effective alternatives to different commercial antibiotics, preservatives in the food and pharmaceutical industries. ObjectivesTo screen and analyze novel bacteriocin-producing bacteria from the fermented food shidal for antibacterial activity against food pathogens and their molecular interactions studied through computationally. MethodsIn this study, a strain Lactobacillus plantarum LA21 was isolated from the fermented food shidal identified based on morphological, biochemical, and 16S rDNA gene sequencing. The potent bacterium was subjected to improve bacteriocins production and characterized. Antimicrobial activity against drug-resistant bacteria and minimum inhibitory concentration (MIC) were determined. The bacteriocin was treated with proteolytic enzymes, and the mechanism of action on food pathogens was analyzed. Molecular docking studies were carried out as GLIDE module in the maestro tool of Schrodinger Software. ResultsBacteriocin was effective against pathogens such as Bacillus pumilus, Bacillus amyloliquefaciens, Staphylococcus aureus, and Listeria monocytogenes, with the most negligible MIC value was detected in L. monocytogenes. Furthermore, the depleted viability of bacterial cells indicated bacteriocin-induced cell lysis in L. monocytogenes via bactericidal activity. In addition, proteolytic enzyme digested bacteriocins revealed bacteriocin-like substances. Finally, molecular docking was performed to study the interactions between the targets and bacteriocins, results in relative intense contact with minimally 3 Å distance. ConclusionsThe characteristic features of these bacteriocin-like molecules revealed that L. plantarum LA21 is a novel bacteriocin-producing bacterial strain to prepare novel antimicrobial drugs, feed additives or preservatives for future use in livestock and food industries.

Fermentation strategies for improving the production of bacteriocin‐like inhibitory substances by Lactobacillus brevis C23 with nutrient supplementation, pH, and temperature variations

In this study, fermentation strategies were carried out for improving the production of bacteriocin-like inhibitory substances by Lactobacillus brevis C23 with nutrient supplementation, pH, and temperature variations, as well as fed-batch cultivation. The BLIS activity was highest (91.52%) in the MRS medium supplemented with 1.5% (w/v) lactose and 1% (w/v) meat extract. pH 5 augmented both the cell biomass and BLIS activity, but incubation temperature only affected growth at 37°C. Fed-batch cultivation strategy that utilized meat extract feeding would positively affect cell growth, whereas BLIS activity was enhanced by lactose feeding. The highest BLIS activity from L. brevis C23 was obtained at the 24 hr mark by feeding the culture medium with lactose (77.73%). The BLIS production of L. brevis C23 fed with meat extract followed a similar trend to lactose (74.20%). Increased BLIS activity in the final solution would reduce the downstream step such as concentrating the purified product. Novelty impact statement Synergic effect of lactose and meat extract enhanced BLIS activity (91.52%) of CFS by Lactobacillus brevis C23 as compared to the control (69.29%) Temperature at 40℃ did not affect the BLIS activity, but it inhibited the cell growth. Fed-batch cultivation strategy that utilized meat extract feeding would positively affect cell growth, whereas BLIS activity was enhanced by lactose feeding.

Respiratory microbiota of healthy broilers can act as reservoirs for multidrug-resistant Escherichia coli

This study aimed at evaluate the presence and to study characteristics of Escherichia coli in the respiratory system microbiota of healthy broilers. Trachea, air sacs, and lungs of 20 broilers were analyzed at 21 days of age, reared in experimental conditions, without receiving antimicrobials. E. coli strains were isolated and identified using conventional bacteriology through morphological and biochemical characterization. The production of bacteriocin-like substances, the presence of virulence-associated genes (VAGs) of APEC (Avian Pathogenic Escherichia coli) predictors, and the antimicrobial susceptibility were evaluated. E. coli was found in 85 % of the animals (17/20), in the trachea, air sacs or lungs; and it was not found in 15 % of the animals (3/20). A total of 34 isolates were recovered, 13 from the air sacs, 13 from the lungs, and 8 from the trachea, which showed no production of bacteriocin-like substances nor virulence genes associated with APEC. Most isolates, 59 % (20/34), showed resistance to at least one of the tested antimicrobials, and six multiresistant strains were identified. The results demonstrated that strains of E. coli were commensal of the respiratory microbiota, and that they did not present pathogenicity to the host, since there were no clinical signs of disease, macroscopic lesions in the organs of the evaluated broilers, production of bacteriocin-like substances, nor virulence-associated genes considered as predictors of APEC in bacteria. These strains of E. coli were mostly susceptible to antimicrobials. However, the occurrence of multidrug-resistant strains suggests that these animals can act as reservoirs of resistant to antimicrobials E. coli.

Isolation, identification and prokaryotic expression of a bacteriocin-like substance from Bacillus licheniformis

The ban on addition of antibiotics in animal feed in China has made the search for new antibiotics substitutes, e.g. bacteriocin, a hot topic in research. The present study successfully isolated an antibacterial substance producing strain of Bacillus sp. from alpaca feces by agar diffusion method, using Escherichia coli, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Listeria monocytogenes as indicator bacteria. The isolated strain was named as B. licheniformis SXAU06 based on colony morphology, Gram staining and 16S rRNA gene sequence. The antibacterial substance was isolated and purified through a series of procedures including (NH4)2SO4 precipitation, chloroform extraction, molecular interception and SDS-PAGE analysis. Bioinformatics analysis of the LC-MS/MS data indicated that the antibacterial substance was a bacteriocin-like substance (BLIS) with an approximate molecular weight of 14 kDa, and it was designated as BLIS_SXAU06. BLIS_SXAU06 exhibited high resistance to treatment of proteinase K, high temperature, high acidity and alkalinity. BLIS_SXAU06 was heterologously expressed in E. coli and the recombinant BLIS_SXAU06 exhibited effective antibacterial activity against S. aureus, S. epidermidis, M. luteus, and L. monocytogenes, showing potential to be investigated further.

Bacteriocin-Like Inhibitory Substance (BLIS) Activity of Enterococcus faecium DB1 Against Biofilm Formation by Clostridium perfringens.

The antibiofilm effect of bacteriocin-like inhibitory substance (BLIS) from Enterococcus faecium DB1 against Clostridium perfringens was investigated in the present study. BLIS of E. faecium DB1 significantly reduced biofilm formation by C. perfringens in a dose-dependent manner for 24 and 48h. In particular, treatment with BLIS of E. faecium DB1 significantly inhibited biofilm formation by C. perfringens on chicken meat and stainless steel coupon surfaces. Moreover, BLIS of E. faecium DB1 decreased the viability of C. perfringens biofilm and planktonic cells, indicating that the reduction of biofilm formation by C. perfringens might be achieved by killing the bacterial cells. Taken together, the present results suggest that BLIS of E. faecium DB1 can be a promising antibiofilm agent to eradicate C. perfringens.

Characterization and profiling of bacteriocin-like substances produced by lactic acid bacteria from cheese samples.

Bacteriocins have become biological weapons against harmful food pathogens and have attracted interest as tools for biopreservation. The aim of this study was to isolate, identify and characterize lactic acid bacterial (LAB) strains from cheese samples, partially purify potential bacteriocins and characterize their antimicrobial activity against pathogens. Bacteriocin-producing organisms were screened by Agar spot assay test. Initially, 25 LAB isolates were isolated from the cheese samples and identified as Lactobacillius spp., among them five strains were able to produce bacteriocin whose antimicrobial activates were analysed by agar-well-diffusion assay test against pathogenic organisms. Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Streptococcus thermophillus and Listeria monocytogens were inhibited, while Enterococcus faecalis, Salmonella typhi, Escherichia coli and Pseudomonas aeruginosa were resistant to the antimicrobial substances from LAB isolates. For optimal production of bacteriocin, LAB broth cultures were harvested at exponential phase. The molecular weights of the bacteriocins are between 7.0–15.0 kDa. The bacteriocins were characterized on the basis of their sensitivity to heat, pH, enzymes, NaCl and treatments with organic solvents. These results revealed that the bacteriocins from Lactobacillius spp. isolated from the cheese might have potential antimicrobial properties and give new insight in the development of bio-preservative agents to prevent and control pathogenic bacterial infection.

Characterization of Enterococcus faecium E86 bacteriocins and their inhibition properties against Listeria monocytogenes and vancomycin-resistant Enterococcus.

In the present scenario of a major demand for new compounds with antimicrobial activity, bacteriocin and bacteriocin-like inhibitory substances (BLIS) are promising tools against deteriorating and pathogenic microorganisms, thus having potential applications in both the food industry and infectious disease control. In the present report, we describe the genetic and phenotypic characteristics of BLIS produced by Enterococcus faecium E86, a strain previously isolated and sequenced by our group, focusing on the structural genes of two bacteriocins identified: enterocin TW21 and enterocin P. Transcription of all four genes associated with the biosynthesis and immunity of enterocin P and enterocin TW21 were confirmed by RT-PCR. However, Sanger sequencing confirmed a truncation of the structural gene of enterocin TW21 due to one base pair deletion (A/T). Thus, although E. faecium E86 was shown to carry two bacteriocinogenic gene clusters, only one cluster encodes a functional bacteriocin, enterocin P. Enterocin P was able to inhibit different strains of Listeria monocytogenes and vancomycin-resistant enterococci (both Enterococcus faecalis and Enterococcus faecium), showing intense bacteriolytic activity, in most cases.