Evaluation of live-culture-producing lacticin 3147 as a treatment for the control of Listeria monocytogenes on the surface of smear-ripened cheese

AIMS: A live Lactococcus lactis culture, producing the two-component broad spectrum bacteriocin lacticin 3147, was assessed for ability to inhibit the food pathogen Listeria monocytogenes on the surface of smear-ripened cheese. METHODS AND RESULTS: In initial experiments, the addition of Listeria to a lacticin 3147-containing fermentate produced with L. lactis DPC4275 (a transconjugant strain derived from L. lactis DPC3147) resulted in at least a 4 log reduction of the pathogen in 30 min. Two separate trials were performed in order to assess the most suitable method for application of the potential protective culture to smear-ripened cheese. In the initial trial, the L. lactis was sprayed onto the surface of the cheese either before or after Listeria was deliberately applied. Application of the culture following Listeria challenge, yielded up to a 1000-fold reduction of the pathogen in contrast to the pretreatment where Listeria numbers were unaffected. In a further trial, three applications of the live lacticin 3147-producing culture was used on a cheese surface containing Listeria. Listeria numbers were found to be up to 100-fold lower than in the cheese treated with L. lactis DPC4268 (control). CONCLUSION: While application of the live lacticin 3147 producer did not give complete elimination of the pathogen the results nonetheless demonstrate the potential of the bioprotectant for improving the safety of smear-ripened cheeses and particularly those that contain low level contamination with Listeria. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of lacticin 3147 as a live-culture can serve as a bioprotectant for the control of L. monocytogenes on the surface of smear-ripened cheese.     

Inhibition of Listeria monocytogenes in cottage cheese manufactured with a lacticin 3147-producing starter culture

The efficacy of using a lacticin 3147-producing starter as a protective culture to improve the safety of cottage cheese was investigated. This involved the manufacture of cottage cheese using Lactococcus lactis DPC4268 (control) and L. lactis DPC4275, a bacteriocin-producing transconjugant strain derived from DPC4268. A number of Listeria monocytogenes strains, including a number of industrial isolates, were assayed for their sensitivity to lacticin 3147. These strains varied considerably with respect to their sensitivity to the bacteriocin. One of the more tolerant strains, Scott A, was used in the cottage cheese study; the cheese was subsequently inoculated with approximately 10(4) L. monocytogenes Scott A g-1. The bacteriocin concentration in the curd was measured at 2560 AU ml-1, and bacteriocin activity could be detected throughout the 1 week storage period. In cottage cheese samples held at 4 degrees C, there was at least a 99.9% reduction in the numbers of L. monocytogenes Scott A in the bacteriocin-containing cheese within 5 d, whereas in the control cheeses, numbers remained essentially unchanged. At higher storage temperatures, the kill rate was more rapid. These results demonstrate the effectiveness of lacticin 3147 as an inhibitor of L. monocytogenes in a food system where post-manufacture contamination by this organism could be problematic.     

Combined action of nisin and carvacrol on Bacillus cereus and Listeria monocytogenes

Nisin, a small antimicrobial protein, was tested for its bactericidal action against Listeria monocytogenes and Bacillus cereus and a typical biphasic reduction of the viable count was observed. The reduction was most fast during the first 10 min of exposure, while the viable count remained stable in the last part of the exposure period. Bacillus cereus was more sensitive towards nisin than L. monocytogenes and the inhibitory effect of nisin was stronger towards cells cultivated and exposed at 8 degrees C than towards cells cultivated and exposed at 20 degrees C. Combining nisin with sublethal doses of carvacrol resulted in an increased reduction in the viable count of both organisms, indicating synergy between nisin and carvacrol. Addition of lysozyme as a third preservative factor increased the synergistic effect between nisin and carvone, especially in the last part of the exposure period.     

Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria

The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels.     

Generation of food-grade lactococcal starters which produce the lantibiotics lacticin 3147 and lacticin 481

Transconjugant lactococcal starters which produce both lantibiotics lacticin 3147 and lacticin 481 were generated via conjugation of large bacteriocin-encoding plasmids. A representative of one of the resultant strains proved more effective at killing Lactobacillus fermentum and inhibiting the growth of Listeria monocytogenes LO28H than either of the single bacteriocin-producing parental strains, demonstrating the potential of these transconjugants as protection cultures for food safety applications.     

Fate of the two-component lantibiotic lacticin 3147 in the gastrointestinal tract

The component peptides of lacticin 3147 were degraded by alpha-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit.     

Overproduction of wild-type and bioengineered derivatives of the lantibiotic lacticin 3147

Lacticin 3147 is a broad-spectrum two-peptide lantibiotic whose genetic determinants are located on two divergent operons on the lactococcal plasmid pMRC01. Here we introduce each of 14 subclones, containing different combinations of lacticin 3147 genes, into MG1363 (pMRC01) and determine that a number of them can facilitate overproduction of the lantibiotic. Based on these studies it is apparent that while the provision of additional copies of genes encoding the biosynthetic/production machinery and the regulator LtnR is a requirement for high-level overproduction, the presence of additional copies of the structural genes (i.e., ltnA1A2) is not.     

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.     

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva

AIMS: To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. METHODS AND RESULTS: Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. CONCLUSION: A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.     

Acid stress in the food pathogen Bacillus cereus

AIMS: The pathogen Bacillus cereus, which is associated with a number of foods including dairy products, was studied for its response to acid stress during the exponential phase. METHODS AND RESULTS: Bacillus cereus was found to adapt to acid stress (pH 4.6) when pre-exposed to a non-lethal, inducing pH of 6.3 or to inducing concentrations of heat, ethanol, salt or hydrogen peroxide. Cells were found to maintain their internal pH at a higher level than the external acid pH and adapted cells had a higher internal pH than unadapted cells. A constitutive acid-sensitive mutant that was also heat- and ethanol-sensitive was found to be capable of high levels of adaptation despite its lack of induction of proteins induced in the wild type by exposure to moderate pH (6.3) values. CONCLUSIONS: A number of proteins were found to be underexpressed in the mutant compared with the wild type at pH 6.3, including some with homology to ribosomal proteins and to the sporulation regulator RapK, while one differentially expressed band contained two proteins, one of which was homologous to the competence regulator CodY. SIGNIFICANCE AND IMPACT OF THE STUDY: The work has implications for the processing of B. cereus-associated foods by acidification. The linked developmental processes of stationary phase, sporulation and possibly competence appear to be involved in the response to acid stress.     

Cross-immunity and immune mimicry as mechanisms of resistance to the lantibiotic lacticin 3147

Lantibiotics are antimicrobial peptides that possess great potential as clinical therapeutic agents. These peptides exhibit many beneficial traits and in many cases the emergence of resistance is extremely rare. In contrast, producers of lantibiotics synthesize dedicated immunity proteins to provide self-protection. These proteins have very specific activities and cross-immunity is rare. However, producers of two peptide lantibiotics, such as lacticin 3147, face the unusual challenge of exposure to two active peptides (α and β). Here, in addition to establishing the contribution of LtnI and LtnFE to lacticin 3147 immunity, investigations were carried out to determine if production of a closely related lantibiotic (i.e. staphylococcin C55) or possession of LtnI/LtnFE homologues could provide protection. Here we establish that not only are staphylococcin C55 producers cross-immune to lacticin 3147, and therefore represent a natural repository of Staphylococcus aureus strains that are protected against lacticin 3147, but that functional immunity homologues are also produced by strains of Bacillus licheniformis and Enterococcus faecium . This result raises the spectre of resistance through immune mimicry, i.e. the emergence of lantibiotic-resistant strains from the environment resulting from the possession/acquisition of immunity gene homologues. These phenomena will have to be considered carefully when developing lantibiotics for clinical application.     

Characterization of a broad range antimicrobial substance from Bacillus cereus

AIMS: The aim of this research was to isolate and characterize an antimicrobial substance from the Bacillus cereus type strain ATCC 14579. METHODS AND RESULTS: A substance with antimicrobial activity was isolated from B. cereus ATCC 14579. The substance was produced during late exponential growth and well into the stationary phase with a maximum 9 h after inoculation. The inhibitory substance was purified by reverse-phase HPLC and shown to be highly active against closely related Bacillus spp. Clinically relevant species such as Staphylococcus aureus and Micrococcus luteus were also inhibited. The substance was characterized as a bacteriocin-like inhibitory substance (BLIS) with a molecular mass of ca 3.4 kDa. The BLIS was very heat stable, and sensitive only to pronase E and proteinase K. Antimicrobial activity was stable and high in the pH range of 2.0-9.0, and relatively unaffected by organic chemicals. CONCLUSIONS: An antimicrobial substance produced by the B. cereus type strain ATCC 14579 was characterized, with a wide spectrum of activity and the potential to be applied as a control agent against pathogenic bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first report of a substance with antimicrobial activity from the B. cereus type strain.     

Detection of Listeria monocytogenes and the toxin listeriolysin O in food

Listeria monocytogenes is an emerging bacterial foodborne pathogen responsible for listeriosis, an illness characterized by meningitis, encephalitis, and septicaemia. Less commonly, infection can result in cutaneous lesions and flu-like symptoms. In pregnant women, the pathogen can cause bacteraemia, and stillbirth or premature birth of the fetus. The mortality rate for those contracting listeriosis is approximately 20%. Currently, the United States has a zero tolerance policy regarding the presence of L. monocytogenes in food, while Canada allows only 100 cfu/g of food. As such, it is essential to be able to detect the pathogen in low numbers in food samples. One of the best ways to detect and confirm the pathogen is through the detection of one of the virulence factors, listeriolysin O (LLO) produced by the microorganism. The LLO-encoding gene (hlyA) is present only in virulent strains of the species and is required for virulence. LLO is a secreted protein toxin that can be detected easily with the use of blood agar or haemolysis assays and it is well characterized and understood. This paper focuses on some of the common methods used to detect the pathogen and the LLO toxin in food products and comments on some of the potential uses and drawbacks for the food industry.     

Occurrence and significance of Bacillus cereus and Bacillus thuringiensis in ready-to-eat food

Among 48,901 samples of ready-to-eat food products at the Danish retail market, 0.5% had counts of Bacillus cereus-like bacteria above 10(4) cfu g(-1). The high counts were most frequently found in starchy, cooked products, but also in fresh cucumbers and tomatoes. Forty randomly selected strains had at least one gene or component involved in human diarrhoeal disease, while emetic toxin was related to only one B. cereus strain. A new observation was that 31 out of the 40 randomly selected B. cereus-like strains could be classified as Bacillus thuringiensis due to crystal production and/or content of cry genes. Thus, a large proportion of the B. cereus-like organisms present in food may belong to B. thuringiensis.     

Heat and salt stress in the food pathogen Bacillus cereus

AIMS: The effects of stresses imposed on bacterial contaminants during food processing and treatment of packaging material were evaluated on the food pathogen Bacillus cereus. METHODS AND RESULTS: Conditions were established which allowed the cells to adapt to heat, ethanol and hydrogen peroxide stresses, but not to osmotic shock. Cross protection between stresses indicated a clear hierarchy of resistance with salt protecting against hydrogen peroxide, which protected against ethanol, which protected against heat shock. The cultures were shown to be most sensitive to heat, ethanol and oxidative stress at mid-exponential phase and to become resistant at stationary phase. Adaptive levels of stressor were found to induce synthesis of general stress and stress-specific proteins and differential accumulation of proteins was demonstrated between heat- or salt-stressed and unstressed cells. CONCLUSIONS: Sequencing revealed that a number of glycolytic enzymes were regulated by heat and osmotic shocks and that the chaperone GroEL was induced by heat shock. SIGNIFICANCE AND IMPACT OF THE STUDY: The implications of the physiological data in designing storage and processing conditions for food are discussed. The identification of stress-regulated proteins reveals a clear role for glycolysis in adaptation to heat shock and osmotic stress.     

Complete alanine scanning of the two-component lantibiotic lacticin 3147: generating a blueprint for rational drug design

Lantibiotics are post-translationally modified antimicrobial peptides which are active at nanomolar concentrations. Some lantibiotics have been shown to function by targeting lipid II, the essential precursor of cell wall biosynthesis. Given that lantibiotics are ribosomally synthesized and amenable to site-directed mutagenesis, they have the potential to serve as biological templates for the production of novel peptides with improved functionalities. However, if a rational approach to novel lantibiotic design is to be adopted, an appreciation of the roles of each individual amino acid (and each domain) is required. To date no lantibiotic has been subjected to such rigorous analysis. To address this issue we have carried out complete scanning mutagenesis of each of the 59 amino acids in lacticin 3147, a two-component lantibiotic which acts through the synergistic activity of the peptides LtnA1 (30 amino acids) and LtnA2 (29 amino acids). All mutations were performed in situ in the native 60 kb plasmid, pMRC01. A number of mutations resulted in the elimination of detectable bioactivity and seem to represent an invariable core within these and related peptides. Significantly however, of the 59 amino acids, at least 36 can be changed without resulting in a complete loss of activity. Many of these are clustered to form variable domains within the peptides. The information generated in this study represents a blue-print that will be critical for the rational design of lantibiotic-based antimicrobial compounds.     

Continuous production of lacticin 3147 and nisin using cells immobilized in calcium alginate

Bacteriocinogenic strains, Lactococcus lactis subsp. lactis DPC 3147 and L. lactis DPC 496, producing lacticin 3147 and nisin, respectively, were immobilized in double-layered calcium alginate beads. These beads were inoculated into MRS broth at a ratio of 1:4 and continuously fermented for 180 h. Free cells were used to compare the effect of immobilization on bacteriocin production. After equilibrium was reached, a flow rate of 580 ml h(-1) was used in the immobilized cell (IC), and 240 ml h(-1) in free-cell (FC) bioreactors. Outgrowth from beads was observed after 18 h. Bacteriocin production peaked at 5120 AU ml(-1) in both IC and FC bioreactors. However, FC production declined after 80 h to 160 AU ml(-1) at the end of the fermentation. Results of this study indicate that immobilization offers the possibility of a more stable and long-term means of producing lacticin 3147 in laboratory media than with free cells.     

Highly selective medium for isolation of Listeria monocytogenes from food

A new selective medium (Al-Zoreky-Sandine listeria medium [ASLM]) was formulated to recover Listeria monocytogenes from food specimens; the medium completely inhibited common food microflora. Recognition of Listeria colonies is evident by black discoloration of the medium due to esculin hydrolysis without need for special illuminating equipment. The medium contains acriflavin, ceftazidime, and moxalactam as selective agents. Compared with Listeria Selective Agar, ASLM was equally effective in recovering L. monocytogenes. However, ASLM inhibited micrococci, enterococci, and gram-negative bacteria, especially a strain that mimicked L. monocytogenes on Listeria Selective Agar. The new medium was able to recover heat injured cells with only 15% less count than the nonselective medium.     

Prevalence and lineages of Listeria monocytogenes in Chinese food products

AIMS: This study was undertaken to investigate the prevalence and lineages of Listeria monocytogenes in different kinds of food products in local Chinese markets. METHODS AND RESULTS: A total of 2686 food samples and 645 water samples were collected and L. monocytogenes was isolated from 2.28% (76 of 3331) of all samples. The prevalence of L. monocytogenes (14 of 290, 4.83%) in raw meat products was significantly higher than that in other raw food products (P < 0.05). Among 844 ready-to-eat (RTE) food samples, 21 samples were positive for L. monocytogenes. RTE packaged food products from two supermarkets had a prevalence ranging from 0.00% to 25.00%. The prevalence of L. monocytogenes in meat products of freshly slaughtered hogs was 0.95% (four of 420), significantly lower than that in raw meat products in the retail markets (P < 0.05). Ten isolates were recovered from 645 water samples, which were collected after hands washing by shopkeepers or waiters. A total of 38 isolates were randomly selected for lineage classification based on the nucleotide variation of actA gene. Eighty percentage of isolates from RTE food products belonged to Lineage II while only 20% belonged to Lineage I. CONCLUSIONS: Food products in Chinese markets are contaminated with L. monocytogenes. Raw meat products have the highest contamination rates among all the raw food samples. RTE food products are more likely to be contaminated with Lineage II strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here show the main contamination sources of L. monocytogenes in Chinese food products.