Enterocin AS-48 Research Articles

Enterocins Produced by Enterococci Isolated from Breast-Fed Infants: Antilisterial Potential.

Enterocins are bacteriocins synthesized by Enterococcus strains that show an interesting antimicrobial effectiveness against foodborne pathogens such as Listeria monocytogenes. The objectives of this study were to identify and analyze the expression of enterocin genes of Enterococcus isolated from breast-fed infants and evaluate their ability to inhibit three human isolates of virulent L. monocytogenes, as well as some probiotic bacteria. The susceptibility of the strains of L. monocytogenes to fifteen antibiotics was tested, detecting their resistance to cefoxitin (constitutively resistant), oxacillin, and clindamycin. The production of enterocins A, B, and P was observed in Enterococcus faecium isolates, while enterocin AS-48 was detected in an Enterococcus faecalis isolate. AS-48 showed antilisterial activity by itself, while the joint action of enterocins A and B or B and P was necessary for inhibiting L. monocytogenes, demonstrating the synergistic effect of those combinations. The presence of multiple enterocin genes does not assure the inhibition of L. monocytogenes strains. However, the expression of multiple enterocin genes showed a good correlation with the inhibition capacity of these strains. Furthermore, the potential beneficial strains of lactobacilli and bifidobacteria examined were not inhibited by any of the enterocins produced individually or in combination, with the exception of Bifidobacterium longum BB536, which was inhibited by enterocin AS-48 and the joint production of enterocins A and B or B and P. The enterocins studied here could be candidates for developing alternative treatments against antibiotic-resistant bacterial infections. Moreover, these selected enterocin-producing E. faecium strains isolated from breast-fed infants could be used as probiotic strains due to their antilisterial effect, as well as the absence of virulence factors.

Engineering circular bacteriocins: structural and functional effects of α-helix exchanges and disulfide introductions in circularin A.

Circular bacteriocins form a distinct group of antimicrobial peptides (AMPs) characterized by their unique head-to-tail ligated circular structure and functional properties. They belong to the ribosomally synthesized and post-translationally modified peptide (RiPP) family. The ribosomal origin of these peptides facilitates rapid diversification through mutations in the precursor genes combined with specific modification enzymes. In this study, we primarily explored the bacteriocin engineering potential of circularin A, a circular bacteriocin produced by Clostridium beijerinckii ATCC 25752. Specifically, we employed strategies involving α-helix replacements and disulfide bond introductions to investigate their effects on both biosynthesis and bioactivity of the bacteriocin. The results show the feasibility of peptide engineering to introduce certain structural properties into circularin A through carefully designed approaches. The introduction of cysteines for potential disulfide bonds resulted in a substantial reduction in bacteriocin biosynthesis and/or bioactivity, indicating the importance of maintaining dynamic flexibility of α-helices in circularin A, while reduction of the potential disulfide in one case increased the activity. The 5 α-helices of circularin A were respectively replaced by corresponding helices from another circular peptide, enterocin AS-48, and modestly active peptides were obtained in a few cases. Overall, this study provides valuable insights into the engineering potential of circular bacteriocins as antimicrobial agents, including their structural and functional restrictions and their suitability as peptide engineering scaffolds. This helps to pave the way for the development of novel antimicrobial peptides with tailored properties based on circular bacteriocins.

COMBATTING METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN THE FOOD INDUSTRY BY HARNESSING THE POWER OF NATURE: A SYSTEMATIC REVIEW

Antibiotic resistance is a critical global health concern, with Methicillin-resistant Staphylococcus aureus (MRSA) posing a significant challenge due to its resistance to commonly used antibiotics. Recent research has revealed the potential of natural compounds and microorganisms in combatting MRSA and other antibiotic-resistant bacteria. In this systematic review, we studied the effect of essential oils, bacteriophages, bacteriocins, and probiotics on S. aureus, including MRSA in particular, in the food industry. Essential oils (EOs) have gained significant attention because of their antimicrobial properties, inhibiting MRSA growth by damaging bacterial cells and inhibiting essential enzymes and compounds. Cinnamon oil liposomes caused the most significant decrease in MRSA populations among our reviewed essential oils. Bacteriophages can lyse the bacterial host. They encode peptidoglycan hydrolases called endolysins that target the bacterial cell wall. In our study, S. aureus phage (containing CHAPLysGH15 and LysGH15), and phage SA11 endolysin (LysSA11) were the most effective against S. aureus. Bacteriocins, antimicrobial peptides produced by bacteria, also show potential in combatting MRSA, mainly by generating organic acids that interfere with bacterial metabolism. According to our review, the most effective bacteriocins against S. aureus were Enterocin AS-48 with phenolic compounds or with 2NPOH, Bacteriocin isolated from Lactobacillus pentosus - Pentocin JL-1, and bacteriocin produced by S. pasteuri RSP-1, respectively. Probiotics can compete with pathogens by producing antimicrobial compounds that disrupt MRSA cell production and ultimately lead to bacterial death. In our review, the most effective probiotics were Streptomyces griseus, Pediococcus acidilactici strains A11 and C12, Lactococcus lactis, and Lactobionic acid respectively. A multi-hurdle approach combining these natural agents has shown promising results in targeting and eliminating MRSA cells. By harnessing the power of nature, we can potentially overcome the challenges posed by MRSA and other antibiotic-resistant bacteria.

Determination of the effect of the bacteriocin enterocin AS-48 on the microbial loads and bacterial diversity of blueberries

Fresh fruits have been involved in transmission of foodborne pathogens. In the present work, five different batches of blueberries were used. One aliquot from each batch was washed with sterile saline solution (SSS) and the other one with a solution of the circular bacteriocin enterocin AS-48 in SSS. Then, the surface microbiota of controls and bacteriocin-treated samples was recovered and used for microbiota analyses, both using viable counts and high-throughput amplicon sequencing. Total aerobic mesophilic loads ranged from 2.70 to 4.09 log CFU/g in most of the samples. Only two samples yielded detectable viable counts on selective media (Enterobacteriaceae, presumptive Salmonella and coliforms), with values ranging from 2.84 to 3.81 log CFU/g. The bacteriocin treatment reduced viable cell counts of total aerobic mesophiles to a range of 1.40–1.88 log CFU/g. No viable cells were detected on selective media. Amplicon sequencing indicated large batch-to-batch variations in the surface microbiota of blueberries and also an effect of the bacteriocin treatment on microbiota composition.

Rationally Designed Minimal Bioactive Domains of AS-48 Bacteriocin Homologs Possess Potent Antileishmanial Properties.

Leishmaniasis, a category I neglected tropical disease, is a group of diseases caused by the protozoan parasite Leishmania species with a wide range of clinical manifestations. Current treatment options can be highly toxic and expensive, with drug relapse and the emergence of resistance. Bacteriocins, antimicrobial peptides ribosomally produced by bacteria, are a relatively new avenue for potential antiprotozoal drugs. Particular interest has been focused on enterocin AS-48, with previously proven efficacy against protozoan species, including Leishmania spp. Sequential characterization of enterocin AS-48 has illustrated that antibacterial bioactivity is preserved in linearized, truncated forms; however, minimal domains of AS-48 bacteriocins have not yet been explored against protozoans. Using rational design techniques to improve membrane penetration activity, we designed peptide libraries using the minimal bioactive domain of AS-48 homologs. Stepwise changes to the charge (z), hydrophobicity (H), and hydrophobic dipole moment (μH) were achieved through lysine and tryptophan substitutions and the inversion of residues within the helical wheel, respectively. A total of 480 synthetic peptide variants were assessed for antileishmanial activity against Leishmania donovani. One hundred seventy-two peptide variants exhibited 50% inhibitory concentration (IC50) values below 20 μM against axenic amastigotes, with 60 peptide variants in the nanomolar range. Nine peptide variants exhibited potent activity against intracellular amastigotes with observed IC50 values of <4 μM and limited in vitro host cell toxicity, making them worthy of further drug development. Our work demonstrates that minimal bioactive domains of naturally existing bacteriocins can be synthetically engineered to increase membrane penetration against Leishmania spp. with minimal host cytotoxicity, holding the promise of novel, potent antileishmanial therapies. IMPORTANCE Leishmaniasis is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. There are three primary clinical forms, cutaneous, mucocutaneous, and visceral, with visceral leishmaniasis being fatal if left untreated. Current drug treatments are less than ideal, especially in resource-limited areas, due to the difficult administration and treatment regimens as well as the high cost and the emergence of drug resistance. Identifying potent antileishmanial agents is of the utmost importance. We utilized rational design techniques to synthesize enterocin AS-48 and AS-48-like bacteriocin-based peptides and screened these peptides against L. donovani using a fluorescence-based phenotypic assay. Our results suggest that bacteriocins, specifically these rationally designed AS-48-like peptides, are promising leads for further development as antileishmanial drugs.

Enterocin Cross-Resistance Mediated by ABC Transport Systems.

In their struggle for life, bacteria frequently produce antagonistic substances against competitors. Antimicrobial peptides produced by bacteria (known as bacteriocins) are active against other bacteria, but harmless to their producer due to an associated immunity gene that prevents self-inhibition. However, knowledge of cross-resistance between different types of bacteriocin producer remains very limited. The immune function of certain bacteriocins produced by the Enterococcus genus (known as enterocins) is mediated by an ABC transporter. This is the case for enterocin AS-48, a gene cluster that includes two ABC transporter-like systems (Transporter-1 and 2) and an immunity protein. Transporter-2 in this cluster shows a high similarity to the ABC transporter-like system in MR10A and MR10B enterocin gene clusters. The aim of our study was to determine the possible role of this ABC transporter in cross-resistance between these two different types of enterocin. To this end, we designed different mutants (Tn5 derivative and deletion mutants) of the as-48 gene cluster in Enterococcus faecalis and cloned them into the pAM401 shuttle vector. Antimicrobial activity assays showed that enterocin AS-48 Transporter-2 is responsible for cross-resistance between AS-48 and MR10A/B enterocin producers and allowed identification of the MR10A/B immunity gene system. These findings open the way to the investigation of resistance beyond homologous bacteriocins.

Synthetic Peptide Libraries Designed From a Minimal Alpha-Helical Domain of AS-48-Bacteriocin Homologs Exhibit Potent Antibacterial Activity.

The circularized bacteriocin enterocin AS-48 produced by Enterococcus sp. exhibits antibacterial activity through membrane disruption. The membrane-penetrating activity of enterocin AS-48 has been attributed to a specific alpha-helical region on the circular peptide. Truncated, linearized forms containing these domains have been shown to preserve limited bactericidal activity. We utilized the amino acid sequence of the active helical domain of enterocin AS-48 to perform a homology-based search of similar sequences in other bacterial genomes. We identified similar domains in three previously uncharacterized AS-48-like bacteriocin genes in Clostridium sordellii, Paenibacillus larvae, and Bacillus xiamenensis. Enterocin AS-48 and homologs from these bacterial species were used as scaffolds for the design of a minimal peptide library based on the active helical domain of each bacteriocin sequence. 95 synthetic peptide variants of each scaffold peptide, designated Syn-enterocin, Syn-sordellicin, Syn-larvacin, and Syn-xiamensin, were designed and synthesized from each scaffold sequence based on defined biophysical parameters. A total of 384 total peptides were assessed for antibacterial activity against Gram-negative and Gram-positive bacteria. Minimal Inhibitory Concentrations (MICs) as low as 15.6 nM could be observed for the most potent peptide candidate tested, with no significant cytotoxicity to eukaryotic cells. Our work demonstrates for the first time a general workflow of using minimal domains of natural bacteriocin sequences as scaffolds to design and rapidly synthesize a library of bacteriocin-based antimicrobial peptide variants for evaluation.

Mutagenicity and genotoxicity assessment of a new biopreservative product rich in Enterocin AS-48

A biopreservative derived from the fermentation of a dairy byproduct by Enterococcus faecalis UGRA10 strains being developed. This product possesses a strong and wide antibacterial spectrum mainly due to the presence of Enterocin AS-48 in its composition. To assess its potential as food additive, the mutagenicicity and genotoxicity has been assayed by means of the bacterial reverse-mutation assay in Salmonella typhimurium TA97A, TA98, TA100, TA102, TA1535 strains (Ames test, OECD 471, 2020) and the micronucleus test (MN) (OECD 487, 2016) in L5178Y/Tk ± cells. The results in the Ames test after exposure to the byproduct (6.75–100 μg/plate) with absence and presence of the metabolic activation system from rat liver (S9 fraction), revealed not mutagenicity at the conditions tested. For the MN test, the exposition to five enterocin AS-48 concentrations (0.2–1 μg/μl) was tested in the absence and presence of S9 fraction, with no evidence of genotoxicity. Negative results in the mutagenicity and genotoxicity assays point out the good safety profile of the byproduct and support its use as additive. Further toxicological studies are required before its approval and commercial application.

Subchronic toxicity study in BALBc mice of enterocin AS-48, an anti-microbial peptide produced by Enterococcus faecalis UGRA10

Few studies have examined the use of animal models to evaluate the in-vivo toxicity of antimicrobial peptides, but such research is essential to their safe use in foods. This study was performed to evaluate any adverse effects of enterocin AS-48, a circular bacteriocin produced by Enterococcus strains, when administered to BALB/c mice at concentrations of 50, 100, and 200 mg/kg in the diet for 90 days. Animals dosed with nisin at a dietary concentration of 200 mg/kg served as a reference treated group.There were no deaths in any of the animal groups, and the AS-48 treatment produced no abnormalities or clinical signs on body weights, food consumption, urinalysis, haematology, or blood biochemistry. Furthermore, there were no significant differences in the weights of liver, spleen, heart, kidneys, and intestines between control mice and those treated with AS-48 or nisin. The histopathological study showed moderate vacuolar degeneration in hepatocytes of some animals fed 100 or 200 mg/kg AS-48 (3/10 and 2/10 respectively). However, this anomaly was lower than in the group treated with nisin (5/10). Conclusively, no toxicologically significant changes were associated in BALB/c mice fed with 50, 100, and 200 mg/kg enterocin AS-48 for 90 days.

Bacterial Inactivation by Using Plastic Materials Activated with Combinations of Natural Antimicrobials

Natural antimicrobials have gained interest as possible inhibitors of biofilm formation. The aim of the present study was to determine the efficacy of antimicrobials derived from essential oils (carvacrol, thymol) plus bacteriocin AS-48 immobilized on two plastic supports (low density polyethylene and polyethylene–polyamide films) on bacterial inactivation. The polyethylene–polyamide vacuum-packaging plastic film activated with a combination of thymol plus enterocin AS-48 was the most effective in reducing the concentrations of viable planktonic and sessile cells for Listeria innocua, Lactobacillus fructivorans, Bacillus coagulans, and Bacillus licheniformis. Results from the study highlight the potential of polyethylene–polyamide film activated with thymol plus enterocin AS-48 for reducing the viable cell concentrations of spoilage Gram-positive bacteria and Listeria in both planktonic and sessile states.

Effect of high hydrostatic pressure and activated film packaging on bacterial diversity of fruit puree

The aim of the present study was to determine the effect of high hydrostatic pressure (HP) and films activated with a combination of enterocin AS-48 and thymol (AF) on the microbial load and bacterial diversity of fruit puree (banana, apple, pear). HP and AF were applied singly, or in combination (AFHP). Samples were chill-stored. HP and AFHP treatments reduced viable counts significantly (P ≤ 0.05). The predominant bacterial groups in the puree were fam. Comamonadaceae (23.17%) and genera Methylobacterium (21.46%), Acidovorax (8.70%) and Sphingomonas (6.63%). Pseudomonas became relevant by the end of storage (10.50%). Most of the AF samples had higher relative abundances of Comamonadaceae, Methylobacterium, Acidovorax and Sphingomonas and lower relative abundance of Pseudomonas. Application of HP treatment markedly reduced the relative abundances of Comamonadaceae, Methylobacterium, Acidovorax and Sphingomonas. Lactobacillales increased in relative abundance upon application of HP treatment but not during storage, while Pseudomonas increased towards the end of storage. The combined treatment (AFHP) achieved greatest reduction in the relative abundances of Comamonadaceae and Methylobacterium. Results indicate that AFHP treatment had greatest effects on the bacterial diversity of the fruit puree and was also the most effective in keeping total aerobic mesophiles and Enterobacteriaceae below detectable levels.

Effects of Enterococcus faecalis UGRA10 and the enterocin AS-48 against the fish pathogen Lactococcus garvieae. Studies in vitro and in vivo

The aim of this study was to evaluate the effects of Enterococcus faecalis UGRA10 and its enterocin AS-48 against the fish pathogen Lactococcus garvieae. The minimum bactericidal concentrations of AS-48 against L. garvieae CECT 5807, 5806, and 5274 were 15.62, 15.62, and 7.81 μg/ml respectively. In broth cultures, enterocin at 100, 50, and 25 μg/ml reduced 108 CFU/ml lactococci after 2, 5, and 10 h, respectively. In co-cultures of UGRA10/L. garvieae at a 1/10 CFU/ml ratio, lactococci were eliminated after 24 h. Studies on UGRA10 biosafety and AS-48 toxicity in R1 cells and in rainbow trout have shown a lack of adverse effects from both the strain and bacteriocin. Trout challenged with L. garvieae and UGRA10 administered in diet 30 days before infection had a cumulative survival rate of 50% compared with 0% for control fish. Trout inoculated with the pathogen and treated by regular dipping in AS-48 baths had a survival rate of 60% after 20 days compared with that of untreated fish (0%). These results indicate the protective effect of the UGRA10 strain and the bacteriocin AS-48 against L. garvieae and the potential of these natural products as alternatives to antibiotics for controlling diseases in aquaculture.

Treatment With High-Hydrostatic Pressure, Activated Film Packaging With Thymol Plus Enterocin AS-48, and Its Combination Modify the Bacterial Communities of Refrigerated Sea Bream (Sparus aurata) Fillets.

The aim of this study was to determine the impact of activated plastic films with thymol and enterocin AS-48 and high-hydrostatic pressure (HP) treatment on the bacterial load and bacterial diversity of vacuum-packaged sea bream fillets under refrigerated storage for 10 days. The activated film and the HP treatment reduced aerobic mesophiles viable counts by 1.46 and 2.36 log cycles, respectively, while the combined treatment achieved a reduction of 4.13 log cycles. HP and combined treatments resulted in longer delays in bacterial growth. Proteobacteria were the dominant phyla in sea bream fillets. The relative abundance of Firmicutes increased by the end of storage both in controls and in samples treated by HP singly or in combination with the activated films. The predominant operational taxonomic units (OTUs) found at time 0 in control samples (Listeria, Acinetobacter, Pseudomonas, Enterobacteriaceae, Chryseobacterium) rapidly changed during storage (with an increase of Vibrio, Photobacterium, and Shewanella together with Cloacibacterium and Lactobacillales by the end of storage). The activated film and the HP treatment induced drastic changes in bacterial diversity right after treatments (with Comamonadaceae, Methylobacterium, Acidovorax, and Sphingomonas as main OTUs) and also induced further modifications during storage. Bacterial diversity in activated film samples was quite homogeneous during storage (with Vibrio, Photobacterium, and Shewanella as main OTUs) and approached control samples. HP treatments (singly or in combination with activated films) determined a high relative abundance of Acinetobacter (followed by Pseudomonas and Shewanella) during early storage as well as a higher relative abundance of lactic acid bacteria by the end of storage. The results indicate that the complex dynamics of bacterial populations in the refrigerated sea bream fillets are markedly influenced by treatment and antimicrobials applied.

Inactivation of Listeria in Foods Packed in Films Activated with Enterocin AS-48 plus Thymol Singly or in Combination with High-Hydrostatic Pressure Treatment

The aim of the present study was to determine the efficacy of films activated with enterocin AS-48 plus thymol singly, or in combination with high-hydrostatic pressure (HHP) on the inactivation of Listeria innocua in sea bream fillets and in fruit puree stored under refrigeration for 10 days. L. innocua proliferated in control fish fillets during storage. The activated film reduced viable Listeria counts in fillets by 1.76 log cycles and prevented growth of survivors until mid-storage. Application of HHP treatment to fillets packed in films without antimicrobials reduced Listeria counts by 1.83 log cycles, but did not prevent the growth of survivors during storage. The combined treatment reduced viable counts by 1.88 log cycles and delayed growth of survivors during the whole storage period. L. innocua survived in puree during storage. The activated film reduced Listeria counts by 1.80 and 2.0 log cycles at days 0 and 3. After that point, Listeria were below the detection limit. No viable Listeria were detected in the purees after application of HHP treatment singly, or in combination with the activated film. Results from the study indicate that the efficacy of activated films against Listeria is markedly influenced by the food type.

Application of enterocin AS-48 as biopreservative in eggs and egg fractions: Synergism through lysozyme

The efficacy of enterocin AS-48 in controlling Bacillus cereus and Staphylococcus aureus in liquid whole egg (LWE), egg white (EW), and egg yolk (EY) at 4 and 28 °C was studied. In LWE, AS-48 yields significant reductions in viable counts of B. cereus with minimum bactericidal concentrations (MBC) of 5 and 0.05 μg/ml respectively at 4 and 28 °C (P < 0.05 and P < 0.01, respectively), notably less than that previously determined in brain heart infusion broth. Results obtained with egg fractions suggest the presence of a compound in EW acting synergistically with AS-48 since, at 4 °C, the MBC for AS-48 was established at 1–2.5 μg/ml. Similar results were obtained for S. aureus in LWE and EW, although the AS-48 MBCs were higher: 5–10 and 1–5 μg/ml respectively at 4 and 28 °C in LWE and 10 μg/ml at 28 °C in EW. In EY, both bacteria showed a much lower susceptibility to AS-48 than in LWE and EW. The combination of lysozyme (8 mg/ml) with AS-48 exerted a synergistic inhibitory effect against B. cereus and S. aureus although this synergy was lower than observed in LWE and EW. This synergy has been also confirmed by electron microscopy observation.

Analysis of the microbiota of refrigerated chopped parsley after treatments with a coating containing enterocin AS-48 or by high-hydrostatic pressure

Parsley can be implicated in foodborne illness, yet chopped parsley is used as an ingredient or garnish for multiple dishes. The aim of the present study was to determine the effect of two different treatments on the bacterial diversity of parsley: (i) coating with a pectin-EDTA solution containing the circular bacteriocin enterocin AS-48, and (ii) treatment by high hydrostatic pressure (HHP) at 600MPa for 8min. Control and treated parsley were stored in trays at 5°C for 10days. Both treatments reduced viable counts by 3.7 log cycles and retarded growth of survivors during storage. The bacterial diversity of the chopped parsley was studied by high throughput sequencing (Illumina Miseq). Bacterial diversity of control samples mainly consists of Proteobacteria (96.87%) belonging to genera Pseudomonas (69.12%), Rheinheimera (8.56%) and Pantoea (6.91%) among others. During storage, the relative abundance of Bacteroidetes (mainly Flavobacterium and Sphingobacterium) increased to 26.66%. Application of the pectin-bacteriocin-EDTA coating reduced the relative abundance of Proteobacteria (63.75%) and increased that of Firmicutes (34.70%). However, the relative abundances of certain groups such as Salmonella, Shigella and Acinetobacter increased at early storage times. Late storage was characterized by an increase in the relative abundance of Proteobacteria, mainly Pseudomonas. Upon application of HHP treatment, the relative abundance of Proteobacteria was reduced (85.88%) while Actinobacteria increased (8.01%). During early storage of HHP-treated samples, the relative abundance of Firmicutes increased. Potentially-pathogenic bacteria (Shigella) only increased in relative abundance by the end of storage. Results of the present study indicate that the two treatments had different effects on the bacterial diversity of parsley. The HHP treatment provided a safer product, since no potentially-pathogenic bacteria were detected until the end of the storage period.

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

We report the feasibility of enterocin AS-48, a circular cationic peptide produced by Enterococcus faecalis, as a new leishmanicidal agent. AS-48 is lethal to Leishmania promastigotes as well as to axenic and intracellular amastigotes at low micromolar concentrations, with scarce cytotoxicity to macrophages. AS-48 induced a fast bioenergetic collapse of L. donovani promastigotes but only a partial permeation of their plasma membrane with limited entrance of vital dyes, even at concentrations beyond its full lethality. Fluoresceinated AS-48 was visualized inside parasites by confocal microscopy and seen to cause mitochondrial depolarization and reactive oxygen species production. Altogether, AS-48 appeared to have a mixed leishmanicidal mechanism that includes both plasma membrane permeabilization and additional intracellular targets, with mitochondrial dysfunctionality being of special relevance. This complex leishmanicidal mechanism of AS-48 persisted even for the killing of intracellular amastigotes, as evidenced by transmission electron microscopy. We demonstrated the potentiality of AS-48 as a new and safe leishmanicidal agent, expanding the growing repertoire of eukaryotic targets for bacteriocins, and our results provide a proof of mechanism for the search of new leishmanicidal bacteriocins, whose diversity constitutes an almost endless source for new structures at moderate production cost and whose safe use on food preservation is well established.

Characteristics and potential applications of circular bacteriocins

Abstract Bacteriocins are ribosomally synthesized peptides or proteins exerting anatagonistic activity toward organisms which are closely related to the producer strain. Circular bacteriocins are produced by Gram-positive bacteria, mainly lactic acid bacteria, and to a lesser extent by Bacillus, Clostridium and Staphylococcus genera. These bacteriocins are characterized by the head-to-tail cyclization of their backbone. The circular nature of these peptides makes them resistant to many proteolytic enzymes and provides great thermal and pH stability. Circular bacteriocins are divided into 2 subgroups based on their physicochemical properties and sequence identity. These bacteriocins are synthesized as linear precursors with a leader sequence which is cleaved off during maturation. The mature circular peptides are composed of 58–70 amino acid residues. Biosynthesis of circular bacteriocins requires three stages: cleavage of the leader sequence, circularization and export out of the cell. Circular bacteriocins have broad antimicrobial activity spectrum, including many food spoilage bacteria and pathogens, such as Listeria, Staphylococcus and Clostridum spp. Circular bacteriocins permeabilize the membrane of sensitive bacteria, causing loss of ions and dissipation of the membrane potential, and finally cell death. Enterocin AS-48 was the first identified circular bacteriocin and is best characterized so far. Circular bacteriocins or bacteriocin-producing lactic acid bacteria have great potential in food preservation, and possibly in pharmaceutical and cosmetic industries. Thanks to their properties, circular bacteriocins could be an alternative not only to preservatives and methods used to provide microbial food safety presently, but also to less stable, linear bacteriocins. 1. Characteristics and classification of circular bacteriocins. 2. Genetics of circular bacteriocins. 3. Biosynthesis of circular bacteriocins. 4. Structure of circular bacteriocins. 5. Modes of action of circular bacteriocins. 6. Enterocin AS-48. 7. Potential applications of circular bacteriocins. 8. Summary

Phenotypic and Genotypic Characterization of Bacteriocinogenic Enterococci Against Clostridium botulinum.

The present study aimed to characterize Enterococcus faecalis (n=-6) and Enterococcus faecium (n=1) isolated from healthy chickens to find a novel perspective probiotic candidate that antagonize Clostridium botulinum types A, B, D, and E. The isolated enterococci were characterized based on phenotypic properties, PCR, and matrix-assisted laser desorption/ionization time of flight (MALDI-TOF). The virulence determinants including hemolytic activity on blood agar, gelatinase activity, sensitivity to vancomycin, and presence of gelatinase (gelE) and enterococcal surface protein (esp) virulence genes were investigated. Also, the presence of enterocin structural genes enterocin A, enterocin B, enterocin P, enterocin L50A/B, bacteriocin 31, enterocin AS48, enterocin 1071A/1071B, and enterocin 96 were assessed using PCR. Lastly, the antagonistic effect of the selected Enterococcus spp. on the growth of C. botulinum types A, B, D, and E was studied. The obtained results showed that four out of six E. faecalis and one E. faecium proved to be free from the tested virulence markers. All tested enterococci strains exhibited more than one of the tested enterocin. Interestingly, E. faecalis and E. faecium significantly restrained the growth of C. botulinum types A, B, D, and E. In conclusion, although, the data presented showed that bacteriocinogenic Enterococcus strains lacking of virulence determinants could be potentially used as a probiotic candidate against C. botulinum in vitro; however, further investigations are still urgently required to verify the beneficial effects of the tested Enterococcus spp. in vivo.

Biofilms formed by microbiota recovered from fresh produce: Bacterial biodiversity, and inactivation by benzalkonium chloride and enterocin AS-48

Microbiota recovered from fresh produce (Romaine lettuce, endives and cucumbers) was allowed to form biofilms on stainless steel coupons. The formed biofilms were treated with benzalkonium chloride (BC) at three different concentrations (0.01, 0.1, and 1.0 g/l), enterocin AS-48 (50 μg/ml) and combinations of BC and enterocin AS-48. The single treatment with bacteriocin had no effect on viability of sessile bacteria. A high concentration of BC (1.0 g/l) was required to achieve 4.1 logs reduction of viable cell counts. The combination of BC (1.0 g/l) and enterocin AS-48 reduced viable cell counts below detectable levels. High-throughput sequencing analysis revealed that the formed biofilms were composed mainly by Proteobacteria of the genera Pseudomonas and Stenotrophomonas. Treatments with enterocin AS-48 and BC at sub-inhibitory concentrations only induced minor changes in the relative abundance of the different bacterial groups associated with biofilms.