Circular and L50-like leaderless enterocins share a common ABC-transporter immunity gene.

Microbes live within complex communities of interacting populations, either free-living in waters and soils or symbionts of animals and plants. Their interactions include the production of antimicrobial peptides (bacteriocins) to antagonize competitors, and these producers must carry their own immunity gene for self-protection. Whether other coexisting populations are sensitive or resistant to the bacteriocin producer will be key for the population dynamics within the microbial community. The immunity gene frequently consists of an ABC transporter to repel its own bacteriocin but rarely protects against a nonrelated bacteriocin. A case where this cross-resistance occurs mediated by a shared ABC transporter has been shown between enterocins MR10A/B and AS-48. The first is an L50-like leaderless enterocin, while AS-48 is a circular enterocin. In addition, L50-like enterocins such as MR10A/B have been found in E. faecalis and E. faecium, but AS-48 appears only in E. faecalis. Thus, using the ABC transporter of the enterocin MR10A/B gene cluster of Enterococcus faecalis MRR10-3 as a cross-resistance model, we aimed to unravel to what extent a particular ABC transporter can be shared across multiple bacteriocinogenic bacterial populations. To this end, we screened the MR10A/B-ABC transporters in available microbial genomes and analyzed their sequence homologies and distribution. Overall, our main findings are as follows: (i) the MR10A/B-ABC transporter is associated with multiple enterocin gene clusters; (ii) the different enterocins associated with this transporter have a saposin-like fold in common; (iii) the Mr10E component of the transporter is more conserved within its associated enterocin, while the Mr10FGH components are more conserved within the carrying species. This is the least known component of the transporter, but it has shown the greatest specificity to its corresponding enterocin. Bacteriocins are now being investigated as an alternative to antibiotics; hence, the wider or narrower distribution of the particular immunity gene should be taken into account for clinical applications to avoid the selection of resistant strains. Further research will be needed to investigate the mechanistic interactions between the Mr10E transporter component and the bacteriocin as well as the specific ecological and evolutionary mechanisms involved in the spread of the immunity transporter across multiple bacteriocins.

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.

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.

The bacteriocin AS-48 requires dimer dissociation followed by hydrophobic interactions with the membrane for antibacterial activity.

The molecular mechanism underlining the antibacterial activity of the bacteriocin AS-48 is not known, and two different and opposite alternatives have been proposed. Available data suggested that the interaction of positively charged amino acids of AS-48 with the membrane would produce membrane destabilization and disruption. Alternatively, it has been proposed that AS-48 activity could rely on the effective insertion of the bacteriocin into the membrane. The biological and structural properties of the AS-48G13K/L40K double mutant were investigated to shed light on this subject. Compared with the wild type, the mutant protein suffered an important reduction in the antibacterial activity. Biochemical and structural studies of AS-48G13K/L40K mutant suggest the basis of its decreased antimicrobial activity. Lipid cosedimentation assays showed that the membrane affinity of AS-48G13K/L40K is 12-fold lower than that observed for the wild type. L40K mutation is responsible for this reduced membrane affinity and thus, hydrophobic interactions are involved in membrane association. Furthermore, the high-resolution crystal structure of AS-48G13K/L40K, together with the study of its dimeric character in solution showed that G13K stabilizes the inactive water-soluble dimer, which displays a reduced dipole moment. Our data suggest that the cumulative effect of these three affected properties reduces AS-48 activity, and point out that the bactericidal effect is achieved by the electrostatically driven approach of the inactive water-soluble dimer towards the membrane, followed by the dissociation and insertion of the protein into the lipid bilayer.

Environmental factors shape the community of symbionts in the hoopoe uropygial gland more than genetic factors.

Exploring processes of coevolution of microorganisms and their hosts is a new imperative for life sciences. If bacteria protect hosts against pathogens, mechanisms facilitating the intergenerational transmission of such bacteria will be strongly selected by evolution. By disentangling the diversity of bacterial strains from the uropygium of hoopoes (Upupa epops) due to genetic relatedness or to a common environment, we explored the importance of horizontal (from the environment) and vertical (from parents) acquisition of antimicrobial-producing symbionts in this species. For this purpose, we compared bacterial communities among individuals in nonmanipulated nests; we also performed a cross-fostering experiment using recently hatched nestlings before uropygial gland development and some nestlings that were reared outside hoopoe nests. The capacity of individuals to acquire microbial symbionts horizontally during their development was supported by our results, since cross-fostered nestlings share bacterial strains with foster siblings and nestlings that were not in contact with hoopoe adults or nests also developed the symbiosis. Moreover, nestlings could change some bacterial strains over the course of their stay in the nest, and adult females changed their bacterial community in different years. However, a low rate of vertical transmission was inferred, since genetic siblings reared in different nests shared more bacterial strains than they shared with unrelated nestlings raised in different nests. In conclusion, hoopoes are able to incorporate new symbionts from the environment during the development of the uropygium, which could be a selective advantage if strains with higher antimicrobial capacity are incorporated into the gland and could aid hosts in fighting against pathogenic and disease-causing microbes.

Special structures of hoopoe eggshells enhance the adhesion of symbiont-carrying uropygial secretion that increase hatching success.

Animals live in a bacterial world, and detecting and exploring adaptations favouring mutualistic relationships with antibiotic-producing bacteria as a strategy to fight pathogens are of prime importance for evolutionary ecologists. Uropygial secretion of European hoopoes (Upupa epops, Linnaeus) contains antimicrobials from mutualistic bacteria that may be used to prevent embryo infection. Here, we investigated the microscopic structure of hoopoe eggshells looking for special features favouring the adhesion of antimicrobial uropygial secretions. We impeded female access to the uropygial gland and compared microscopic characteristics of eggshells, bacterial loads of eggs and of uropygial secretion, and hatching success of experimental and control females. Then, we explored the link between microbiological characteristics of uropygial secretion and these of eggs of hoopoes, as well as possible fitness benefits. The microscopic study revealed special structures in hoopoes' eggshells (crypts). The experimental prevention of females' gland access demonstrated that crypts are filled with uropygial secretion and that symbiotic enterococci bacteria on the eggshells come, at least partially, from those in the female's uropygial gland. Moreover, the experiment resulted in a higher permeability of eggshells by several groups of bacteria and in elimination of the positive relationships detected for control nests between hatching success and density of symbiotic bacteria, either in the uropygial secretion of females or on the eggshell. The findings of specialized crypts on the eggshells of hoopoes, and of video-recorded females smearing secretion containing symbiotic bacteria at a high density onto the eggshells strongly support a link between secretion and bacteria on eggs. Moreover, the detected associations between bacteria and hatching success suggest that crypts enhancing the adhesion of symbiont-carrying uropygial secretion likely protect embryos against infections.

Genomic Characterization of Piscicolin CM22 Produced by Carnobacterium maltaromaticum CM22 Strain Isolated from Salmon (Salmo salar).

Carnobacterium maltaromaticum is a species of lactic acid bacteria (LAB) that has been isolated from various natural environments. It is well-known for producing a diverse spectrum of bacteriocins with potential biotechnological applications. In the present study, a new psychrotolerant strain of C. maltaromaticum CM22 is reported, isolated from a salmon gut sample and producing a variant of the bacteriocin piscicolin 126 that has been named piscicolin CM22. After identification by 16S rRNA gene, this strain has been genomically characterized by sequencing and assembling its complete genome. Moreover, its bacteriocin was purified and characterized. In vitro tests demonstrated that both the strain and its bacteriocin possess antimicrobial activity against several Gram-positive bacteria of interest in human and animal health, such as Listeria monocytogenes, Clostridium perfringens, or Enterococcus faecalis. However, this bacteriocin did not produce any antimicrobial effect on Gram-negative species. The study of its genome showed the genetic structure of the gene cluster that encodes the bacteriocin, showing a high degree of homology to the gene cluster of piscicolin 126 described in other C. maltaromaticum. Although more studies are necessary concerning its functional properties, this new psychrotolerant strain C. maltaromaticum CM22 and its bacteriocin could be considered an interesting candidate with potential application in agri-food industry.

Advances in the preclinical characterization of the antimicrobial peptide AS-48.

Antimicrobial resistance is a natural and inevitable phenomenon that constitutes a severe threat to global public health and economy. Innovative products, active against new targets and with no cross- or co-resistance with existing antibiotic classes, novel mechanisms of action, or multiple therapeutic targets are urgently required. For these reasons, antimicrobial peptides such as bacteriocins constitute a promising class of new antimicrobial drugs under investigation for clinical development. Here, we review the potential therapeutic use of AS-48, a head-to-tail cyclized cationic bacteriocin produced by Enterococcus faecalis. In the last few years, its potential against a wide range of human pathogens, including relevant bacterial pathogens and trypanosomatids, has been reported using in vitro tests and the mechanism of action has been investigated. AS-48 can create pores in the membrane of bacterial cells without the mediation of any specific receptor. However, this mechanism of action is different when susceptible parasites are studied and involves intracellular targets. Due to these novel mechanisms of action, AS-48 remains active against the antibiotic resistant strains tested. Remarkably, the effect of AS-48 against eukaryotic cell lines and in several animal models show little effect at the doses needed to inhibit susceptible species. The characteristics of this molecule such as low toxicity, microbicide activity, blood stability and activity, high stability at a wide range of temperatures or pH, resistance to proteases, and the receptor-independent effect make AS-48 unique to fight a broad range of microbial infections, including bacteria and some important parasites.

Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO) Reduces Vibrio Populations and Increases Body Weight of European Seabass (Dicentrarchus labrax) Juveniles.

The global demand for fish products is continuously increasing as the population grows, and aquaculture plays an important role in supplying this demand. However, industrial antibiotic misuse has contributed to the spread of antimicrobial resistance among pathogenic bacteria, therefore, several antibiotic alternatives have been proposed. In this study, we have analyzed the effects of Allium-derived propyl propane thiosulfonate (PTSO) in European seabass juveniles' growth and performance. These effects were tested by measuring the body weight and analyzing the gut microbiome of fish after 89 days of feeding trial. The relative abundance of potentially pathogenic Vibrio in the foregut and hindgut of supplemented fish decreased, while Pseudomonas and Kocuria increased significantly. Alpha diversity indices significantly decreased in both gut regions of fish fed with Allium-derived PTSO supplemented diet, as well as between bacterial community composition. These results may indicate a positive effect of the supplementation in the diet with Allium-derived PTSO, reducing potentially pathogenic Vibrio and increasing body weight at the end of the experiment (89 days). However, this supplementation with Allium-derived PTSO produces changes in the diversity and composition of microbial communities, so further experiments would be necessary to explore bacterial community composition and health relationship.

AS-48 bacteriocin: close to perfection.

Bacteriocin AS-48 is an intriguing molecule because of its unique structural characteristics, genetic regulation, broad activity spectrum, and potential biotechnological applications. It was the first reported circular bacteriocin and has been undoubtedly the best characterized for the last 25 years. Thus, AS-48 is the prototype of circular bacteriocins (class IV), for which the structure and genetic regulation have been elucidated. This review discusses the state-of-the-art in genetic engineering with regard to this circular protein, with the use of site-directed mutagenesis and circular permutation. Mutagenesis studies have been used to unravel the role of (a) different residues in the biological activity, underlining the relevance of several residues involved in membrane interaction and the low correlation between stability and activity and (b) three amino acids involved in maturation, providing information on the specificity of the leader peptidase and the circularization process itself. To investigate the role of circularity in the stability and biological properties of the enterocin AS-48, two different ways of linearization have been attempted: in vitro by limited proteolysis experiments and in vivo by circular permutation in the structural gene as-48A. The results summarized here show the significance of circularization on the secondary structure, potency and, especially, the stability of AS-48 and point as well to a putative role of the leader peptide as a protecting moiety in the pre-proprotein. Taken all together, the data available on circular bacteriocins support the idea that AS-48 has been engineered by nature to make a remarkably active and stable protein with a broad spectrum of activity.

Beneficial Shifts in the Gut Bacterial Community of Gilthead Seabream (Sparus aurata) Juveniles Supplemented with Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO).

This study analyzes the potential use of an Allium-derived compound, propyl propane thiosulfonate (PTSO), as a functional feed additive in aquaculture. Gilthead seabream (Sparus aurata) juveniles had their diet supplemented with this Allium-derived compound (150 mg/kg of PTSO) and were compared with control fish. The effects of this organosulfur compound were tested by measuring the body weight and analyzing the gut microbiota after 12 weeks. The relative abundance of potentially pathogenic Vibrio and Pseudomonas in the foregut and hindgut of supplemented fish significantly decreased, while potentially beneficial Lactobacillus increased compared to in the control fish. Shannon's alpha diversity index significantly increased in both gut regions of fish fed with a PTSO-supplemented diet. Regarding beta diversity, significant differences between treatments only appeared in the hindgut when minority ASVs were taken into account. No differences occurred in body weight during the experiment. These results indicate that supplementing the diet with Allium-derived PTSO produced beneficial changes in the intestinal microbiota while maintaining the productive parameters of gilthead seabream juveniles.

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.

Antimicrobial Activity of the Circular Bacteriocin AS-48 against Clinical Multidrug-Resistant Staphylococcus aureus.

The treatment and hospital-spread-control of methicillin-resistant Staphylococcus aureus (MRSA) is an important challenge since these bacteria are involved in a considerable number of nosocomial infections that are difficult to treat and produce prolonged hospitalization, thus also increasing the risk of death. In fact, MRSA strains are frequently resistant to all ß-lactam antibiotics, and co-resistances with other drugs such as macrolides, aminoglycosides, and lincosamides are usually reported, limiting the therapeutical options. To this must be added that the ability of these bacteria to form biofilms on hospital surfaces and devices confer high antibiotic resistance and favors horizontal gene transfer of genetic-resistant mobile elements, the spreading of infections, and relapses. Here, we genotypically and phenotypically characterized 100 clinically isolated S. aureus for their resistance to 18 antibiotics (33% of them were OXA resistant MRSA) and ability to form biofilms. From them, we selected 48 strains on the basis on genotype group, antimicrobial-resistance profile, and existing OXA resistance to be assayed against bacteriocin AS-48. The results showed that AS-48 was active against all strains, regardless of their clinical source, genotype, antimicrobial resistance profile, or biofilm formation capacity, and this activity was enhanced in the presence of the antimicrobial peptide lysozyme. Finally, we explored the effect of AS-48 on formed S. aureus biofilms, observing a reduction in S. aureus S-33 viability. Changes in the matrix structure of the biofilms as well as in the cell division process were observed with scanning electron microscopy in both S-33 and S-48 S. aureus strains.

Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum.

Phytobiotics (bioactive compounds extracted from plants) are one of the explored alternatives to antibiotics in poultry and livestock due to their antimicrobial activity and its positive effects on gut microbiota and productive properties. In this study, we supplemented a product based on garlic and onion compounds in the diet to laying hens at the beginning of their productive life (from 16 to 20 weeks post-hatching). The experimental group showed a significant increase in the number of eggs laid and in their size, produced in one month compared to the control. This increase in production was accompanied by microbiota changes in the ileum and cecum by means of high throughput sequencing analyses. These bacterial shifts in the ileum were mainly the result of compositional changes in the rare biosphere (unweighted UniFrac), while in the cecum, treatment affected both majority and minority bacterial groups (weighted and unweighted UniFrac). These changes in the microbiota suggest an improvement in food digestibility. The relative abundance of Lactococcus in the ileum and Lactobacillus in the cecum increased significantly in the experimental group. The relative abundance of these bacterial genera are known to have positive effects on the hosts. These results are very promising for the use of these compounds in poultry for short periods.

Allium Extract Implements Weaned Piglet's Productive Parameters by Modulating Distal Gut Microbiota.

Antimicrobial resistance (AMR) has risen as a global threat for human health. One of the leading factors for this emergence has been the massive use of antibiotics growth-promoter (AGPs) in livestock, enhancing the spread of AMR among human pathogenic bacteria. Thus, several alternatives such as probiotics, prebiotics, or phytobiotics have been proposed for using in animal feeding to maintain or improve productive levels while diminishing the negative effects of AGPs. Reducing the use of antibiotics is a key aspect in the pig rearing for production reasons, as well as for the production of high-quality pork, acceptable to consumers. Here we analyze the potential use of Allium extract as an alternative. In this study, weaned piglets were fed with Allium extract supplementation and compared with control and antibiotic (colistin and zinc oxide) treated piglets. The effects of Allium extract were tested by analyzing the gut microbiome and measuring different productive parameters. Alpha diversity indices decreased significantly in Allium extract group in caecum and colon. Regarding beta diversity, significant differences between treatments appeared only in caecum and colon. Allium extract and antibiotic piglets showed better values of body weight (BW), average daily weight gain (ADG), and feed conversion ratio (FCR) than control group. These results indicate that productive parameters can be implemented by modifying the gut microbiota through phytobiotics such as Allium extract, which will drive to drop the use of antibiotics in piglet diet.

Insights into the functionality of the putative residues involved in enterocin AS-48 maturation.

AS-48 is a 70-residue, α-helical, cationic bacteriocin produced by Enterococcus faecalis and is very singular in its circular structure and its broad antibacterial spectrum. The AS-48 preprotein consists of an N-terminal signal peptide (SP) (35 residues) followed by a proprotein moiety that undergoes posttranslational modifications to yield the mature and active circular protein. For the study of the specificity of the region of AS-48 that is responsible for maturation, three single mutants have been generated by site-directed mutagenesis in the as-48A structural gene. The substitutions were made just in the residues that are thought to constitute a recognition site for the SP cleavage enzyme (His-1, Met1) and in those involved in circularization (Met1, Trp70). Each derivative was expressed in the enterococcal JH2-2 strain containing the necessary native biosynthetic machinery for enterocin production. The importance of these derivatives in AS-48 processing has been evaluated on the basis of the production and structural characterization of the corresponding derivatives. Notably, only two of them (Trp70Ala and Met1Ala derivatives) could be purified in different forms and amounts and are characterized for their bactericidal activity and secondary structure. We could not detect any production of AS-48 in JH2-2(pAM401-81(His-1Ile)) by using the conventional chromatographic techniques, despite the high efficiency of the culture conditions applied to produce this enterocin. Our results underline the different important roles of the mutated residues in (i) the elimination of the SP, (ii) the production levels and antibacterial activity of the mature proteins, and (iii) protein circularization. Moreover, our findings suggest that His-1 is critically involved in cleavage site recognition, its substitution being responsible for the blockage of processing, thereby hampering the production of the specific protein in the cellular culture supernatant.

Genetic features of circular bacteriocins produced by Gram-positive bacteria.

This review highlights the main genetic features of circular bacteriocins, which require the co-ordinated expression of several genetic determinants. In general terms, it has been demonstrated that the expression of such structural genes must be combined with the activity of proteins involved in maturation (cleavage/circularization) and secretion outside the cell via different transporter systems, as well as multifaceted immunity mechanisms essential to ensuring the bacteria's self-protection against such strong inhibitors. Several circular antibacterial peptides produced by Gram-positive bacteria have been described to date, including enterocin AS-48, from Enterococcus faecalis S-48 (the first one characterized), gassericin A, from Lactobacillus gasseri LA39, and a similar one, reutericin 6, from Lactobacillus reuteri LA6, butyrivibriocin AR10, from the ruminal anaerobe Butyrivibrio fibrisolvens AR10, uberolysin, from Streptococcus uberis, circularin A, from Clostridium beijerinckii ATCC 25752, and subtilosin A, from Bacillus subtilis. We summarize here the progress made in the understanding of their principal genetic features over the last few years, during which the functional roles of circular proteins with wide biological activity have become clearer.

Synergy of the Bacteriocin AS-48 and Antibiotics against Uropathogenic Enterococci.

The genus Enterococcus comprises a ubiquitous group of Gram-positive bacteria that can cause diverse health care-associated infections. Their genome plasticity enables easy acquisition of virulence factors as well as antibiotic resistances. Urinary tract infections (UTIs) and catheter-associated UTIs are common diseases caused by enterococci. In this study, Enterococcus strains isolated from UTIs were characterized, showing that the majority were E. faecalis and contained several virulence factors associated to a better colonization of the urinary tract. Their susceptibility against the bacteriocin AS-48 and several antibiotics was tested. AS-48 is a potent circular bacteriocin that causes bacterial death by pore formation in the cell membrane. The interest of this bacteriocin is based on the potent inhibitory activity, the high stability against environmental conditions, and the low toxicity. AS-48 was active at concentrations below 10 mg/L even against antibiotic-resistant strains, whereas these strains showed resistance to, at least, seven of the 20 antibiotics tested. Moreover, the effect of AS-48 combined with antibiotics commonly used to treat UTIs was largely synergistic (with up to 100-fold MIC reduction) and only occasionally additive. These data suggest AS-48 as a potential novel drug to deal with or prevent enterococcal infections.

Assay of enterocin AS-48 for inhibition of foodborne pathogens in desserts.

Enterocin AS-48 was tested against Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes in different kinds of desserts. The highest activity against S. aureus was detected in baker cream. However, in yogurt-type soy-based desserts and in gelatin pudding, AS-48 (175 arbitrary units [AU]/g) reduced viable cell counts of S. aureus by only 1.5 to 1.8 log units at most. The efficacy of AS-48 in puddings greatly depended on inoculum size, and viable S. aureus counts decreased below detection levels within 24 h for inocula lower than 4 to 5.5 log CFU/g. For L. monocytogenes, bacteriocin concentrations of 52.5 to 87.5 AU/g reduced viable counts below detection levels and avoided regrowth of survivors. The lowest activity was detected in yogurt-type desserts. For B. cereus, viable cell counts were reduced below detection levels for bacteriocin concentrations of 52.5 AU/g in instant pudding without soy or by 175 AU/g in the soy pudding. In gelatin pudding, AS-48 (175 AU/g) reduced viable cell counts of B. cereus below detection levels after 8 h at 10 degrees C or after 48 h at 22 degrees C. Bacteriocin addition also inhibited gelatin liquefaction caused by the proteolytic activity of B. cereus.

Antibacterial protection by enterocin AS-48 in sport and energy drinks with less acidic pH values.

The low pH and acid content found in sports and energy drinks are a matter of concern in dental health. Raising the pH may solve this problem, but at the same time increase the risks of spoilage or presence of pathogenic bacteria. In the present study, commercial energy drinks were adjusted to pH 5.0 and challenged with Listeria monocytogenes (drinks A to F), Staphylococcus aureus, Bacillus cereus, and Bacillus licheniformis (drink A) during storage at 37 degrees C. L. monocytogenes was able to grow in drink A and survived in drinks D and F for at least 2 days. Addition of enterocin AS-48 (1 microg/ml final concentration) rapidly inactivated L. monocytogenes in all drinks tested. S. aureus and B. cereus also survived quite well in drink A, and were completely inactivated by 12.5 microg/ml enterocin AS-48 after 2 days of storage or by 25 microg/ml bacteriocin after 1 day. B. licheniformis was able to multiply in drink A, but it was completely inactivated by 5 microg/ml enterocin AS-48 after 2 days of storage or by 12.5 microg/ml bacteriocin after 1 day. Results from the present study suggest that enterocin AS-48 could be used as a natural preservative against these target bacteria in less acidic sport and energy drinks.