Hervé Prévost

The Continuing Story of Class IIa Bacteriocins

SUMMARY Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.

Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag.

Aims:  The aim of this study was to isolate and identify bacteriocin‐producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mares milk), and to purify and characterize bacteriocins produced by these LAB.

Identification of the Enterococcus faecalis Tyrosine Decarboxylase Operon Involved in Tyramine Production

ABSTRACT Screening of a library of Enterococcus faecalis insertional mutants allowed isolation of a mutant affected in tyramine production. The growth of this mutant was similar to that of the wild-type E. faecalis JH2-2 strain in Maijala broth, whereas high-performance liquid chromatography analyses showed that tyramine production, which reached 1,000 μg ml−1 for the wild-type strain, was completely abolished. Genetic analysis of the insertion locus revealed a gene encoding a decarboxylase with similarity to eukaryotic tyrosine decarboxylases. Sequence analysis revealed a pyridoxal phosphate binding site, indicating that this enzyme belongs to the family of amino acid decarboxylases using this cofactor. Reverse transcription-PCR analyses demonstrated that the gene (tdc) encoding the putative tyrosine decarboxylase of E. faecalis JH2-2 is cotranscribed with the downstream gene encoding a putative tyrosine-tyramine antiporter and with the upstream tyrosyl-tRNA synthetase gene. This study is the first description of a tyrosine decarboxylase gene in prokaryotes.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Selection and evaluation of seafood-borne psychrotrophic lactic acid bacteria as inhibitors of pathogenic and spoilage bacteria.

In this study, inhibitory psychrotrophic lactic acid bacteria were isolated and investigated for future use in biopreservation of seafood products. Screening of 5575 colonies isolated from various seafood products resulted in the selection of 132 colonies presenting inhibitory properties. Among them, 52 isolates had characteristics of LAB and showed growth at 15 degrees C but not at 30 degrees C. The inhibition spectrum of these 52 isolates against 14 target strains (Gram-positive and -negative) showed inhibition of typical seafood spoiling and pathogenic bacteria and enabled the formation of seven interesting clusters. Sequencing of the 16S rRNA gene of a representative isolate from each cluster identified three Leuconostoc gelidum, two Lactococcus piscium, one Lactobacillus fuchuensis and one Carnobacterium alterfunditum. Theses strains did not produce histamine nor tyramine, and showed no particular antibiotic resistance profile. Growth rate as a function of temperature was tested for one L. piscium and one L. gelidum isolate and confirmed their psychrotrophic behavior. One out of seven isolates showed bacteriocin-like activity. The inhibition mechanisms of the other isolates are still unknown but may be due to competition for substrate. Absence of a bacteriocin-like component could be a positive point to gain rapid authorization for food application in France. This collection of LAB is now ready for testing on products.

Lactobacillus salivarius: Bacteriocin and probiotic activity

Lactic acid bacteria (LAB) antimicrobial peptides typically exhibit antibacterial activity against food-borne pathogens, as well as spoilage bacteria. Therefore, they have attracted the greatest attention as tools for food biopreservation. In some countries LAB are already extensively used as probiotics in food processing and preservation. LAB derived bacteriocins have been utilized as oral, topical antibiotics or disinfectants. Lactobacillus salivarius is a promising probiotic candidate commonly isolated from human, porcine, and avian gastrointestinal tracts (GIT), many of which are producers of unmodified bacteriocins of sub-classes IIa, IIb and IId. It is a well-characterized bacteriocin producer and probiotic organism. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. This review gives an up-to-date overview of all L. salivarius strains, isolated from different origins, known as bacteriocin producing and/or potential probiotic.

Study of the bacterial ecosystem in tropical cooked and peeled shrimps using a polyphasic approach.

The characterization of the microbial ecosystem of cooked tropical shrimps was carried out using a polyphasic approach. First, culture-dependent methods were used for bacterial enumeration and the phenotypic and molecular identification of bacterial isolates. Then, culture-independent methods, including PCR-TTGE (V3 region of the 16S rRNA gene), provided a fingerprinting of bacterial DNA directly extracted from shrimps. Two batches of cooked and peeled tropical shrimps were stored at 5 and 15 degrees C for 5 and 3 weeks, respectively. Trained panelists carried out a sensory evaluation and microbiological enumerations were performed. When spoilage of samples was perceived, several colonies were isolated from the total viable count media. Thus, 137 bacterial strains were identified by phenotypic and molecular tests. Lactic acid bacteria (LAB) constituted the major group with the most represented genera being Carnobacterium (C. divergens, C. maltaromaticum and indiscernible C. alterfunditum/pleistocenium), Vagococcus (indiscernible V. carniphilus/fluvialis) and Enterococcus (E. faecalis and E. faecium). The other groups corresponded to Brochothrix thermosphacta and Enterobacteriaceae (Serratia liquefaciens). In PCR-TTGE profiles some of DNA fragments were assigned to those of standard strains (S. liquefaciens, B. thermosphacta, E. faecalis, C. divergens and C. maltaromaticum) or identified isolates from culture-dependent analysis (E. faecium). Other additional informations were provided by fragment cloning (Psychrobacter sp, Citrobacter gillenii and Firmicute). In conclusion, TTGE is an excellent tool to monitor the evolution of the microbial ecosystem in seafood products.

Biodiversity of Listeria monocytogenes sensitivity to bacteriocin-producing Carnobacterium strains and application in sterile cold-smoked salmon.

Aims:  The aim of this study was to demonstrate the inhibitory capacity of Carnobacterium strains against a collection of Listeria monocytogenes strains in cold‐smoked salmon (CSS).

Heterologous Expression and Purification of Active Divercin V41, a Class IIa Bacteriocin Encoded by a Synthetic Gene in Escherichia coli

Divercin V41, a class IIa bacteriocin with strong antilisterial activity, is produced by Carnobacterium divergens V41. To express a recombinant version of divercin V41, we constructed a synthetic gene that encodes the mature divercin V41 peptide and then overexpressed the gene in pET-32b by using the T7 RNA polymerase promoter in the Escherichia coli Origami (DE3)(pLysS) strain. The DvnRV41 peptide was expressed as a translational fusion protein with thioredoxin and accumulated in the cell cytoplasm in a soluble anti-Listeria active form. The fusion protein was then purified and cleaved to obtain pure, soluble, folded DvnRV41 (462 microg per 20 ml of culture). This paper describes the first design of a synthetic bacteriocin gene and the first bacteriocin expressed in the E. coli cytoplasm.

Differentiation of Closely Related Carnobacterium Food Isolates Based on 16S-23S Ribosomal DNA Intergenic Spacer Region Polymorphism

ABSTRACT A novel strategy for identification of Carnobacterium food isolates based on restriction fragment length polymorphism (RFLP) of PCR-amplified 16S-23S ribosomal intergenic spacer regions (ISRs) was developed. PCR amplification from all Carnobacterium strains studied always yielded three ISR amplicons, which were designated the small ISR (S-ISR), the medium ISR (M-ISR), and the large ISR (L-ISR). The lengths of these ISRs varied from one species to another. Carnobacterium divergens NCDO 2763T and C. mobile DSM 4849T generated one major S-ISR band (ca. 400 bp) and minor M-ISR and L-ISR bands (ca. 500 and ca. 600 bp, respectively). The ISRs amplified from C. gallinarum NCFB 2766T and C. piscicola NCDO 2762T were larger (S-ISR, ca. 600 bp; M-ISR, ca. 700 bp; and L-ISR, ca. 800 bp). The L-ISR contained two tDNAs coding for tRNAIle and tRNAAla genes. The M-ISR included one tRNAAla gene, and the S-ISR did not contain a tDNA gene. The RFLP scheme devised involves estimation of variable PCR product sizes together with HinfI, TaqI, and HindIII restriction analysis. Forty-two isolates yielded four unique band patterns that correctly resolved these isolates into four Carnobacterium species. This method is very suitable for rapid, low-cost identification of a wide variety of Carnobacterium species without sequencing.