Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum

Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms.Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACD^Cgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l-asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.     

Production of Active Chimeric Pediocin AcH in Escherichia coli in the Absence of Processing and Secretion Genes from the Pediococcus pap Operon

Minimum requirements have been determined for synthesis and secretion of the Pediococcus antimicrobial peptide, pediocin AcH, in Escherichia coli. The functional mature domain of pediocin AcH (Lys⁺¹ to Cys⁺⁴⁴) is targeted into the E. coli sec machinery and secreted to the periplasm in active form when fused in frame to the COOH terminus of the secretory protein maltose-binding protein (MBP). The PapC-PapD specialized secretion machinery is not required for secretion of the MBP-pediocin AcH chimeric protein, indicating that in Pediococcus, PapC and PapD probably are required for recognition and processing of the leader peptide rather than for translocation of the mature pediocin AcH domain across the cytoplasmic membrane. The chimeric protein displays bactericidal activity, suggesting that the NH₂ terminus of pediocin AcH does not span the phospholipid bilayer in the membrane-interactive form of the molecule. However, the conserved Lys⁺¹-Tyr-Tyr-Gly-Asn-Gly-Val⁺⁷-sequence at the NH₂ terminus is important because deletion of this sequence abolishes activity. The secreted chimeric protein is released into the culture medium when expressed in a periplasmic leaky E. coli host. The MBP fusion-periplasmic leaky expression system should be generally advantageous for production and screening of the activity of bioactive peptides.     

Detection of the pediocin gene <Emphasis Type="Italic">pedA</Emphasis> in strains from human faeces by real-time PCR and characterization of <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis>UVA1

Background  

Bacteriocin-producing lactic acid bacteria are commonly used as natural protective cultures. Among them, strains of the genus Pediococcus are particularly interesting for their ability to produce pediocin, a broad spectrum antimicrobial peptide with a strong antagonistic activity against the food-borne pathogen Listeria monocytogenes. Furthermore, there is increasing interest in isolating new bacteriocin-producing strains of human intestinal origin that could be developed for probiotic effects and inhibition of pathogenic bacteria in the gut. In this work, we typed a new strain, co-isolated from baby faeces together with a Bifidobacterium thermophilum strain, and characterized its proteinaceous compound with strong antilisterial activity.     

Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon

Lactic acid bacteria (LAB) are widely used in the food industry. Pediococcus spp. belong to the LAB group and include several species that are essential for the quality of fermented food. Pediococcus pentosaceus is the species that is most frequently isolated from fermented food and beverages but its uncontrolled growth during food fermentation processes can contribute to undesired flavours. Hence, the characterisation of these bacteria at the strain level is of great importance for the quality of fermented products. Despite their importance, misidentification at the species level is common for members of the genus Pediococcus. To clarify the taxonomic relationships among strains, a multilocus sequencing approach was developed for the characterisation of a collection of 29 field strains, 1 type strain and 1 reference strain of P. pentosaceus isolated from food. These strains were also tested for several phenotypic properties of technological interest and for the production of bacteriocins. The chromosomal operon involved in the synthesis of the bacteriocin penocin was also investigated. The present study enabled a good genomic characterisation, identifying 17 sequence types, with an overview of phenotypic characteristics related to different technological abilities, and also provides a thorough characterisation of the operon involved in penocin production.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Fermenting Cucumber, a Potential Source for the Isolation of Pediocin-Like Bacteriocin Producers

Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin. These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic strains.     

Cloning Vectors Based on Cryptic Plasmids Isolated from Lactic Acid Bacteria:Their Characteristics and Potential Applications in Biotechnology

Lactic acid bacteria (LAB) are Gram positive bacteria, widely distributed in nature, and industrially important as they are used in a variety of industrial food fermentations. The use of genetic engineering techniques is an effective means of enhancing the industrial applicability of LAB. However, when using genetic engineering technology, safety becomes an essential factor for the application of improved LAB to the food industry. Cloning and expression systems should be derived preferably from LAB cryptic plasmids that generally encode genes for which functions can be proposed, but no phenotypes can be observed. However, some plasmid-encoded functions have been discovered in cryptic plasmids originating from Lactobacillus, Streptococcus thermophilus, and Pediococcus spp. and can be used as selective marker systems in vector construction. This article presents information concerning LAB cryptic plasmids, and their structures, functions, and applications. A total of 134 cryptic plasmids collated are discussed.     

Effect of Selected Types of Beer on Bacteria of the Genus Arcobacter

The genus Arcobacter is related to the well-known human pathogen, Campylobacter jejuni, and has been linked to human diseases. In this study, the survival of Arcobacter spp. in various concentrations of ethanol, in various samples of beers, and in a model stomach has been investigated. For most of these bacteria, a concentration of 10 % ethanol was determined to be the minimum inhibitory concentration. The fact that these organisms are able to survive under these conditions may have an impact in the food processing industry. We studied the activity of beer against arcobacters. These bacteria were killed in all samples of beer within 30 min. A model stomach, containing a food matrix and a synthetic gastric fluid, was used to deduce the effect of beer against Arcobacter spp. during food consumption. Complete inactivation of all monitored arcobacters was detected usually within 15 min. However, the presence of beer does not potentiate the effect of gastric fluid against these bacteria. This is apparently the first study focusing upon the effect of beer on Arcobacter spp.     

Fluorescence-Based Comparative Evaluation of Bactericidal Potency and Food Application Potential of Anti-listerial Bacteriocin Produced by Lactic Acid Bacteria Isolated from Indigenous Samples

The aim of the present study was to ascertain the potency of anti-listerial bacteriocin produced by lactic acid bacteria (LAB) isolated from indigenous samples of dahi, dried fish, and salt-fermented cucumber. A total of 231 LAB isolates were obtained from the samples, of which 51 isolates displayed anti-listerial activity. The anti-listerial LAB were identified by PCR as Lactobacillus sp., Pediococcus sp., Enterococcus sp., and Lactococcus sp. PCR also enabled the detection of Class IIa bacteriocin-encoding genes such as enterocin A, pediocin, and plantaricin A in some of the LAB isolates. The culture filtrate from anti-listerial LAB isolates demonstrated bacteriocin-like inhibitory substance (BLIS) against common Gram-positive pathogenic bacteria such as Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus, and partial characterization of BLIS confirmed the production of bacteriocin by the LAB isolates. Sensitive fluorescence-based assays employing specific probes indicated the comparative potencies of the bacteriocin and clearly revealed the membrane-targeted anti-listerial activity of the purified bacteriocin produced by selected LAB isolates. The food application potential of plantaricin A produced by a native isolate Lactobacillus plantarum CRA52 was evidenced as the bacteriocin suppressed the growth of Listeria monocytogenes Scott A inoculated in paneer samples that were stored at 8?°C for 5?days.     

Digestion of Herring by Indigenous Bacteria in the Minke Whale Forestomach

Northeastern Atlantic minke whales (Balaenoptera acutorostrata) have a multichambered stomach system which includes a nonglandular forestomach resembling that of ruminants. Bacteria from the forestomachs of herring-eating whales were enumerated and isolated in an anaerobic rumen-like culture medium (M8W medium). The total viable population of anaerobic bacteria ranged from 73 × 10⁷ to 145 × 10⁸/ml of forestomach fluid (n = 4). Lactobacillus spp. (19.7%), Streptococcus spp. (35.9%), and Ruminococcus spp. (12.8%) were the most common of the bacterial strains (n = 117) isolated by use of M8W medium from the forestomach fluid population of two minke whales. Most of the isolates stained gram positive (93.2%), 62.4% were cocci, and all strains were strictly anaerobic. The population of lipolytic bacteria in one animal, enumerated by use of a selective lipid medium, constituted 89.7% of the viable population. The total viable population of anaerobic bacteria in freshly caught and homogenized herring (Clupea harengus) ranged from 56.7 to 95.0 cells per gram of homogenized prey (n = 3) when M8W medium was used. Pediococcus spp. (30.6%) and Aerococcus spp. (25.0%) were most common of the bacterial strains (n = 72) isolated from the homogenized herring. Most of the bacterial strains were gram positive (80.6%), and 70.8% were cocci. Unlike the forestomach bacterial population, as many as 61.1% of the strains from the herring were facultatively anaerobic. All bacterial strains isolated from the prey had phenotypic patterns different from those of strains isolated from the dominant bacterial population in the forestomach, indicating that the forestomach microbiota is indigenous. Scanning electron microscopic examinations revealed large numbers of bacteria, surrounded by a glycocalyx, attached to partly digested food particles in the forestomach. These data support the hypothesis that symbiotic microbial digestion occurs in the forestomach and that the bacteria are indigenous to minke whales.     

The Genus Lactobacillus: A Taxonomic Update

Lactic Acid Bacteria (LAB) are a functional group of microorganisms comprising Gram-positive, catalase negative bacteria that produce lactic acid as the major metabolic end-product of carbohydrate fermentation. Among LAB, Lactobacillus is the genus including a high number of GRAS species (Generally Recognized As Safe) and many strains are among the most important bacteria in food microbiology and human nutrition, due to their contribution to fermented food production or their use as probiotics. From a taxonomic point of view, the genus Lactobacillus includes at present (October 2012), 152 validly described species, and it belongs to the family Lactobacillaceae together with genus Pediococcus, with whom it is phylogenetically intermixed. The updated phylogenetic analysis based on 16S rRNA gene sequence revealed that the family is divided into 15 groups of three or more species, 4 couples and 10 single lines of descents. In addition, other taxonomically relevant information for Lactobacillus species was collected. This study aims at updating the taxonomy of the genus Lactobacillus, presenting the phylogenetic structure of the Lactobacillaceae and discussing the clusters as possible nuclei of genera to be described in the future. It is expected that scientists and producers in the field of probiotics could benefit from information reported here about the correct identification procedures and nomenclature of beneficial strains of lactobacilli.     

Discrimination of wine lactic acid bacteria by Raman spectroscopy

Species of Lactobacillus, Pediococcus, Oenococcus, and Leuconostoc play an important role in winemaking, as either inoculants or contaminants. The metabolic products of these lactic acid bacteria have considerable effects on the flavor, aroma, and texture of a wine. However, analysis of a wine’s microflora, especially the bacteria, is rarely done unless spoilage becomes evident, and identification at the species or strain level is uncommon as the methods required are technically difficult and expensive. In this work, we used Raman spectral fingerprints to discriminate 19 strains of Lactobacillus, Pediococcus, and Oenococcus. Species of Lactobacillus and Pediococcus and strains of O. oeni and P. damnosus were classified with high sensitivity: 86–90 and 84–85%, respectively. Our results demonstrate that a simple, inexpensive method utilizing Raman spectroscopy can be used to accurately identify lactic acid bacteria isolated from wine.     

How the interaction of Listeria monocytogenes and Acanthamoeba spp. affects growth and distribution of the food borne pathogen

Listeria monocytogenes is a foodborne opportunistic pathogen capable to switch from an environmental saprophyte to a potentially fatal human pathogen. The fact that the pathogen maintains the genes suitable for an elaborate infectious process indicates that these genes are required to survive in the environment. However, no environmental host reservoir for L. monocytogenes has been identified so far. The similarity of free-living, bacteria-scavenging amoebae to macrophages led to the hypothesis that protozoa may represent the missing link in the ecology and pathology of L. monocytogenes. Consequently, numerous studies have been published reporting on the potential of Acanthamoeba spp. to serve as host for a variety of pathogenic bacteria. However, the data on the interaction of L. monocytogenes with Acanthamoeba spp. are inconsistent and relatively little information on the impact of this interaction on growth and distribution of the foodborne pathogen is currently available. Hence, this review focuses on the interaction of L. monocytogenes and Acanthamoeba spp. affecting survival and growth of the foodborne pathogen in natural and man-made environments, in order to highlight the potential impact of this interplay on food safety and human health.     

Arabinoxylan Oligosaccharide Hydrolysis by Family 43 and 51 Glycosidases from Lactobacillus brevis DSM 20054

Due to their potential prebiotic properties, arabinoxylan-derived oligosaccharides [(A)XOS] are of great interest as functional food and feed ingredients. While the (A)XOS metabolism of Bifidobacteriaceae has been extensively studied, information regarding lactic acid bacteria (LAB) is still limited in this context. The aim of the present study was to fill this important gap by characterizing candidate (A)XOS hydrolyzing glycoside hydrolases (GHs) identified in the genome of Lactobacillus brevis DSM 20054. Two putative GH family 43 xylosidases (XynB1 and XynB2) and a GH family 43 arabinofuranosidase (Abf3) were heterologously expressed and characterized. While the function of XynB1 remains unclear, XynB2 could efficiently hydrolyze xylooligosaccharides. Abf3 displayed high specific activity for arabinobiose but could not release arabinose from an (A)XOS preparation. However, two previously reported GH 51 arabinofuranosidases from Lb. brevis were able to specifically remove α-1,3-linked arabinofuranosyl residues from arabino-xylooligosaccharides (AXHm3 specificity). These results imply that Lb. brevis is at least genetically equipped with functional enzymes in order to hydrolyze the depolymerization products of (arabino)xylans and arabinans. The distribution of related genes in Lactobacillales genomes indicates that GH 43 and, especially, GH 51 glycosidase genes are rare among LAB and mainly occur in obligately heterofermentative Lactobacillus spp., Pediococcus spp., members of the Leuconostoc/Weissella branch, and Enterococcus spp. Apart from the prebiotic viewpoint, this information also adds new perspectives on the carbohydrate (i.e., pentose-oligomer) metabolism of LAB species involved in the fermentation of hemicellulose-containing substrates.     

Cloning and Expression of the Pediocin Operon in Streptococcus thermophilus and Other Lactic Fermentation Bacteria

Production of pediocin in Pediococcus acidilactici is associated with pMBR1.0, which encodes prepediocin, a pediocin immunity protein, and two proteins involved in secretion and precursor processing. These four genes are organized as an operon under control of a single promoter. We have constructed shuttle vectors that contain all four structural genes, the chromosomal promoter ST_P2201 from Streptococcus thermophilus, and repA from the 2-kbp S. thermophilus plasmid pER8. The recombinant plasmid, pPC318, expressed and secreted active pediocin in Escherichia coli. Streptococcus thermophilus, Lactococcus lactis subsp. lactis, and Enterococcus faecalis were electrotransformed with pPC418, a modified vector fitted with an erythromycin resistance tracking gene. Pediocin was produced and secreted in each of the lactic acid bacteria, and production was stable for up to ten passages. The expression of pediocin in dairy fermentation microbes has important implications for bacteriocins as food preservatives in dairy products. Received: 7 June 1999 / Accepted: 6 July 1999     

Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2

The lactic acid bacteria (LAB) are of great interest because of their food grade quality and industrial importance. In the recent past, the pediocin PA-1 like bacteriocin was found to be synthesized in cross-species and cross-genera. Hence, the present work was carried out in order to determine the transfer of plasmid encoded pediocin PA-1 like bacteriocin among LAB. The objective of this study is to demonstrate the dissemination of bacteriocin-encoded plasmid from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Enterococcus faecalis JH2-2 under in vitro (filter mating method) and in situ (soymilk model) conditions. The fermentation of the soymilk was determined by the selected pediocin producers. E. faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the native pediocin producer P. acidilactici NCIM 5424 transferred the bacteriocin under both the methods used. The in situ method gave more transfer frequency, ranging from 10^?7 to 10^?4 transconjugants per recipient cell. No conjugal transfer by L. plantarum Acr2 was observed. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the obtained transconjugants. The results suggest the horizontal gene transfer (HGT) and the natural spread of pediocin PA-1-like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1-like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and construction of novel cultures.     

Lactose and galactose uptake by genetically engineered Pediococcus species

The ability to utilize lactose is requisite for lactic acid bacteria used as starters in the dairy industry. Modern genetic recombination techniques have facilitated the introduction of the lactose-positive phenotype into bacteria such as Pediococcus species, which traditionally have not been used as dairy starters. This study investigated lactose and galactose uptake along with phospho-β-galactosidase activity in pediococci that had been transformed with a Latococcus lactis lactose plasmid. Lactose-positive transformants, Pediococcus acidilactici SAL and Pediococcus pentosaceus SPL-2, demonstrated an ability to accumulate [¹⁴C]lactose at a rate greater than the Lactococcus lactis control. Phospho-β-galactosidase activity was also higher in transformants versus Lactococcus lactis. Studies of [³H]galactose uptake suggested that a wild-type galactose transport system and the introduced lactose phosphotransferase system both functioned in galactose uptake by Pediococcus spp. transformants. Significantly lower levels of free galactose were detected in milk fermented with Lactobacillus helveticus LH100 and SAL or SPL-2 than in milk fermented with a LH100 plus Streptococcus thermophilus TA061 control starter blend. Received: 16 September 1997 /  Received revision: 11 November 1997 / Accepted: 21 November 1997     

Production of Pediocin PA-1 by Lactococcus lactis Using the Lactococcin A Secretory Apparatus

The class II bacteriocins pediocin PA-1, from Pediococcus acidilactici, and lactococcin A, from Lactococcus lactis subsp. lactis bv. diacetylactis WM4 have a number of features in common. They are produced as precursor peptides containing similar amino-terminal leader sequences with a conserved processing site (Gly-Gly at positions −1 and −2). Translocation of both bacteriocins occurs via a dedicated secretory system. Because of the strong antilisterial activity of pediocin PA-1, its production by lactic acid bacteria strains adapted to dairy environments would considerably extend its application in the dairy industry. In this study, the lactococcin A secretory system was adapted for the expression and secretion of pediocin PA-1. A vector containing an in-frame fusion of sequences encoding the lcnA promoter, the lactococcin A leader, and the mature pediocin PA-1, was introduced into L. lactis IL1403. This strain is resistant to pediocin PA-1 and encodes a lactococcin translocation apparatus. The resulting L. lactis strains secreted a bacteriocin with an antimicrobial activity of approximately 25% of that displayed by the parental pediocin-producing P. acidilactici 347. A noncompetitive indirect enzyme-linked immunosorbent assay with pediocin PA-1-specific antibodies and amino-terminal amino acid sequencing confirmed that pediocin PA-1 was being produced by the heterologous host.Bacteriocins of lactic acid bacteria have received considerable attention in recent years due to their potential application in the food industry as natural preservatives. Most interest has focused on lantibiotics (class I bacteriocins), e.g., nisin, and small heat-stable non-lanthionine-containing bacteriocins (class II) (22, 23). A major subgroup of class II bacteriocins (IIa) has been given the generic name of pediocin family (28) after its most extensively studied member, pediocin PA-1. Members of this class have a number of features in common, including a very strong antimicrobial activity against Listeria species (28). The food-borne pathogen Listeria monocytogenes is a major concern in the dairy industry since it can grow in a variety of dairy products at low temperature and pH (13). Although a pediocin PA-1-producing Lactobacillus plantarum strain has recently been isolated (12), this bacteriocin is generally produced by Pediococcus acidilactici strains of meat origin (3, 16, 18, 29, 31). Because of its antilisterial activity, the expression of pediocin PA-1 in strains of dairy origin would be highly desirable.Pediocin PA-1 production, immunity, and secretion are determined by an operon containing four genes (26). The structural gene, pedA, encodes the pediocin PA-1 precursor, pedB specifies immunity, and the pedC and pedD gene products are membrane-bound proteins required for secretion of the active peptide (39). Homologs of these genes have been described for related peptides. Biosynthesis of the well-characterized class II bacteriocin, lactococcin A, produced by strains of Lactococcus lactis also involves four genes (20, 36, 40). In addition to the structural gene (lcnA) and immunity gene (lciA), there are two genes (lcnC and lcnD) whose products together form a transport system dedicated to the translocation of lactococcin through the host membrane. The LcnC protein belongs to the family of ATP-binding cassette transporter proteins (40), and LcnD acts as an accessory protein (14). These two proteins have considerable homology to PedD and PedC, respectively (39), suggesting that the latter proteins play a similar role in the transport of active pediocin. The two bacteriocins also share the double glycine-processing site found in many lactic acid bacteria class II bacteriocins, some lantibiotics, and the Escherichia coli bacteriocin, colicin V (17).Van Belkum et al. (38) have recently investigated the role of leader sequences of the class II bacteriocins in the recognition of the precursor peptide by the dedicated translocation machinery of the host organism. By constructing hybrid genes, they demonstrated that the leader peptides of leucocin A, lactococcin A, and colicin V, which are cleaved at the Gly-Gly (positions −2 and −1) site, can direct the secretion of the nonrelated bacteriocin divergicin A. Our studies have focused on the class II bacteriocins pediocin PA-1 and lactococcin A. Since these peptides have a number of features in common, it might be expected that a pediocin PA-1 precursor could be secreted and processed by using the lactococcin A translocation machinery. L. lactis IL1403 is a plasmid-free strain that does not produce bacteriocin but contains chromosomal copies of genes analogous to lcnC and lcnD (33, 40). In addition, the natural resistance of this strain to pediocin PA-1 (8) makes it an ideal candidate for a production host to investigate the expression of pediocin PA-1 in lactococci.This paper describes the development of an expression system geared to the production of heterologous peptides in L. lactis. Testing the system with pediocin PA-1 involved the construction of a vector containing an in-frame fusion between sequences encoding the lactococcin A leader and the structural part of mature pediocin PA-1. The hybrid genes were introduced into L. lactis IL1403, and the ability of these strains to produce and secrete pediocin PA-1 was investigated.     

Characterization and Antilisterial Effect of Phosphatidylcholine Nanovesicles Containing the Antimicrobial Peptide Pediocin

Encapsulation may provide increased stability and antimicrobial efficiency to bacteriocins. In this work, the antilisterial peptide pediocin was encapsulated in nanovesicles prepared from partially purified soybean phosphatidylcholine. The maintenance of antimicrobial activity and properties of free and encapsulated pediocin was observed during 13 days at 4 °C, and after this period, the encapsulated pediocin retained 50 % its initial activity. The maintenance of the bioactive properties of free and encapsulated pediocin was observed against different species of Listeria, inhibiting Listeria monocytogenes, Listeria innocua and Listeria ivanovii. The size of vesicles containing pediocin was determined by dynamic light scattering as an average of 190 nm, with little change throughout the observation period. Polydispersity index values were around 0.201 and are considered satisfactory, indicating an adequate size distribution of liposomes. The efficiency of encapsulation was 80 %. Considering these results, the protocol used was appropriate for the encapsulation of this bacteriocin. Results demonstrate the production of stable nanoparticulate material. The maintenance of the properties of pediocin encapsulated in liposomes is fundamental to prospect the stability in different conditions of the food matrix.     

Epidemiology of Pathogenic Enterobacteria in Humans,Livestock, and Peridomestic Rodents in Rural Madagascar

Background

Among the families of enteric bacteria are globally important diarrheal agents. Despite their potential for zoonotic and environmental transmission, few studies have examined the epidemiology of these pathogens in rural systems characterized by extensive overlap among humans, domesticated and peridomestic animals. We investigated patterns of infection with Enterotoxigenic Escherichia coli, Shigella spp., Salmonella enterica, Vibrio cholerae, and Yersinia spp. (enterocolitica, and pseudotuberculosis) in Southeastern Madagascar where the potential for the aforementioned interactions is high. In this pilot project we conducted surveys to examine behaviors potentially associated with risk of infection and if infection with specific enterobacteria species was associated with diarrheal disease.

Methodology/Principal Findings

PCR was conducted on DNA from human, livestock, and rodent fecal samples from three villages. Overall, human prevalence was highest (77%), followed by rodents (51%) and livestock (18%). Rodents were ∼2.8 times more likely than livestock to carry one of the bacteria. The incidence of individual species varied between villages, with the observation that, E. coli and Shigella spp. were consistently associated with co-infections. As an aggregate, there was a significant risk of infection linked to a water source in one village. Individually, different pathogens were associated with certain behaviors, including: those who had used medication, experienced diarrhea in the past four weeks, or do not use toilets.

Conclusions/Significance

Different bacteria were associated with an elevated risk of infection for various human activities or characteristics. Certain bacteria may also predispose people to co-infections. These data suggest that a high potential for transmission among these groups, either directly or via contaminated water sources. As these bacteria were most prevalent in humans, it is possible that they are maintained in humans and that transmission to other species is infrequent. Further studies are needed to understand bacterial persistence, transmission dynamics, and associated consequences in this and similar systems.