Michael L. Chikindas

Bacteriocins: safe, natural antimicrobials for food preservation

Bacteriocins are antibacterial proteins produced by bacteria that kill or inhibit the growth of other bacteria. Many lactic acid bacteria (LAB) produce a high diversity of different bacteriocins. Though these bacteriocins are produced by LAB found in numerous fermented and non-fermented foods, nisin is currently the only bacteriocin widely used as a food preservative. Many bacteriocins have been characterized biochemically and genetically, and though there is a basic understanding of their structure-function, biosynthesis, and mode of action, many aspects of these compounds are still unknown. This article gives an overview of bacteriocin applications, and differentiates bacteriocins from antibiotics. A comparison of the synthesis. mode of action, resistance and safety of the two types of molecules is covered. Toxicity data exist for only a few bacteriocins, but research and their long-time intentional use strongly suggest that bacteriocins can be safely used.

Temperature- and Surfactant-Induced Membrane Modifications That Alter Listeria monocytogenes Nisin Sensitivity by Different Mechanisms

ABSTRACT Nisin interacts with target membranes in four sequential steps: binding, insertion, aggregation, and pore formation. Alterations in membrane composition might influence any of these steps. We hypothesized that cold temperatures (10°C) and surfactant (0.1% Tween 20) in the growth medium would influence Listeria monocytogenes membrane lipid composition, membrane fluidity, and, as a result, sensitivity to nisin. Compared to the membranes of cells grown at 30°C, those of L. monocytogenes grown at 10°C had increased amounts of shorter, branched-chain fatty acids, increased fluidity (as measured by fluorescence anisotropy), and increased nisin sensitivity. When 0.1% Tween 20 was included in the medium and the cells were cultured at 30°C, there were complex changes in lipid composition. They did not influence membrane fluidity but nonetheless increased nisin sensitivity. Further investigation found that these cells had an increased ability to bind radioactively labeled nisin. This suggests that the modification of the surfactant-adapted cell membrane increased nisin sensitivity at the binding step and demonstrates that each of the four steps can contribute to nisin sensitivity.

Functional analysis of the pediocin operon of Pediococcus acidilactici PAC1.0: PedB is the immunity protein and PedD is the precursor processing enzyme

The bacteriocin pediocin PA‐1 operon of Pediococcus acidilactici PAC1.0 encompasses four genes: pedA, pedB, pedC and pedD. Transcription of the operon results in the formation of two overlapping transcripts, probably originating from a single promoter upstream of pedA. The major transcript comprises pedA, pedB, and pedC, while a minor transcript encompasses all of these genes and pedD. By deletion analysis and overexpression of pedB in Pediococcus pentosaceus we demonstrate that this gene encodes the pediocin PA‐1 immunity protein. Prepediocin is active in Escherichia coli and when pedA was expressed concomitantly with pedD both the precursor and the mature form of pediocin were observed intracellularly. Extracellular pediocin was only detected if both pedC and pedD were present. The N‐terminal domains of PedD and a subgroup of bacteriocin ABC‐transporters are conserved. Expression of only this domain of PedD in cells producing prepediocin was sufficient for prepediocin processing. From these results we conclude that both PedC and PedD are essential for pediocin transport, and that PedD is capable of processing prepediocin.

Isolation of the Bacillus subtilis antimicrobial peptide subtilosin from the dairy product-derived Bacillus amyloliquefaciens

Aims:  To purify and characterize an antimicrobial protein (bacteriocin) isolated from the dairy product‐derived Bacillus amyloliquefaciens.

EFFECTIVE CONTROL OF LISTERIA MONOCYTOGENES BY COMBINATION OF NISIN FORMULATED AND SLOWLY RELEASED INTO A BROTH SYSTEM

In order to identify conditions for efficient food preservation by nisin, the sensitivity of Listeria monocytogenes to this preservative was studied under the following three model conditions: (1) the instantaneous addition of nisin into broth medium to simulate the formation of nisin in foods, (2) the slow delivery of nisin solution into broth medium using a pump to simulate the slow release of nisin from packaging materials to foods, (3) a combination of the two delivery methods. Based on the following results, we conclude that the antimicrobial effectiveness of nisin strongly depends on its mode of delivery. The instantaneous and slow methods for adding nisin inhibited L. monocytogenes, but over time of exposure, L. monocytogenes developed tolerance to nisin. Our data indicate that cells treated with instantaneously added nisin developed resistance to higher concentrations of nisin (200 IU/ml), compared to cells treated with slowly added nisin at the same total amount of the antimicrobial. Further studies indicated that nisin-tolerant cells recovered from treatments in which 200 IU/ml nisin was added instantaneously were likely to be mutants, which became resistant to the bacteriocin. In contrast, when 200 IU/ml of the antimicrobial was added slowly to the cells, only a temporary tolerance was developed; these cells became nisin-sensitive after passage through nisin-free medium. Due to the development of nisin-resistant cells, excessive amounts of nisin in the model system did not further inhibit L. monocytogenes. These results signify that excess nisin in foods does not necessarily improve the efficiency of controlling L. monocytogenes. Our data suggest that the combination of packaging material containing nisin used in conjunction with nisin-containing foods will provide the most effective means of preventing L. monocytogenes growth.

Characterization and heterologous expression of a class IIa bacteriocin, plantaricin 423 from Lactobacillus plantarum 423, in Saccharomyces cerevisiae

Lactobacillus plantarum 423 produces a small heat-stable antimicrobial protein designated plantaricin 423. This protein is bactericidal for many Gram-positive foodborne pathogens and spoilage bacteria, including Listeria spp., Staphylococcus spp., Pediococcus spp., Lactobacillus spp., etc. The DNA sequence of the plantaricin 423-encoding region on plasmid pPLA4 revealed a four open reading frame (ORF) operon structure similar to pediocin PA-1/AcH from Pediococcus acidilactici and coagulin from Bacillus coagulans I(4). The first ORF, plaA, encodes a 56-amino acid prepeptide consisting of a 37-amino acid mature molecule, with a 19-amino acid N-terminal leader peptide. The second ORF, plaB, encodes a putative immunity protein with protein sequence similarities to several bacteriocin immunity proteins. The plaC and plaD genes are virtually identical to pedC and pedD of the pediocin PA-1 operon, as well as coaC and coaD of the coagulin operon. Plantaricin 423 was cloned on a shuttle vector under the control of a yeast promoter and heterologously produced in Saccharomyces cerevisiae.

The aetiology of bacterial vaginosis

Bacterial vaginosis (BV) is the most common vaginal infection among women of childbearing age. This condition is notorious for causing severe complications related to the reproductive health of women. Five decades of intense research established many risk factors for acquisition of BV; however, because of the complexity of BV and lack of a reliable animal model for this condition, its exact aetiology remains elusive. In this manuscript, we use a historical perspective to critically review the development of major theories on the aetiology of BV, ultimately implicating BV‐related pathogens, healthy vaginal microbiota, bacteriophages and the immune response of the host. None of these theories on their own can reliably explain the epidemiological data. Instead, BV is caused by a complex interaction of multiple factors, which include the numerous components of the vaginal microbial ecosystem and their human host. Many of these factors are yet to be characterized because a clear understanding of their relative contribution to the aetiology of BV is pivotal to the formulation of an effective treatment for and prophylaxis of this condition.

Carbon Dioxide and Nisin Act Synergistically on Listeria monocytogenes

ABSTRACT This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nisr) cells grown in broth at 4°C. Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nisrcells. Wild-type cells grown in 100% CO2 were two to five times longer than cells grown in air. Nisin (2.5 μg/ml) did not decrease the viability of Nisr cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO2. There was a quantifiable synergistic action between nisin and CO2 in the wild-type strain. The MIC of nisin for the wild-type strain grown in the presence of 2.5 μg of nisin per ml increased from 3.1 to 12.5 μg/ml over 35 days, but this increase was markedly delayed for cultures in CO2. This synergism between nisin and CO2 was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes. Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO2 and nisin occurs at the cytoplasmic membrane. Liposomes made from cells grown in a CO2 atmosphere were even more sensitive to nisin action. Liposomes made from cells grown at 4°C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30°C. Cells grown in the presence of 100% CO2 and those grown at 4°C had a greater proportion of short-chain fatty acids. The synergistic action of nisin and CO2 is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action.

The synergistic effect of nisin and lactoferrin on the inhibition of Listeria monocytogenes and Escherichia coli O157:H7

Aims:  The goal of this study was to determine whether nisin and lactoferrin would act synergistically to inhibit the growth of Listeria monocytogenes and Escherichia coli O157:H7.

Enterocin P selectively dissipates the membrane potential of Enterococcus faecium T136

ABSTRACT Enterocin P is a pediocin-like, broad-spectrum bacteriocin which displays a strong inhibitory activity against Listeria monocytogenes. The bacteriocin was purified from the culture supernatant of Enterococcus faecium P13, and its molecular mechanism of action against the sensitive strain E. faecium T136 was evaluated. Although enterocin P caused significant reduction of the membrane potential (ΔΨ) and the intracellular ATP pool of the indicator organism, the pH gradient (ΔpH) component of the proton motive force (Δp) was not dissipated. By contrast, enterocin P caused carboxyfluorescein efflux from E. faecium T136-derived liposomes.