Activity Of Bacteriocin-like Inhibitory Substances Research Articles

Stability of Bacteriocin-Like Inhibitory Substance (BLIS) Produced by Pediococcus acidilactici kp10 at Different Extreme Conditions.

Nowadays, bacteriocin industry has substantially grown replacing the role of chemical preservatives in enhancing shelf-life and safety of food. The progress in bacteriocin study has been supported by the emerging of consumer demand on the applications of natural food preservatives. Since food is a complex ecosystem, the characteristics of bacteriocin determine the effectiveness of their incorporation into the food products. Among four commercial media (M17 broth, MRS broth, tryptic soy broth, and nutrient broth) tested, the highest growth of Pediococcus acidilactici kp10 and bacteriocin-like-inhibitory substance (BLIS) production were obtained in the cultivation with M17. BLIS production was found to be a growth associated process where the production was increased concomitantly with the growth of producing strain, P. acidilactici kp10. The antimicrobial property of BLIS against three indicator microorganisms (Listeria monocytogenes, Escherichia coli, and Staphylococcus aureus) remained stable upon heating at 100°C but not detectable at 121°C. The BLIS activity was also observed to be stable and active at a wide pH range (pH 2 to pH 7). The BLIS activity remained constant at -20°C and -80°C for 1 month of storage. However, the activity dropped after 3 and 6 months of storage at 4°C, -20°C, and -80°C with more than 80% reduction. The ability of bacteriocin from P. acidilactici kp10 to inhibit food-borne pathogens while remaining stable and active at extreme pH and temperature is of potential interest for future applications in food preservatives.

Bacteriocin-like inhibitory activities of seven Lactobacillus delbrueckii subsp. bulgaricus strains against antibiotic susceptible and resistant Helicobacter pylori strains.

In this study, anti-Helicobacter pylori activity of seven Lactobacillus delbrueckii subsp. bulgaricus (GLB) strains was evaluated by four cell-free supernatant (CFS) types. The GLB strains produced heat-stable bacteriocin-like inhibitory substances (BLISs) with a strong anti-H. pylori activity and some neutralized, catalase- and heat-treated CFSs inhibited >83% of the test strains. Bacteriocin-like inhibitory substance production of GLB strains can render them valuable probiotics in the control of H. pylori infection.

Inhibitory substances production by Lactobacillus plantarum ST16Pa cultured in hydrolyzed cheese whey supplemented with soybean flour and their antimicrobial efficiency as biopreservatives on fresh chicken meat

Cheese whey, the main byproduct of the dairy industry, is one of the most worrisome types of industrial waste, not only because of its abundant annual global production but also because it is a notable source of environmental pollution. However, cheese whey can serve as a raw material for the production of biocomposites. In this context, in this study, we assayed the production of a bacteriocin-like inhibitory substance (BLIS) and lactate by culturing Lactobacillus plantarum ST16Pa in hydrolyzed fresh cheese whey. The process was improved by studying the enzymatic hydrolysis of cheese whey as well as its supplementation with soybean flour under microaerophilic or anaerobic conditions. Thus, the highest values of BLIS (7367.23 arbitrary units [AU]/mL) and lactate yield (Ylactate/lactose=1.39g/g) were achieved after addition of 10g/L soybean flour in microaerophilia. These conditions were successfully scaled up in a bioreactor because during complete anaerobiosis at 150rpm, L. plantarum ST16Pa attained considerable cell growth (3.14g/L), lactate concentration (14.33g/L), and BLIS activity (8082.56AU/mL). In addition, the cell-free supernatant resulting from this bioprocess showed high biopreservative efficiency in chicken breast fillets artificially contaminated with Enterococcus faecium 711 during 7days of refrigerated storage, thus indicating the potential use of this BLIS as a biopreservative in the food industry.

Potential Factors Enabling Human Body Colonization by Animal Streptococcus dysgalactiae subsp. equisimilis Strains

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a pyogenic, Lancefield C or G streptococcal pathogen. Until recently, it has been considered as an exclusive animal pathogen. Nowadays, it is responsible for both animal infections in wild animals, pets, and livestock and human infections often clinically similar to the ones caused by group A streptococcus (Streptococcus pyogenes). The risk of zoonotic infection is the most significant in people having regular contact with animals, such as veterinarians, cattlemen, and farmers. SDSE is also prevalent on skin of healthy dogs, cats, and horses, which pose a risk also to people having contact with companion animals. The main aim of this study was to evaluate if there are features differentiating animal and human SDSE isolates, especially in virulence factors involved in the first stages of pathogenesis (adhesion and colonization). Equal groups of human and animal SDSE clinical strains were obtained from superficial infections (skin, wounds, abscesses). The presence of five virulence genes (prtF1, prtF2, lmb, cbp, emm type) was evaluated, as well as ability to form bacterial biofilm and produce BLIS (bacteriocin-like inhibitory substances) which are active against human skin microbiota. The study showed that the presence of genes coding for fibronectin-binding protein and M protein, as well as BLIS activity inhibiting the growth of Corynebacterium spp. strains might constitute the virulence factors which are necessary to colonize human organism, whereas they are not crucial in animal infections. Those virulence factors might be horizontally transferred from human streptococci to animal SDSE strains, enabling their ability to colonize human organism.

Determination of Acquired Resistance Profiles of Pseudomonas aeruginosa Isolates and Characterization of an Effective Bacteriocin-Like Inhibitory Substance (BLIS) Against These Isolates.

BackgroundThe emergence of pan-drug resistant strains (PDR) of Pseudomonas aeruginosa has led to renewed efforts to identify alternative agents, such as bacteriocins and bacteriocin-like inhibitory substances (BLISs).ObjectivesThe aims of this study were to determine the acquired resistance profiles of multidrug-resistant (MDR), extensively drug-resistant (XDR), and PDR P. aeruginosa isolates based on the revised definitions of the CDC and ECDC and to screen and characterize effective BLISs against these isolates.Patients and MaterialsIn a cross-sectional study, 96 P. aeruginosa strains were isolated during a 12-month period. The resistance profiles of these isolates were determined as MDR, XDR, and PDR, and the data were analyzed using WHONET5.6 software. A BLIS against the P. aeruginosa strains was characterized based on its physicochemical properties, size, growth curves, and production profiles.ResultsAmong the 96 isolates of P. aeruginosa, 2 (2.1%), 94 (97.9%), and 63 (65.6%) were non-MDR, MDR, and XDR, respectively, and 1 (1.1%) was PDR. The most effective antibiotics against these isolates were polymyxins and fosfomycin. A BLIS isolated from the P. aeruginosa DSH22 strain had potent activity against 92 (95.8%) of the 96 isolates. The BLIS was heat stable, (up to 100°C for 10 min), UV stable, and active within a pH range of 3 - 9. The activity of BLIS disappeared when treated with trypsin, proteinase K, and pepsin, indicating its proteinous nature. Based on its size (25 kDa), the BLIS may belong to the large colicin-like bacteriocin family. BLIS production started in the midexponential phase of growth, and the maximum level (2700 AU/mL) occurred in the late-stationary phase after 25 hours of incubation at 30°C.ConclusionsThis BLIS with broad-spectrum activity may be a potential agent for the treatment or control of drug-resistant strains of P. aeruginosa infection.

Aqueous two-phase flotation for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10.

An aqueous two-phase flotation (ATPF) system based on polyethylene glycol (PEG) and sodium citrate (NaNO3C6H5O7·2H2O) was considered for primary recovery of bacteriocin-like inhibitory substance (BLIS) from Pediococcus acidilactici Kp10. The effects of ATPF parameters namely phase composition, tie-line length (TLL), volume ratio between the two phases (VR), amount of crude load (CL), pH, nitrogen gas flow rate (FR) and flotation time (FT) on the performance of recovery were evaluated. BLIS was mainly concentrated into the upper PEG-rich phase in all systems tested so far. The optimum conditions for BLIS purification, which composed of PEG 8000/sodium citrate, were: TLL of 42.6, VR of 0.4, CL of 22% (w/w), pH 7, average FT of 30min and FR of 20mL/min. BLIS was partially purified up to 5.9-fold with a separation efficiency of 99% under this optimal conditions. A maximum yield of BLIS activity of about 70.3% was recovered in the PEG phase. The BLIS from the top phase was successfully recovered with a single band in SDS-gel with molecular weight of about 10-15kDa. ATPF was found to be an effective technique for the recovery of BLIS from the fermentation broth of P. acidilactici Kp10.

Antimicrobial activity of a UV-stable bacteriocin-like inhibitory substance (BLIS) produced by Enterococcus faecium strain DSH20 against vancomycin-resistant Enterococcus (VRE) strains

The narrow spectrum of action of most bacteriocins is an important limitation for their application as antimicrobial agents. The current study describes a novel bacteriocin-like inhibitory substance (BLIS) that display extended spectrum antimicrobial activity against vancomycin-resistant Enterococcus (VRE) strains. Acquired resistance profiles of Enterococcus isolates determined based on the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) definition as multidrug-resistant (MDR), extensively drug-resistant (XDR) and pandrug resistant (PDR). BLIS activity of Enterococcus isolates was investigated against Enterococcus faecalis (E. faecalis) ATCC 29212 as the indicator strain and clinical isolates including VRE, methicillin resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria containing Pseudomonas aeruginosa (P. aeruginosa), Klebsiella, Acinetobacter, and Escherichia coli (E. coli). Among 273 Enterococcus isolates, 27 and 2 VRE isolates, respectively, were XDR and PDR and eight isolates had BLIS activity against the indicator strain. One of these isolates, identified as E. faecium strain DSH20 based on its phenotypical and biochemical properties, as well as its 16S rRNA gene sequence, had potent BLIS production against all 29 VRE strains, but had no activity against MRSA, P. aeruginosa, Klebsiella, Acinetobacter, and E. coli strains. It was heat stable up to 121°C for 15 minutes (autoclave condition), active within the pH range of 3-9 and had UV stability, but its activity disappeared by treatment with proteinase K, pepsin, and trypsin, demonstrating its proteinaceous nature. It was designated as an approximately 35kDa peptide using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. This peptide is a potential agent for use as an alternative antibacterial agent for the treatment of drug-resistant strains of VRE infection.

Application of a bacteriocin-like inhibitory substance producing Enterococcus durans E204 strain, isolated from camel milk, to control Listeria monocytogenes CECT 4032 in goat jben

Enterococcus durans E204 is a bacteriocin-like inhibitory substance (BLIS)-producing strain. It was isolated from camel’s milk in Morocco. Enterococcus durans shows interesting features such as the susceptibility to vancomycin and other clinically relevant antibiotics. In this study, we have shown that E. durans lacks haemolytic activity. Its performance as a protective culture was evaluated against Listeria monocytogenses CECT 4032 in goat jben, a Moroccan traditional fresh cheese. Ten liters of goats’ milk, originating from one farm in M’diq (northern Morocco), were pasteurised and used to make 12 small cheeses. Enterococcus durans E204 at approximately 106 CFU/ml was added in the preparation of six jben samples and six others were made without E. durans. Before packaging, two groups of four jbens were contaminated with brine solutions at 106 and 104 CFU/ml of L. monocytogenes. During storage, E. durans E204 was given time to multiply in jben and produce BLIS. In jben batches prepared with E. durans, Listeria counts decreased progressively from the beginning of storage and were undetectable at 8 and 6 days post-contamination with 106 and 104 CFU/ml, respectively. On the other hand, a substantial amount of BLIS activity (240 AU/ml) was detected from the start of the experiment and was stable during the storage. These data suggest that E. durans E204 can be used as a protective culture to inhibit growth of L. monocytogenes in cheese.

Influence of baking enzymes on antimicrobial activity of five bacteriocin-like inhibitory substances produced by lactic acid bacteria isolated from Lithuanian sourdoughs

To evaluate the effect of four different baking enzymes on the inhibitory activity of five bacteriocin-like inhibitory substances (BLIS) produced by lactic acid bacteria (LAB) isolated from Lithuanian sourdoughs. The overlay assay and the Bioscreen methods revealed that the five BLIS exhibited an inhibitory effect against spore germination and vegetative outgrowth of Bacillus subtilis, the predominant species causing ropiness in bread. The possibility that the observed antibacterial activity of BLIS might be lost after treatment with enzymes used for baking purposes was also examined. The enzymes tested; hemicellulase, lipase, amyloglucosidase and amylase had little or no effect on the majority of the antimicrobial activities associated with the five BLIS studied. This study suggests a potential application in the sourdough baking industry for these antimicrobial producing LAB strains in the control of B. subtilis spore germination and vegetative outgrowth.

Antimicrobial activity and partial characterization of bacteriocin-like inhibitory substances (BLIS) produced by Bifidobacterium infantis BCRC 14602

Bifidobacterium infantis BCRC 14602 was found to produce a bacteriocin-like inhibitory substance (BLIS) with inhibitory activities against a wide range of Gram- positive and Gram-negative bacteria. An activity level of 400 AU/ml in the middle of the exponential phase (i.e. 6 h) and maximum activity (1600 AU/ml) at the beginning of the stationary phase (i.e. 16 h) was recorded in MRS broth at 37 °C. BLIS was partially purified by a two-step purification protocol resulting in a specific activity of 31,605 AU/mg and a purification fold of 120. Based on Tricine–SDS–PAGE, the BLIS is approximately 3.0 kDa in size. Complete inactivation of BLIS activity was observed after treatment with proteolytic enzymes, but not with catalase, α-amylase and lipase. The adsorption of the BLIS to the producer cells was strongly affected by the pH of the broth culture of which 100% adsorption to the killed cells occured between pH 6.0 and 7.0, whereas at pH values below 6.0 and above 7.0, the adsorption ratio decreased to 43 and 60%, respectively. BLIS showed high temperature stability up to 121 °C for 15 min with no loss in its activity, and had pH stability in the range of 4–10. The temperature and heat stability of BLIS makes it useful for applications in food processing technologies and food safety control applications.

The role of environmental factors and medium composition on bacteriocin-like inhibitory substances (BLIS) production by Enterococcus mundtii strains

Bacteriocin-like inhibitory substances (BLIS)-producers Enterococcus mundtii WGWT1-1A, WGW11.2, WGJ20.1, WGJ40.2 and WGK53 from raw material origin were subjected to a study for the characterization of antimicrobial compound production under several growth conditions, including different cultivation media, growth temperatures, pHs, different concentrations and sources of nitrogen compounds, carbohydrates and other nutritional factors, and in the presence of different percentages of ethanol and NaCl. The five E. mundtii strains showed different behaviors. However, in all cases, MRS and sour dough bacteria (SDB) were found as the optimal media for BLIS production. In general, the higher BLIS production was observed with pH in the range 6.0–8.0 and, except 45 °C, the temperature did not show a defining effect. Low or no BLIS activity was detected after growth without nitrogen sources and carbohydrates. Absence of Tween 80, triammoniun citrate, K 2HPO 4, MgSO 4 and MnSO 4 did not affect BLIS activity levels. Except for a strain (WGWT1-1A), ethanol did not play a negative role in BLIS expression, while NaCl determined decrease of BLIS activity, proportional with concentration. The above strains did not contain plasmids, hence, BLIS expression is encoded by chromosomal DNA.

Inhibition of Vibrio parahaemolyticus by a bacteriocin-like inhibitory substance (BLIS) produced by Vibrio mediterranei 1

The aim of this research was to identify and partially purify new bacteriocin-like substances from strains of halophilic 'non-cholera' vibrios isolated from food sources. Forty-five halophilic Vibrio spp. strains were screened for antimicrobial production. Vibrio mediterranei 1, a nonpathogenic strain, showed antimicrobial activity towards Vibrio parahaemolyticus spp. and related species. The bacteriocin-like inhibitory substance (BLIS), released by the bacteria into growth media, was concentrated by ultrafiltration and characterized. BLIS was sensitive to proteinase K, was stable in the pH range 5-9, was resistant to organic solvents and was heat stable up to 75 degrees C. Initial purification of BLIS by size exclusion chromatography showed an apparent molecular mass of 63-65 kDa. This study reports the ability of V. mediterranei 1 to produce a bacteriocin-like substance inhibiting growth of V. parahaemolyticus spp. and other closely related bacteria. The strong activity of BLIS towards the human and fish pathogen V. parahaemolyticus and the persistence of antimicrobial properties under a variety of different conditions suggest its potential application in food microbiology.

Characterization of bacteriocin-like inhibitory substances (BLIS) from sourdough lactic acid bacteria and evaluation of their in vitro and in situ activity.

To identify and characterize bacteriocion-producing lactic acid bacteria (LAB) in sourdoughs and to compare in vitro and in situ bacteriocin activity of sourdough- and nonsourdough LAB. Production of antimicrobial compounds by 437 Lactobacillus strains isolated from 70 sourdoughs was investigated. Five strains (Lactobacillus pentosus 2MF8 and 8CF, Lb. plantarum 4DE and 3DM and Lactobacillus spp. CS1) were found to produce distinct bacteriocin-like inhibitory substances (BLIS). BLIS-producing Lactococcus lactis isolated from raw barley showed a wider inhibitory spectrum than sourdough LAB, but they did not inhibit all strains of the key sourdough bacterium Lb. sanfranciscensis. Antimicrobial production by Lb. pentosus 2MF8 and Lc. lactis M30 was also demonstrated in situ. BLIS production by sourdough LAB appears to occur at a low frequency, showing limited inhibitory spectrum when compared with BLIS-producing Lc. lactis. Nevertheless, they are active BLIS producers under sourdough and bread-making conditions. The activity of BLIS has been demonstrated in situ. It may influence the complex sourdough microflora and support the implantation and stability of selected insensitive bacteria, such as Lb. sanfranciscensis, useful to confer good characteristics to the dough.

Bacteriocin-like inhibitory substance (BLIS) production by the normal flora of the nasopharynx: potential to protect against otitis media?

The normal bacterial flora of the upper airways provides an important barrier to invading pathogens. This study investigated the production of bacteriocin-like inhibitory substances (BLIS) by streptococci isolated from the nasopharyngeal flora of children who either do or do not experience recurrent acute otitis media (AOM). Twenty children with recurrent AOM and 15 controls were tested. Swabs from the nasopharynx were evaluated for streptococci having BLIS activity against two representative strains of each of the AOM pathogens Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis. Streptococci displaying strong BLIS activity were characterized further and tested for known streptococcal bacteriocin structural genes. Sixty-five per cent of children had nasopharyngeal streptococcal isolates that were inhibitory to strains of one or more of the AOM pathogens. Six children (17 %) had streptococci that demonstrated strong BLIS activity against strains of at least three of the pathogenic species. Three of these inhibitory isolates were Streptococcus salivarius, two were S. pneumoniae and one was S. pyogenes. The inhibitory S. salivarius and S. pyogenes were shown to have structural genes for known streptococcal bacteriocins. No statistically significant difference was found between the two groups of children with respect to the presence of inhibitory streptococci in their nasopharyngeal floras. The finding of S. salivarius with strong inhibitory activity against several AOM pathogens in the nasopharyngeal flora of children is unique. Although there is no clear evidence from the present study that these organisms protect against AOM, their low pathogenicity and strong in-vitro BLIS production capability indicate that they should be incorporated in future trials of bacteriotherapy for recurrent AOM.

Diverse Activity Spectra of Bacteriocin–Like Inhibitory Substances Having Activity against Mutans Streptococci

Mutans streptococci (MS) are known to be causative agents of dental caries. It has been suggested that these cariogenic bacteria could be eliminated from dental plaque by application of bacteriocins or bacteriocin–like inhibitory substances (BLIS). In the present study 272 bacterial strains of the genera Streptococcus, Enterococcus and Staphylococcus were tested for their production of BLIS activity against MS by use of a deferred antagonism test on agar media supplemented with either whole blood or yeast extract. Although only 14.3% of the test strains displayed anti–MS activity, the inhibitory agents produced by these strains were characterised by considerable diversity in the range of their inhibitory action against both MS and other common oral streptococcal species. It is suggested that combinations of relatively specifically targeted anti–MS BLIS may have potential application to the prevention of dental caries.

Bacteriocin activity and probiotic activity of Aeromonas media

Three strains of Aeromonas media (161, A164 and A199) were shown to be active in-vitro producers of bacteriocin-like inhibitory substances (BLIS). For example, the producer strain, Aer. media A199, displayed antagonistic activity against all strains tested of Aer. caviae, Aer. hydrophila, Aer. salmonicida, Aer. veronii var. sobria, Listonella anguillarum, Photobacterium damsella, eight species of Vibrio and Yersinia ruckeri. Because of this wide-ranging activity against fish/shellfish pathogens, A199 was chosen for the probiotic work. By contrast, however, the BLIS produced by A199 did not inhibit the growth of Enterococcus seriolicida. The aim of the project was to ascertain whether or not the activity observed in vitro could be repeated in vivo. The ability of BLIS-producing strain A199 to act as a probiotic was assessed on the host animal, Crassostrea gigas, by testing whether or not strain A199 could prevent death of the oyster larvae when challenged with V. tubiashit. Whereas larvae, challenged with the Vibrio, died within 5 days, the presence of both the pathogen and the probiotic strain, together, did not affect the viability of the larvae over the same time period; the viability of larvae challenged with A199 alone was also unaffected when compared with the viability of unchallenged larvae (controls). These findings have important, economic implications for those engaged in the oyster producing industry where heavy losses can be experienced as a result of an infectious outbreak. At this stage, the association between BLIS activity and probiotic activity is circumstantial and, hence, future work will involve the use of non-BLIS-producing strains of Aer. media and BLIS-negative variants of the producer. Moreover, extension of the project will involve the use of other BLIS-producing strains (A161, and A164), hosts (salmon, crayfish, scallops and abalone) and pathogens.