Genetic and phenotypic assessment of the antimicrobial activity of three potential probiotic lactobacilli against human enteropathogenic bacteria.

Introduction: Lactobacilli are avid producers of antimicrobial compounds responsible for their adaptation and survival in microbe-rich matrices. The bactericidal or bacteriostatic ability of lactic acid bacteria (LAB) can be exploited for the identification of novel antimicrobial compounds to be incorporated in functional foodstuffs or pharmaceutical supplements. In this study, the antimicrobial and antibiofilm properties of Lactiplantibacillus pentosus L33, Lactiplantibacillus plantarum L125 and Lacticaseibacillus paracasei SP5, previously isolated form fermented products, were examined, against clinical isolates of Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Enteritidis and Escherichia coli. Methods: The ability of viable cells to inhibit pathogen colonization on HT-29 cell monolayers, as well as their co-aggregation capacity, were examined utilizing the competitive exclusion assay. The antimicrobial activity of cell-free culture supernatants (CFCS) was determined against planktonic cells and biofilms, using microbiological assays, confocal microscopy, and gene expression analysis of biofilm formation-related genes. Furthermore, in vitro analysis was supplemented with in silico prediction of bacteriocin clusters and of other loci involved in antimicrobial activity. Results: The three lactobacilli were able to limit the viability of planktonic cells of S. aureus and E. coli in suspension. Greater inhibition of biofilm formation was recorded after co-incubation of S. enterica with the CFCS of Lc. paracasei SP5. Predictions based on sequence revealed the ability of strains to produce single or two-peptide Class II bacteriocins, presenting sequence and structural conservation with functional bacteriocins. Discussion: The efficiency of the potentially probiotic bacteria to elicit antimicrobial effects presented a strain- and pathogen-specific pattern. Future studies, utilizing multi-omic approaches, will focus on the structural and functional characterization of molecules involved in the recorded phenotypes.

BADASS: BActeriocin-Diversity ASsessment Software.

BACKGROUND: Bacteriocins are defined as thermolabile peptides produced by bacteria with biological activity against taxonomically related species. These antimicrobial peptides have a wide application including disease treatment, food conservation, and probiotics. However, even with a large industrial and biotechnological application potential, these peptides are still poorly studied and explored. BADASS is software with a user-friendly graphical interface applied to the search and analysis of bacteriocin diversity in whole-metagenome shotgun sequencing data. RESULTS: The search for bacteriocin sequences is performed with tools such as BLAST or DIAMOND using the BAGEL4 database as a reference. The putative bacteriocin sequences identified are used to determine the abundance and richness of the three classes of bacteriocins. Abundance is calculated by comparing the reads identified as bacteriocins to the reads identified as 16S rRNA gene using SILVA database as a reference. BADASS has a complete pipeline that starts with the quality assessment of the raw data. At the end of the analysis, BADASS generates several plots of richness and abundance automatically as well as tabular files containing information about the main bacteriocins detected. The user is able to change the main parameters of the analysis in the graphical interface. To demonstrate how the software works, we used four datasets from WMS studies using default parameters. Lantibiotics were the most abundant bacteriocins in the four datasets. This class of bacteriocin is commonly produced by Streptomyces sp. CONCLUSIONS: With a user-friendly graphical interface and a complete pipeline, BADASS proved to be a powerful tool for prospecting bacteriocin sequences in Whole-Metagenome Shotgun Sequencing (WMS) data. This tool is publicly available at https://sourceforge.net/projects/badass/ .

From Cheese Whey Permeate to Sakacin A: A Circular Economy Approach for the Food-Grade Biotechnological Production of an Anti-Listeria Bacteriocin.

Cheese Whey Permeate (CWP) is the by-product of whey ultrafiltration for protein recovery. It is highly perishable with substantial disposal costs and has serious environmental impact. The aim of the present study was to develop a novel and cheap CWP-based culture medium for Lactobacillus sakei to produce the food-grade sakacin A, a bacteriocin exhibiting a specific antilisterial activity. Growth conditions, nutrient supplementation and bacteriocin yield were optimized through an experimental design in which the standard medium de Man, Rogosa and Sharpe (MRS) was taken as benchmark. The most convenient formulation was liquid CWP supplemented with meat extract (4 g/L) and yeast extract (8 g/L). Although, arginine (0.5 g/L) among free amino acids was depleted in all conditions, its supplementation did not increase process yield. The results demonstrate the feasibility of producing sakacin A from CWP. Cost of the novel medium was 1.53 €/L and that of obtaining sakacin A 5.67 €/106 AU, with a significant 70% reduction compared to the corresponding costs with MRS (5.40 €/L, 18.00 €/106 AU). Taking into account that the limited use of bacteriocins for food application is mainly due to the high production cost, the obtained reduction may contribute to widening the range of applications of sakacin A as antilisterial agent.

Pharmacological, Toxicological, and Dose Range Assessment of OG716, a Novel Lantibiotic for the Treatment of Clostridium difficile-Associated Infection.

Lantibiotics present an attractive scaffold for the development of novel antibiotics. We report here a novel lantibiotic for the treatment of Clostridium difficile infection. The lead compounds were selected from a library of over 700 single- and multiple-substitution variants of the lantibiotic mutacin 1140 (MU1140). The best performers in vitro and in vivo were further used to challenge Golden Syrian hamsters orally in a Golden Syrian hamster model of Clostridium difficile-associated disease (CDAD) in a dose-response format, resulting in the selection of OG716 as the lead compound. This lantibiotic was characterized by a 50% effective dose of 23.85 mg/kg of body weight/day (10.97 µmol/kg/day) in this model. Upon oral administration of the maximum feasible dose (≥1,918 mg/kg/day), no observable toxicities or side effects were noted, and no effect on intestinal motility was observed. Compartmentalization to the gastrointestinal tract was confirmed. MU1140-derived variants offer a large pipeline for the development of novel antibiotics for the treatment of several indications and are particularly attractive considering their novel mechanism of action. Based on the currently available data, OG716 has an acceptable profile for further development for the treatment of CDAD.

Production of Recombinant Antimicrobial Polymeric Protein Beta Casein-E 50-52 and Its Antimicrobial Synergistic Effects Assessment with Thymol.

Accelerating emergence of antimicrobial resistance among food pathogens and consumers' increasing demands for preservative-free foods are two contemporary challenging aspects within the food industry. Antimicrobial packaging and the use of natural preservatives are promising solutions. In the present study, we used beta-casein-one of the primary self-assembly proteins in milk with a high polymeric film production capability-as a fusion partner for the recombinant expression of E 50-52 antimicrobial peptide in Escherichia coli. The pET21a-BCN-E 50-52 construct was transformed to E. coli BL21 (DE3), and protein expression was induced under optimized conditions. Purified protein obtained from nickel affinity chromatography was refolded under optimized dialysis circumstances and concentrated to 1600 µg/mL fusion protein by ultrafiltration. Antimicrobial activities of recombinant BCN-E 50-52 performed against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, Staphylococcus aureus, Aspergillus flavus, and Candida albicans. Subsequently, the synergistic effects of BCN-E 50-52 and thymol were assayed. Results of checkerboard tests showed strong synergistic activity between two compounds. Time-kill and growth kinetic studies indicated a sharp reduction of cell viability during the first period of exposure, and SEM (scanning electron microscope) results validated the severe destructive effects of BCN E 50-52 and thymol in combination on bacterial cells.

In Vivo Assessment of Immunogenicity and Toxicity of the Bacteriocin TSU4 in BALB/c Mice.

Bacteriocin TSU4 is a novel antimicrobial peptide isolated from Catla catla gut isolate Lactobacillus animalis TSU4. It has been reported for its potential antimicrobial activity against fish pathogens and food spoilage bacteria. In vivo safety evaluation is necessary to determine its immunogenicity, toxicity, and importance in real-life applications. The present study was designed to evaluate the immunogenicity, acute and sub-chronic toxicity of bacteriocin TSU4 in BALB/c mice to ensure its safety in industrial application. Male BALB/c mice were administered intraperitoneally for immunogenicity assessment, by oral gavage with 50, 100, and 200 mg/kg/body weight for acute test and 0.5 mg/kg/day dose of bacteriocin TSU4 for sub-chronic toxicity test. Neither mortality nor any infections were observed during experimental period. There was no major increase in antibody titer during the immunogenicity test, and no mortality was observed during acute or sub-chronic toxicity tests. The LD50 value of bacteriocin TSU4 was found to be higher than 200 ± 0.45 mg/kg. No significant change in the serum biochemical markers, histopathological analysis and visual observation in spleen sizes was observed. These findings revealed that bacteriocin TSU4 is a non-immunogenic, safe, non-toxic, and could be a potential candidate for industrial applications in food preservation and aquaculture industries.

Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish.

In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50 kDa in 12 % Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40 °C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23 ± 1.15 µg of maltose liberated mg-1 protein ml-1 of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25 % bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.

Short communication: In vivo screening platform for bacteriocins using Caenorhabditis elegans to control mastitis-causing pathogens.

This study aimed to develop an in vivo screening platform using Caenorhabditis elegans to identify a novel bacteriocin for controlling the mastitis-causing pathogen Staphylococcus aureus strain RF122 in dairy cows. Using Bacillus spp. isolated from traditional Korean foods, we developed a direct in vivo screening platform that uses 96-well plates and fluorescence image analysis. We identified a novel bacteriocin produced by Bacillus licheniformis strain 146 (lichenicin 146) with a high in vivo antimicrobial activity using our liquid C. elegans-Staph. aureus assay. We also determined the characteristics of lichenicin 146 using liquid chromatography-mass spectrometry and confirmed that it shared homologous sequences with bacteriocin family proteins. In addition, RNA-sequencing analysis revealed genes encoding cell surface or membrane proteins (SAB0993c, SAB0150, SAB0994c, and SAB2375c) that are involved in the bactericidal activity of lichenicin 146 against Staph. aureus strain RF122 infection as well as those encoding transcriptional regulators (SAB0844c and SAB0133). Thus, our direct in vivo screening platform facilitates simple, convenient, cost-effective, and reliable screening of potential antimicrobial compounds with applications in the dairy field.

Lanthipeptides: chemical synthesis versus in vivo biosynthesis as tools for pharmaceutical production.

Lanthipeptides (also called lantibiotics for those with antibacterial activities) are ribosomally synthesized post-translationally modified peptides having thioether cross-linked amino acids, lanthionines, as a structural element. Lanthipeptides have conceivable potentials to be used as therapeutics, however, the lack of stable, high-yield, well-characterized processes for their sustainable production limit their availability for clinical studies and further pharmaceutical commercialization. Though many reviews have discussed the various techniques that are currently employed to produce lanthipeptides, a direct comparison between these methods to assess industrial applicability has not yet been described. In this review we provide a synoptic comparison of research efforts on total synthesis and in vivo biosynthesis aimed at fostering lanthipeptides production. We further examine current applications and propose measures to enhance product yields. Owing to their elaborate chemical structures, chemical synthesis of these biomolecules is economically less feasible for large-scale applications, and hence biological production seems to be the only realistic alternative.

Strategies to increase the hygienic and economic value of fresh fish: Biopreservation using lactic acid bacteria of marine origin.

In this work we describe the development of a biopreservation strategy for fresh fish based on the use of bacteriocinogenic LAB of marine origin. For this purpose, two multibacteriocinogenic LAB strains, Lactobacillus curvatus BCS35 and Enterococcus faecium BNM58, previously isolated from fish and fish products were selected owing to their capability to inhibit the growth of several fish-spoilage and food-borne pathogenic bacteria. Two commercially important fish species were chosen, young hake (Merluccius merluccius) and megrim (Lepidorhombus boscii), and the specimens were acquired at the Marín (Pontevedra, Spain) retail fish market, after one night in the chilled hold of a near-shore fishing vessel. The biopreservation potential and the application strategies of these two LAB strains were first tested at a laboratory scale, where several batches of fresh fish were inoculated with: (i) the multibacteriocinogenic LAB culture(s) as protective culture(s); and/or (ii) their cell-free culture supernatant(s) as food ingredient(s), and (iii) the lyophilized bacteriocin preparation(s) as lyophilized food ingredient(s). All batches were stored in polystyrene boxes, permanently filled with ice at 0-2 °C, for 14 days. Microbiological analyses, as well as sensorial analyses, were carried out during the biopreservation trials. Subsequently, Lb. curvatus BCS35 was selected to up-scale the trials, and combinations of the three application methods were assayed. For this purpose, this strain was grown in a semi-industrial scale fermentor (150l) in modified MRS broth, and three batches of fresh fish were inoculated with the protective culture and/or food ingredient, and stored on ice in a chilled chamber at 0-2 °C at the Marín retail fish market for 14 days. Microbiological analyses were carried out during the storage period, showing that when Lb. curvatus BCS35 culture or the corresponding cell-free culture supernatant was used as protective culture or food ingredient, respectively, bacterial counts were significantly lower than those of the untreated control batches, both for young hake and megrim. In addition, the presence of Listeria spp. in megrim was inhibited in both analyses. The effect of protective culture or food ingredient on the sensory characteristics of fish was evaluated by an official fish appraiser from the Marín retail fish market, who concluded that all the biopreserved batches were worth a higher price in the fish market than the respective control batches, demonstrating that the multibacteriocinogenic strain of marine origin Lb. curvatus BCS35 may be considered as a suitable candidate for its application as fresh fish biopreservative.

In vivo Toxicity Assessment of Antimicrobial Peptides (AMPs LR14) Derived from Lactobacillus plantarum Strain LR/14 in Drosophila melanogaster.

Lactic acid bacteria are known to produce antimicrobial peptides (AMPs) such as bacteriocins which can be employed to control pathogens and food spoilage microorganisms. However, their possible role as toxic agents against a eukaryotic system still remains unexplored. The present study deals with the in vivo evaluation of acute toxic effect of AMPs LR14, a mixture of AMPs isolated from Lactobacillus plantarum LR/14 on Drosophila melanogaster. The fly was used as a model system to measure the extent of toxicity of these peptides. The results showed that concentrations below 10 mg/ml are not significantly effective. When exposed to 10 mg/ml of AMPs LR14, acute toxic effect and a significant delay in the developmental cycle of the fly could be observed. Also, the weight and size of the flies were significantly reduced upon ingestion of these peptides. Higher concentrations (beyond 15 mg/ml) exerted a strong larvicidal effect. Detailed analysis on larval tissues and adult germ cells of the insect revealed deformity in cellular architecture, DNA fragmentation, and premature apoptosis, confirming that the peptides have a dose-dependent toxic property. Our studies provide the first information on the role of AMPs LR14 as an insecticidal agent.

Active polymers containing Lactobacillus curvatus CRL705 bacteriocins: effectiveness assessment in Wieners.

Bacteriocins from lactic acid bacteria have potential as natural food preservatives. In this study two active (synthetic and gluten) films were obtained by the incorporation of lactocin 705 and lactocin AL705, bacteriocins produced by Lactobacillus curvatus CRL705 with antimicrobial activity against spoilage lactic acid bacteria and Listeria. Antimicrobial film effectiveness was determined in Wieners inoculated with Lactobacillus plantarum CRL691 and Listeria innocua 7 (10(4)CFU/g) stored at 5°C during 45days. Active and control (absence of bacteriocins) packages were prepared and bacterial counts in selective media were carried out. Visual inspection and pH measurement of Wieners were also performed. Typical growth of both inoculated microorganisms was observed in control packages which reached 10(6)-10(7)CFU/g at the end of storage period. In the active packages, L. innocua 7 was effectively inhibited (2.5 log cycles reduction at day 45), while L. plantarum CRL691 was only slightly inhibited (0.5 log cycles) up to the second week of storage, then counts around 10(6)-10(7)CFU/g were reached. Changes in pH values from 6.3 to 5.8 were produced and gas formation was observed in active and control packages. The low inhibitory effectiveness against lactic acid bacteria is in correlation with the low activity observed for lactocin 705 in the presence of fat; Wieners fat content (20-30%) may adversely affect antimicrobial activity. This study supports the feasibility of using polymers activated with L. curvatus CRL705 bacteriocins to control Listeria on the surface of Wieners and highlights the importance of evaluating antimicrobial packaging systems for each particular food application.

Assessment of the bacteriocinogenic potential of marine bacteria reveals lichenicidin production by seaweed-derived Bacillus spp.

The objectives of this study were (1) to assess the bacteriocinogenic potential of bacteria derived mainly from seaweed, but also sand and seawater, (2) to identify at least some of the bacteriocins produced, if any and (3) to determine if they are unique to the marine environment and/or novel. Fifteen Bacillus licheniformis or pumilus isolates with antimicrobial activity against at least one of the indicator bacteria used were recovered. Some, at least, of the antimicrobials produced were bacteriocins, as they were proteinaceous and the producers displayed immunity. Screening with PCR primers for known Bacillus bacteriocins revealed that three seaweed-derived Bacillus licheniformis harbored the bli04127 gene which encodes one of the peptides of the two-peptide lantibiotic lichenicidin. Production of both lichenicidin peptides was then confirmed by mass spectrometry. This is the first definitive proof of bacteriocin production by seaweed-derived bacteria. The authors acknowledge that the bacteriocin produced has previously been discovered and is not unique to the marine environment. However, the other marine isolates likely produce novel bacteriocins, as none harboured genes for known Bacillus bacteriocins.

New developments and applications of bacteriocins and peptides in foods.

There is an increased desire for sophisticated foods, whereby consumers harbor higher expectations of health-promoting benefits above basic nutrition. Moreover, there is a move from the adulteration of foods with chemical preservatives toward biopreservation. Such expectations have led scientists to identify novel approaches to satisfy both demands, which utilize bacteriocin and peptide-based solutions. The best known examples of biopreservation involve bacteriocins. However, with the exception of nisin, bacteriocins have received limited use in the food industry. Peptides can be added to foods to improve consumer health. Some of the best known examples are angiotensin I-converting enzyme (ACE)-inhibitory peptides, which inhibit ACE, a key enzyme involved in blood pressure (BP) regulation. To be effective, these peptides must be bioavailable, but by their nature, peptides are degraded by digestion with proteolytic enzymes. This review critically discusses the use and potential of peptides and bacteriocins in food systems in terms of safety, quality, and improvement of human health.

Assessment of the in vitro efficacy of the novel antimicrobial peptide CECT7121 against human Gram-positive bacteria from serious infections refractory to treatment.

BACKGROUND: Resistant Gram-positive bacteria are causing increasing concern in clinical practice. This work investigated the efficacy of AP-CECT7121 (an antimicrobial peptide isolated from an environmental strain of Enterococcus faecalis CECT7121) against various pathogenic Gram-positive bacteria. METHODS: Strains were isolated from intensive care unit patients unresponsive to standard antibiotic treatments. Inhibitory activity of AP-CECT7121 was assessed using the agar-well diffusion method. The most resistant isolates from each species screened (Enterococcus faecium, Enterococcus faecalis,Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Clostridium perfringens and Clostridium difficile) were further examined in time-killing curve studies. RESULTS: These bactericidal kinetic experiments demonstrated a rapid killing effect with no viable bacteria being detected within 30 and 90 min for enterococcal and streptococcal strains and 180 min for community-acquired methicillin-resistant S. aureus and C. perfringens: viable counts for C. difficile were threefold decreased after 90 min. CONCLUSIONS: AP-CECT7121 may provide a novel strategy for treating potentially fatal clinical infections in hospitalized patients.

Real-time assessment of inflammation and treatment response in a mouse model of allergic airway inflammation.

Eosinophils are multifunctional leukocytes that degrade and remodel tissue extracellular matrix through production of proteolytic enzymes, release of proinflammatory factors to initiate and propagate inflammatory responses, and direct activation of mucus secretion and smooth muscle cell constriction. Thus, eosinophils are central effector cells during allergic airway inflammation and an important clinical therapeutic target. Here we describe the use of an injectable MMP-targeted optical sensor that specifically and quantitatively resolves eosinophil activity in the lungs of mice with experimental allergic airway inflammation. Through the use of real-time molecular imaging methods, we report the visualization of eosinophil responses in vivo and at different scales. Eosinophil responses were seen at single-cell resolution in conducting airways using near-infrared fluorescence fiberoptic bronchoscopy, in lung parenchyma using intravital microscopy, and in the whole body using fluorescence-mediated molecular tomography. Using these real-time imaging methods, we confirmed the immunosuppressive effects of the glucocorticoid drug dexamethasone in the mouse model of allergic airway inflammation and identified a viridin-derived prodrug that potently inhibited the accumulation and enzyme activity of eosinophils in the lungs. The combination of sensitive enzyme-targeted sensors with noninvasive molecular imaging approaches permitted evaluation of airway inflammation severity and was used as a model to rapidly screen for new drug effects. Both fluorescence-mediated tomography and fiberoptic bronchoscopy techniques have the potential to be translated into the clinic.

Inhibition of Listeria innocua and Brochothrix thermosphacta in vacuum-packaged meat by addition of bacteriocinogenic Lactobacillus curvatus CRL705 and its bacteriocins.

AIMS: To evaluate the inhibition effectiveness of Lactobacillus curvatus CRL705 used as a bioprotective culture and of its bacteriocins, lactocin 705 and lactocin AL705, against Listeria innocua, Brochothrix thermosphacta and indigenous lactic acid bacteria (LAB) in vacuum-packaged meat stored at 2 degrees C. METHODS AND RESULTS: The live culture of Lact. curvatus CRL705 as well as synthetic lactocin 705 and purified lactocin AL705 were shown to be similarly effective in preventing the growth of B. thermosphacta and L. innocua in meat discs in contrast to control samples in which these micro-organisms grew rapidly, their numbers increasing by 3.0- and 2.1-log cycles respectively. In addition, indigenous LAB population showed a lower growth rate in the presence of lactocin 705. Bacteriocin activity was detected in the meat discs during 36 days at 2 degrees C irrespective of the biopreservation strategy applied. Changes in pH were not significantly different in meat discs treated with the protective culture when compared with control samples. CONCLUSIONS: Lactobacillus curvatus CRL705 and the produced bacteriocins, lactocin 705 and lactocin AL 705, were effective in inhibiting L. innocua and B. thermosphacta. The use of the bioprotective culture in refrigerated vacuum-packaged fresh meat would be more feasible from an economic and legal point of view. SIGNIFICANCE AND IMPACT OF THE STUDY: Establishment of biopreservation as a method to ensure the microbiological safety of vacuum-packaged fresh meat at 2 degrees C.

Data mining and characterization of a novel pediocin-like bacteriocin system from the genome of Pediococcus pentosaceus ATCC 25745.

The genome of Pediococcus pentosaceus ATCC 25745 contains a gene cluster that resembles a regulated bacteriocin system. The gene cluster has an operon-like structure consisting of a putative pediocin-like bacteriocin gene (termed penA) and a potential immunity gene (termed peiA). Genetic determinants involved in bacteriocin transport and regulation are also found in proximity to penA and peiA but the so-called accessory gene involved in transport and the inducer gene involved in regulation are missing. Consequently, this bacterium is a poor bacteriocin producer. To analyse the potency of the putative bacteriocin operon, the two genes penA-peiA were heterologously expressed in a Lactobacillus sakei host that contains the complete apparatus for gene activation, maturation and externalization of bacteriocins. It was demonstrated that the heterologous host expressing penA and peiA produced a strong bacteriocin activity; in addition, the host became immune to its own bacteriocin, identifying the gene pair penA-peiA as a potent bacteriocin system. The novel pediocin-like bacteriocin, termed penocin A, has an isotopic mass [M+H]+ of 4684.6 Da as determined by mass spectrometry; this value corresponds well to the expected size of the mature 42 aa peptide containing a disulfide bridge. The bacteriocin is heat-stable but protease-sensitive and has a calculated pI of 9.45. Penocin A has a relatively broad inhibition spectrum, including pathogenic Listeria and Clostridium species. Immediately upstream of the regulatory genes reside some features that resemble remnants of a disrupted inducer gene. This degenerate gene was restored and shown to encode a double-glycine leader-containing peptide. Furthermore, expression of the restored gene triggered high bacteriocin production in P. pentosaceus ATCC 25745, thus confirming its role as an inducer in the pen regulon.

Frequency of bacteriocin resistance development and associated fitness costs in Listeria monocytogenes.

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in Listeria monocytogenes to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10(-6). However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10(-7) to 10(-2). Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10 degrees C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 x 10(-8)). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5 degrees C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5 degrees C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.

A comparative study on the use of flow cytometry and colony forming units for assessment of the antibacterial effect of bacteriocins.

Flow cytometry was investigated as a rapid method to determine the antibacterial effect of the bacteriocins nisin, pediocin PA-1, and sakacin A on the indicator organisms Lactobacillus reuteri DSM 12246, Lactobacillus sakei NCFB 2714 and Lactobacillus sakei DSM 20017, respectively. Fluorescence intensities of the cells were measured by flow cytometry upon exposure to bacteriocins after staining with carboxyfluorescein diacetate (cFDA) and were compared to the number of colony forming units (CFU). The fluorescence index (FI) of the bacterial populations decreased when exposed to the bacteriocins. For the different bacteriocins the pattern of decreases in FI and colony forming units differed at equal bacteriostatic concentrations. FI was the most sensitive measure of bacteriocin activity, i.e. the decrease in FI was observed at lower bacteriocin concentrations than decrease in CFU. It was demonstrated that the decrease in FI was caused by rapid leakage of carboxyfluorescein from cells exposed to pediocin. Cells showing severe leakage after pediocin treatment could be detected as CFU when transferred to a rich medium. Such a repair was less pronounced for cells exposed to sakacin and very limited for cells exposed to nisin. The influence of temperature and NaCl in combination with pediocin on FI and CFU of Lactobacillus sakei NCFB 2714 was examined at conditions relevant to foods. At all temperatures (5, 10, 20 and 37 degrees C) and NaCl concentrations (0, 2 and 4% w/v) investigated the flow cytometric measurements were significantly more sensitive compared to CFU. Both methods showed that the inhibitory effect of pediocin increased with increasing temperatures and decreased with increasing NaCl concentrations.