Fast and Reproducible 96-Well Plate-Based Method for the Evaluation of the Antigerminative Potential of Plant Extracts and Phytotoxic Compounds.

With the aim of evaluating the antigerminative activity of plant extracts, a miniaturized assay using 96-well plates (WP assay) was developed and compared to the long-established assay using Petri dishes (PD assay). The WP assay yielded results comparable to those of the PD assay using an ethanolic extract of the Himalayan balsam and lawsone as a standard. It also allowed the needed volume of the test solution to be cut by half and the number of required cress seeds to be cut by more than 1.5. The WP assay was then successfully applied to various extracts of Himalayan balsam, molecules (2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate, and 2-methoxy-1,4-naphthoquinone (2-MNQ)) and target seeds (radish, lettuce, and wheat). By being adapted to a 96-well plate format, the antigerminative WP assay is a promising alternative to the PD assay. Besides, its convenience and low resource consumption make it ready for accelerated and high-throughput screening, as well as automation.

Maltaricin CPN, a new class IIa bacteriocin produced by Carnobacterium maltaromaticum CPN isolated from mould-ripened cheese.

AIMS: The purpose of this study was to isolate, characterize and determine the structure and the antibacterial activities of a bacteriocin produced by Carnobacterium maltaromaticum CPN, a strain isolated from unpasteurized milk Camembert cheese. METHODS AND RESULTS: This bacteriocin, termed maltaricin CPN, was produced at higher amounts in MRS broth at temperatures between 15°C and 25°C. It was purified to homogeneity from culture supernatant by using a simple method consisting of cation-exchange and reversed-phase chromatographies. Mass spectrometry showed that maltaricin was a 4427·29 Da bacteriocin. Its amino acid sequence was determined by Edman degradation which showed that it had close similarity with bacteriocins of the class IIa. Maltaricin CPN consisted in fact of 44 unmodified amino acids including two cysteine residues at positions 9 and 14 linked by a disulphide bond. The antimicrobial activity of maltaricin CPN covered a range of bacteria, with strong activity against many species of Gram-positive bacteria, especially the food-borne pathogen Listeria monocytogenes, but no activity against Gram-negative ones. CONCLUSIONS: In the studied conditions, C. maltaromaticum CPN produced a new class IIa bacteriocin with strong anti-Listeria activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study covers the purification and the structural characterization of a new bacteriocin produced by strain C. maltaromaticum CPN isolated from Camembert cheese. Its activity against strains of L. monocytogenes and higher production rates at relatively low temperatures show potential technological applications to improve the safety of refrigerated food.

Investigation of potential markers of acid resistance in Lactobacillus plantarum by comparative proteomics.

AIMS: To investigate cell determinants of resistance to gastric acidity in Lactobacillus plantarum using comparative proteomics. METHODS AND RESULTS: Among ten Lact. plantarum strains that were tested for their acid resistance in vitro, three strains with different phenotypes were selected for comparative proteomic analysis: LC 804 (resistant), CIP A159 (intermediate) and CECT 4185 (sensitive). Constitutive differences between whole-cell proteomes of the three strains were studied. Among the differentially expressed proteins between strains, 17 have previously been reported to be involved in acid resistance processes. The effect of a low-pH exposure on these proteomic patterns was investigated, which led to the identification of five putative determinants of acid resistance (heat-shock protein GrpE, methionine synthase and 30S ribosomal protein S2) or sensitivity (phosphotransacetylase and adenylosuccinate synthase). Analysis also revealed three additional proteins involved in cell envelope biogenesis (3-oxoacyl-synthase II, dTDP-glucose 4,6-dehydratase and dTDP-4-dehydrorhamnose 3,5-epimerase) likely to be key factors of intrinsic acid tolerance in Lact. plantarum. CONCLUSIONS: The approach used in this study enabled the identification of potential markers of acid tolerance in Lact. plantarum, which may serve for phenotyping and screening purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work represents a further step towards the identification of bacterial biomarkers for each particular probiotic feature.

Resistance to simulated gastrointestinal conditions and adhesion to mucus as probiotic criteria for Bifidobacterium longum strains.

Eight Bifidobacterium longum strains, including reported probiotic strains (commercial and noncommercial), collection strains, and laboratory isolates, were investigated for their ability to adhere to mucin as well as their ability to tolerate acid and bile. Strains could be discriminated based on their sensitivity at pH values of 2.0 to 2.5 and bile concentrations of 0.5% to 2.0%. B. longum NCC 2705, a strain known for its probiotic properties, showed the highest resistance to gastrointestinal conditions, whereas the commercial probiotic strains B. longum BB 536 and SP 07/3 were the least resistant. In parallel, the human isolate B. longum BIF 53 showed the highest adhesion to mucin, whereas the commercial probiotic strains B. longum W 11, BB 536, and SP 07/3 were the least adhesive. The bacterial adhesion to mucin of strains B. longum NCC 2705 and BIF 53 could be reduced by lysozyme, indicating that cell-wall components are involved in the adhesion process. These results showed that there is no obvious link between adhesion and resistance to gastrointestinal conditions and the probiotic status of the studied strains. This calls for a definition of conditions for in vitro tests that better predict the in vivo functionality of probiotic strains.

Differential immunomodulatory properties of Bifidobacterium logum strains: relevance to probiotic selection and clinical applications.

Modulation of host immunity is one of the proposed benefits of the consumption of probiotics. Nonetheless, comparative studies on the immunological properties that support the selection of strains of the same species for specific health benefits are limited. In this study, the ability of different strains of Bifidobacterium longum to induce cytokine production by peripheral blood mononuclear cells (PBMCs) has been evaluated. Live cells of all B. longum strains greatly stimulated regulatory cytokine interleukin (IL)-10 and proinflammatory cytokine tumour necrosis factor (TNF)-alpha production. Strains of the same species also induced specific cytokine patterns, suggesting that they could drive immune responses in different directions. The probiotic strain B. longum W11 stimulated strongly the production of T helper 1 (Th1) cytokines while B. longum NCIMB 8809 and BIF53 induced low levels of Th1 cytokines and high levels of IL-10. The effects of cell-surface components obtained by sonication of B. longum strains overall confirm the effects detected by stimulation of PBMCs with live cells, indicating that these components are important determinants of the immunomodulatory activity of B. longum. Genomic DNA of some strains stimulated the production of the Th1 and pro-inflammatory cytokines, interferon (IFN)-gamma and TNF-alpha, but not that of IL-10. None of the cell-free culture supernatants of the studied strains was able to induce TNF-alpha production, suggesting that the proinflammatory component of these strains is associated mainly with structural cell molecules. The results suggest that despite sharing certain features, some strains can perform a better functional role than others and their careful selection for therapeutic use is desirable.

Biochemical and genetic evidence for production of enterocins A and B by Enterococcus faecium WHE 81.

Enterococcus faecium WHE 81, isolated from cheese, has been reported to produce a bacteriocin called "enterocin 81" [J. Appl. Microbiol. 85 (1998) 521.]. Purification of "enterocin 81" was carried out using ammonium sulfate precipitation, desalting on ODP-90 reverse-phase column, and purification through SP Sepharose HP cation exchange and C2/C18 reverse-phase chromatographies. The antimicrobial was eluted from the C2/C18 column as four individually active fractions, designated A81, B81, C81 and D81. The purification procedure used proved particularly efficient for the bacteriocin in fraction D81, with a yield of 46%, while only 3.8% the bacteriocin in fraction B81 could be collected. MALDI-TOF mass spectrometry of the bacteriocins in fractions B81 and D81 showed respective masses of 4,833.0 and 5,462.2 Da. Amino acid sequencing of the two peptides revealed two class-II bacteriocins whose sequences were similar to those of enterocin A and enterocin B, respectively. Using proper primers, chromosomal fragments of 212 and 216 bp enclosing bacteriocin structural genes were PCR-amplified. Cloning of the amplicons and their sequencing revealed two genes with sequences identical to the structural genes of enterocins A and B, respectively. It was therefore clearly established that E. faecium WHE 81 produces bacteriocins respectively identical to enterocins A and B. Our results, combined with data from previous reports, suggest that the two bacteriocins may be widespread among enterococcal strains and may play an important role in controlling the growth of pathogens and other undesirable bacteria in certain fermented food products.

Natural variation in susceptibility of Listeria strains to class IIa bacteriocins.

Thirty-one Listeria strains were tested for sensitivity to four class IIa bacteriocins, namely, enterocin A, mesentericin Y105, divercin V41, and pediocin AcH, and to nisin A. Class IIa bacteriocins displayed surprisingly similar antimicrobial patterns ranging from highly susceptible to fully resistant strains, whereas nisin A showed a different pattern in which all Listeria strains were inhibited. Particularly, it was observed that the strain Listeria monocytogenes V7 could not be inhibited by any of the class lIa bacteriocins tested. These observations suggest that Listeria strains resistant to the whole range of class IIa bacteriocins may occur in natural environments, which could be of great concern with regard to the use of these peptides as food preservatives.

Anti-Listeria effect of enterocin A, produced by cheese-isolated Enterococcus faecium EFM01, relative to other bacteriocins from lactic acid bacteria.

Enterocin A produced by Enterococcus faecium EFM01 displayed a narrow antimicrobial spectrum, mainly directed against Listeria spp. In particular, the bacteriocin was extremely active against 13 Listeria monocytogenes strains. This high specificity of action of enterocin A for Listeria spp. relative to lactic acid bacteria, together with its broad range of activity from pH 4.0 to pH 9.0, are factors which may be of great interest with respect to the potential antilisterial use of this bacteriocin in fermented foods. Assessment of the effect of enterocin A concentration on the extent and kinetics of bactericidal activity on L. monocytogenes Lm 6 (107 cfu ml-1 in culture broth), suggested that viability losses of higher than 5 log10, and time intervals necessary for maximum loss of viability of less than 2 h, could not be obtained. Moreover, it was shown that both parameters are closely dependent on the Listeria strain used. On the other hand, at concentrations inducing destruction of approximately 2 log10 cycles, maximum loss of viability was achieved within time intervals which varied widely from one lactic acid bacteria bacteriocin to another.

Class IIa bacteriocins: biosynthesis, structure and activity.

In the last decade, a variety of ribosomally synthesized antimicrobial peptides or bacteriocins produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into fundamental aspects of biology and biochemistry such as producer self protection, membrane-protein interactions, and protein modification and secretion. Moreover, it has become evident that these peptides may be developed into useful antimicrobial additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their activities and potential applications. They have first attracted particular attention as listericidal compounds and are now believed to be the next in line if more bacteriocins are to be approved in the future. The present review attempts to provide an insight into general knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances.

Class IIa bacteriocins from lactic acid bacteria: antibacterial activity and food preservation.

In the last decade, a variety of ribosomally synthesized antimicrobial peptides, or bacteriocins, produced by lactic acid bacteria have been identified and characterized. As a result of these studies, insight has been gained into various fundamental aspects of biology and biochemistry such as bacteriocin processing and secretion, mechanisms of cell immunity, and structure-function relationships. In parallel, there has been a growing awareness that bacteriocins may be developed into useful antimicrobial food additives. Class IIa bacteriocins can be considered as the major subgroup of bacteriocins from lactic acid bacteria, not only because of their large number, but also because of their significant biological activities and potential applications. The present review provides an overview of the knowledge available for class IIa bacteriocins and discusses common features and recent findings concerning these substances. The activity and potential food applications of class IIa bacteriocins are a major focus of this review.

Antilisterial activity of enterocin 81, a bacteriocin produced by Enterococcus faecium WHE 81 isolated from cheese.

Enterocin 81, a bacteriocin produced by Enterococcus faecium WHE 81 previously isolated from cheese, exhibited a very narrow spectrum of activity, which is mainly directed against enterococci and Listeria spp. including Listeria monocytogenes. Enterocin 81 activity, which was extremely rapid with maximal effect achieved within 30 min, could not be detected after treatment with various proteolytic enzymes. This activity was bactericidal in nature and induced an important efflux of intracellular material, which was visualized under electron microscopy as filaments coming out of L. monocytogenes cells. However, enterocin 81 did not display bacterial lysis on sensitive cells, as no changes in cell morphology were detected following the bactericidal action. Furthermore, this bacteriocin was shown to be equally active at pH values ranging from 4.0 to 8.0, which, along with the narrow activity spectrum, are two factors of paramount interest with regards to possible use of this bacteriocin in fermented foods.

Inhibition of Listeria monocytogenes in a smear-surface soft cheese by Lactobacillus plantarum WHE 92, a pediocin AcH producer.

The anti-Listeria monocytogenes activity of Lactobacillus plantarum WHE 92, a pediocin AcH producer, was investigated in Munster cheese, a smear-surface soft cheese. The appearance of L. monocytogenes in the cheese, which naturally occurs solely in the crust and never before 1 week of ripening, could be prevented by spraying a cell suspension of L. plantarum WHE 92 (ca. 10(5) CFU/ml) on the cheese surface at the beginning of the ripening period. L. monocytogenes was sometimes detected at low levels (<5.0 x 10(1) CFU/g) after 7 to 11 days of ripening. However, this pathogen not be able to grow, nor did it survive the presence of L. plantarum WHE 92 in any of the samples examined until the end of the ripening not be able to grow, nor did it survive the presence of than 10(4) CFU/g in control samples. In other respects, L. plantarum WHE 92, which exists naturally in Munster cheese, did not adversely affect the evolution of the ripening process. This procedure has allowed manufacturers to successfully put an antilisterial treatment into practice in their ripening rooms.

Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese.

Among 1,962 bacterial isolates from a smear-surface soft cheese (Munster cheese) screened for activity against Listeria monocytogenes, six produced antilisterial compounds other than organic acids. The bacterial strain WHE 92, which displayed the strongest antilisterial effect, was identified at the DNA level as Lactobacillus plantarum. The proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action of the antilisterial compound produced by this bacterium suggested that it was a bacteriocin. Purification to homogeneity and sequencing of this bacteriocin showed that it was a 4.6-kDa, 44-amino-acid peptide, the primary structure of which was identical to that of pediocin AcH produced by different Pediococcus acidilactici strains. We report the first case of the same bacteriocin appearing naturally with bacteria of different genera. Whereas the production of pediocin AcH from P. acidilactici H was considerably reduced when the final pH of the medium exceeded 5.0, no reduction in the production of pediocin AcH from L. plantarum WHE 92 was observed when the pH of the medium was up to 6.0. This fact is important from an industrial angle. As the pH of dairy products is often higher than 5.0, L. plantarum WHE 92, which develops particularly well in cheeses, could constitute an effective means of biological combat against L. monocytogenes in this type of foodstuff.