Growth inhibition of Listeria monocytogenes by a nonbacteriocinogenic Carnobacterium piscicola

AIMS: This study elucidates the mechanisms by which a nonbacteriocinogenic Carnobacterium piscicola inhibits growth of Listeria monocytogenes. METHODS AND RESULTS: Listeria monocytogenes was exposed to live cultures of a bacteriocin-negative variant of C. piscicola A9b in co-culture, in a diffusion chamber system, and to a cell-free supernatant. Suppression of maximum cell density (0-3.5 log units) of L. monocytogenes was proportional to initial levels of C. pisciola (10(3)-10(7) CFU ml(-1)). Cell-to-cell contact was not required to cause inhibition. The cell-free C. piscicola supernatant caused a decrease in L. monocytogenes maximum cell density, which was abolished by glucose addition but not by amino acid, vitamin or mineral addition. The fermentate also gave rise to a longer lag phase and a reduction in growth rate. These effects were independent of glucose and may have been caused by acetate production by C. piscicola. 2D gel-electrophoretic patterns of L. monocytogenes exposed to C. piscicola or to L. monocytogenes fermentate did not differ. Treatment with C. piscicola fermentate resulted in down-regulation (twofold) of genes involved in purine- or pyrimidine metabolism, and up-regulation (twofold) of genes from the regulon for vitamin B12 biosynthesis and propanediol and ethanolamine utilization. CONCLUSIONS: A nonbacteriocinogenic C. piscicola reduced growth of L. monocytogenes partly by glucose depletion. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mechanism of microbial interaction enhances prediction of growth in mixed communities as well as use of bioprotective principles for food preservation.     

Mode of action of piscicocin CS526 produced by Carnobacterium piscicola CS526

AIMS: Piscicocin CS526 is a unique class IIa bacteriocin produced by Carnobacterium piscicola CS526. The mode of action against the sensitive strain Listeria monocytogenes IID581 was evaluated. METHODS AND RESULTS: Piscicocin CS526 was adsorbed on both sensitive and insensitive gram-positive and gram-negative bacterial cells. Treatment of L. monocytogenes cells with trypsin, lipase and Triton X-100 did not reduce subsequent adsorption of piscicocin CS526. The activity of piscicocin CS526 against L. monocytogenes cells was bactericidal rather than bacteriostatic, but did not cause bacteriolysis. Piscicocin CS526 induced the efflux of K+ ions from the target cells which cause dissipation of the transmembrane potential (DeltaPsi) of the cell membrane. Moreover, after exposure to piscicocin CS526, intracellular adenosine 5'-triphosphate (ATP) level of the target cells rapidly reduced without leakage of ATP from the cells, indicating that ATP depletion occurred in the cells. CONCLUSIONS: Pore formation by piscicocin CS526 caused a rapid efflux of small molecules such as K+ from the indicator cells and dissipation of proton motive force (PMF), which lead to the cell death. SIGNIFICANCE AND IMPACT OF THE STUDY: Molecular mechanism of action of piscicocin CS526 is very similar to that of other pediocin-like bacteriocins, although piscicocin CS526 possesses a unique N-terminal sequence in which Val is substituted for by Leu in the amino acid at position 7.     

Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp. lactis as potential protective adjuncts to control Listeria monocytogenes in dry-fermented sausages

AIM: Study of the effectiveness of in situ bacteriocin production by lactic acid bacteria (LAB) to control Listeria monocytogenes in dry-fermented sausages. METHODS AND RESULTS: Two bacteriocin-producing strains: Lactococcus lactis subsp. lactis LMG21206 and Lactobacillus curvatus LBPE were grown in a pilot scale fermentor and lyophilized to be directly used in dry sausage fermentation. A commercial starter culture (Bel'meat SL-25) not inhibitory to L. monocytogenes (Bac- starter) was mixed (1 : 1) with each of the two lyophilized bacteriocin-producing strains to obtain starters active against the pathogen (Bac+ starter). Anti-Listeria effectiveness of the Bac+ starters was studied in dry-fermented sausages. The meat batter was experimentally contaminated with a mixture of four different strains of L. monocytogenes (10(2)-10(3) CFU g(-1)). The results showed that L. monocytogenes did not grow in any of the contaminated batches, but no significant decrease (P > 0.05) was observed either in the positive control (no added starter culture) or in samples fermented with the Bac- starter culture during the fermentation period and up to 15 days of drying. When the Bac+ starter contained Lb. curvatus LBPE, cell counts of L. monocytogenes decreased to below the detectable limit (<10 CFU g(-1)) after 4 h of fermentation and no survivors could be recovered by enrichment beyond day 8 of drying. When the Bac+ starter culture containing Lc. lactis LMG21206 was used, a decrease in Listeria counts to below the detectable limit was achieved after 15 days of drying. CONCLUSIONS: The bacteriocin-producing strains studied may be used as adjunct cultures for sausage fermentations to control the occurrence and survival of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Addition of the Bac+ strains, especially the Lb. curvatus strain would provide an additional hurdle to enhance the control of L. monocytogenes in fermented meat products.     

Purification and characterization of a novel class IIa bacteriocin, piscicocin CS526, from surimi-associated Carnobacterium piscicola CS526

The bacteriocin piscicocin CS526 was inactivated by proteolytic enzymes, was stable at 100 degrees C for 30 min, had a pH range of 2 to 8, and was active against Enterococcus, Listeria, Pediococcus, and Leuconostoc. The N-terminal sequence was YGNGL, not the YGNGV consensus motif common in class IIa bacteriocins (alternate residues underlined). The molecular mass of piscicocin CS526, which had a bactericidal mode of action, was approximately 4,430 Da.     

Control of Listeria monocytogenes in a biofilm by competitive-exclusion microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37 degrees C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10(3) CFU of L. monocytogenes/ml and 10(5) CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37 degrees C for 24 h, 15 degrees C for 14 days, 8 degrees C for 21 days, and 4 degrees C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37 degrees C, two at 15 and 8 degrees C, and three at 4 degrees C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4 degrees C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log(10) CFU of L. monocytogenes/cm(2)). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37 degrees C.     

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). METHODS AND RESULTS: Three bacteriocin-producing strains, Carnobacterium divergens V41, C. piscicola V1 and C. piscicola SF668, were screened for their antilisterial activity against a collection of 57 L. monocytogenes strains selected from the French smoked salmon industry, using an agar spot test. All the Listeria strains were inhibited but three different groups could be distinguished differing in sensitivity to the three Carnobacterium strains. However, C. divergens V41 always had the highest inhibitory effect. The antilisterial capacity was then tested in sterile CSS blocks co-inoculated with Carnobacterium spp. and mixtures of L. monocytogenes strains. C. divergens V41 was the most efficient strain, maintaining the level of L. monocytogenes at <50 CFU g(-1) during the 4 weeks of vacuum storage at 4 and 8 degrees C, whatever the sensitivity of the set of L. monocytogenes strains. CONCLUSIONS: C. divergens V41 may be a good candidate for biopreservation in CSS. SIGNIFICANCE AND IMPACT OF THE STUDY: A biopreservation strategy for CSS against the risk of L. monocytogenes was investigated using bacteriocin-producing lactic acid bacteria.     

Anti-bacterial activity of Lactobacillus plantarum strain SK1 against Listeria monocytogenes is due to lactic acid production

AIMS: The aim of this research was to investigate the potential of Lactobacillus plantarum strain SK1 for use as a biological control agent against Listeria monocytogenes and determine its mechanism of anti-listerial activity. METHODS AND RESULTS: Co-growth of Lact. plantarum SK1 and L. monocytogenes UMCC98 in MRS broth showed that anti-listerial activity of Lact. plantarum SK1 occurred during late log/early stationary phase of growth. This coincided with a reduction in broth pH to 4.26. Evidence obtained from the analysis of cell-free culture filtrates of strain SK1 grown in MRS broth using thin-layer chromatography and growth of L. monocytogenes in pH-adjusted culture filtrates suggested that the anti-listerial activity was due to lactic acid production alone. Trials of Lact. plantarum SK1 on radishes stored at 5 degrees C showed that it had statistically significant (P < 0.05) anti-listerial activity. CONCLUSIONS: The anti-listerial activity of Lact. plantarum SK1 was due to lactic acid production alone. A small-scale trial on radishes stored at 5 degrees C showed it to have significant anti-listerial activity in planta. SIGNIFICANCE AND IMPACT OF THE STUDY: This organism has potential as a biological control agent for L. monocytogenes.     

Specific molecular detection of Carnobacterium piscicola SF668 in cold smoked salmon

AIMS: To establish a rapid and reliable multiplex PCR (mPCR)-based method allowing specific identification of Carnobacterium piscicola SF668 during storage of cold smoked salmon (CSS). METHODS AND RESULTS: CSS was inoculated with C. piscicola SF668 and stored at 4 degrees C. Samples were withdrawn at regular time intervals and analysed by counting the number of viable cells. About 25-100% of colonies grown on Elliker plates were subjected to mPCR amplification. The results show that strains presumably identified as C. piscicola SF668 were predominant over the test period. CONCLUSIONS: mPCR is a powerful tool to study competitiveness of C. piscicola SF668, which inhibits the growth of Listeria monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study demonstrates the importance of molecular methods in studying competitiveness of strains with potential food applications.     

Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin AcH during storage at 4 or 25 degrees C.

Exudative fluids were collected from packages of five brands of all-beef wieners and inoculated to contain 10(4) to 10(5) CFU of a three-strain (Scott A, V7, and 101M) mixture of Listeria monocytogenes per ml. Listeriae were inactivated (decrease of 0.61 to 3.8 log10 CFU/ml) in all five exudates held at 4 degrees C for 29 days. L. monocytogenes grew (increase of 1.7 to 3.6 log10 CFU/ml) in two of five exudates held at 25 degrees C for 6 days. Exudate was inoculated with a derivative of Pediococcus acidilactici H (designated JBL1095) or treated with pediocin AcH (a bacteriocin) as a novel approach to control the growth of L. monocytogenes in wiener exudates. Initially, pediocin AcH caused rapid death (decrease of 0.74 log10 CFU/ml in 2 h) of L. monocytogenes in exudate held at 4 degrees C, but thereafter the inactivation was similar to that in control exudate (L. monocytogenes only) or exudate containing L. monocytogenes plus JBL1095. At 25 degrees C, L. monocytogenes grew in the presence of JBL1095 during the first 64 h of incubation, but thereafter the numbers of the pathogen decreased appreciably (5.84 log10 CFU/ml in 3 days). In the presence of pediocin AcH, there was a gradual decrease in numbers of L. monocytogenes throughout the storage period at 25 degrees C. These data indicate that added biopreservatives can potentiate and amplify the intrinsic listeriostatic or listericidal activity of wiener exudate.     

Behavior of Listeria monocytogenes in wiener exudates in the presence of Pediococcus acidilactici H or pediocin AcH during storage at 4 or 25 degrees C

Exudative fluids were collected from packages of five brands of all-beef wieners and inoculated to contain 10(4) to 10(5) CFU of a three-strain (Scott A, V7, and 101M) mixture of Listeria monocytogenes per ml. Listeriae were inactivated (decrease of 0.61 to 3.8 log10 CFU/ml) in all five exudates held at 4 degrees C for 29 days. L. monocytogenes grew (increase of 1.7 to 3.6 log10 CFU/ml) in two of five exudates held at 25 degrees C for 6 days. Exudate was inoculated with a derivative of Pediococcus acidilactici H (designated JBL1095) or treated with pediocin AcH (a bacteriocin) as a novel approach to control the growth of L. monocytogenes in wiener exudates. Initially, pediocin AcH caused rapid death (decrease of 0.74 log10 CFU/ml in 2 h) of L. monocytogenes in exudate held at 4 degrees C, but thereafter the inactivation was similar to that in control exudate (L. monocytogenes only) or exudate containing L. monocytogenes plus JBL1095. At 25 degrees C, L. monocytogenes grew in the presence of JBL1095 during the first 64 h of incubation, but thereafter the numbers of the pathogen decreased appreciably (5.84 log10 CFU/ml in 3 days). In the presence of pediocin AcH, there was a gradual decrease in numbers of L. monocytogenes throughout the storage period at 25 degrees C. These data indicate that added biopreservatives can potentiate and amplify the intrinsic listeriostatic or listericidal activity of wiener exudate.     

Selection and identification of a Listeria monocytogenes target strain for pulsed electric field process optimization

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23 degrees C for 144 micro s, inactivation ranged from 0.7 to 3.7 log(10) CFU/ml. Inactivation by 72- micro s PEF treatments at 37 degrees C ranged from 0.3 to 2.5 log(10) CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.     

The contribution of bacteriocin to inhibition of Listeria monocytogenes by Carnobacterium piscicola strains in cold-smoked salmon systems

AIMS: To study the importance of bacteriocin production for the antilisterial effect of a bacteriocinogenic Carnobacterium piscicola strain A9b on growth of Listeria monocytogenes in broth and cold-smoked salmon systems. METHODS AND RESULTS: Acriflavin treatment of strain A9b resulted in loss of bacteriocin production and of immunity to carnobacteriocin B2. Two plasmids present in the wild-type were lost in the variant that was also more sensitive to bavaricin and leucocin A than the wild-type indicating cross-resistance to class IIa bacteriocins. The growth rate of the bac- mutant was higher than that of the wild-type at 5 and 37 degrees C but not at 25 or 30 degrees C. In salmon juice the maximum cell density of L. monocytogenes was suppressed 3 and 6 log by co-culture with C. piscicola A9b bac- and bac+, respectively, as compared with the control. Sterile filtered cultures of C. piscicola A9b bac- caused a limited suppression of the maximum cell density of L. monocytogenes similar to that observed when sterile buffer was added in equal amounts. Semi-purified carnobacteriocin B2 caused a 3.5 log decline in viable cell count after 6 day of incubation in cold-smoked salmon juice at 5 degrees C. High resistance level to carnobacteriocin B2 was observed for L. monocytogenes cells exposed to semi-purified and in situ produced carnobacteriocin B2. CONCLUSIONS: The presence of bacteriocin production in C. piscicola enhances its inhibition of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the emergence of resistance, a bacteriocin negative lactic acid bacteria may be more suited for practical use as a bioprotective agent against L. monocytogenes in ready-to-eat foods.     

Survival and heat resistance of Listeria monocytogenes after exposure to alkali and chlorine

A strain of Listeria monocytogenes isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59 degrees C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21 degrees C. Cells of L. monocytogenes incubated at 37 degrees C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56 degrees C than were cells stored in TPB at pH 7.3. The alkaline-stress response in L. monocytogenes may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 +/- 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log(10) CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log(10) CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56 degrees C than cells treated for 5 min. Contamination of foods by L. monocytogenes cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of L. monocytogenes against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log(10)-unit reductions is not compromised in these cells.     

Growth of Listeria monocytogenes at different pH values in uncultured whey or whey cultured with Penicillium camemberti

Wheys from making Camembert cheese were either uncultured or cultured with Penicillium camemberti, adjusted to pH 5.0, 5.2, 5.4, 5.6, 6.2, and 6.8, and filter sterilized. Whey samples were inoculated to contain 100 to 500 Listeria monocytogenes (strains Scott A, V7, CA, or OH) cfu/mL and incubated at 6 degrees C. Counts of L. monocytogenes were obtained by surface plating appropriate dilutions on Tryptose Agar. Listeria monocytogenes failed to grow at or below pH 5.4; except for strains Scott A and OH which grew in cultured whey at pH 5.4 and attained populations of 7.8 x 10(3) and 5.4 x 10(4) cfu/mL, respectively, after 35 d of storage. In uncultured whey at pH 5.6, 6.2, and 6.8, populations of L. monocytogenes increased from 7.20 to 7.81, 7.51 to 8.23, and 7.48 to 8.08 log10 cfu/mL, respectively, after 35 d of storage at 6 degrees C. In cultured whey at pH 5.6, 6.2, and 6.8, numbers of L. monocytogenes increased from 7.53 to 8.13, 7.82 to 8.55, and 7.95 to 8.80 log10 cfu/mL, respectively, after 35 d of storage. Generation times for L. monocytogenes at 6 degrees C in uncultured whey at pH 5.6, 6.2, and 6.8 ranged between 25.3 and 31.6 h, 14.8 and 21.1 h, and 14.0 and 19.4 h, respectively, depending on the Listeria strain. In contrast, generation times were significantly (p less than 0.05) shorter in cultured whey and ranged between 16.6 and 27.4 h, 10.3 and 16.6 h, and 17.4 and 16.3 h at pH values of 5.6, 6.2, and 6.8, respectively.     

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.     

Enhanced control of Listeria monocytogenes by in situ-produced pediocin during dry fermented sausage production.

To determine whether pediocin is produced and has effective antilisterial activity during food fermentation, six sausage fermentation trials were conducted with antibiotic-resistant, pediocin-producing (Bac+) Pediococcus acidilactici PAC 1.0 (Strr Rifr) and an isogenic pediocin-negative (Bac-) derivative used as a control. Meat was inoculated (ca. 10(5) CFU/g) with a composite of five Listeria monocytogenes strains, each electrotransformed with pGK12 (Cmr Emr). P. acidilactici and L. monocytogenes populations were selectively enumerated by plating on media with antibiotics. This study indicated that the dry sausage fermentation process can reduce L. monocytogenes populations. Effective inactivation of L. monocytogenes was observed when the pH at the end of the fermentation portion of the process was less than 4.9. Pediocin was responsible for part of the antilisterial activity during the fermentation in each of the six trials. Furthermore, inhibition of L. monocytogenes during drying was enhanced in the presence of pediocin in the three trials in which L. monocytogenes could be detected throughout the drying process. Thus, pediocin production contributed to an increase in safety during both the fermentation and drying portions of sausage manufacturing.     

Influence of the natural microbial flora on the acid tolerance response of Listeria monocytogenes in a model system of fresh meat decontamination fluids

Depending on its composition and metabolic activity, the natural flora that may be established in a meat plant environment can affect the survival, growth, and acid tolerance response (ATR) of bacterial pathogens present in the same niche. To investigate this hypothesis, changes in populations and ATR of inoculated (10(5) CFU/ml) Listeria monocytogenes were evaluated at 35 degrees C in water (10 or 85 degrees C) or acidic (2% lactic or acetic acid) washings of beef with or without prior filter sterilization. The model experiments were performed at 35 degrees C rather than lower (8.0 log CFU/ml) by day 1. The pH of inoculated water washings decreased or increased depending on absence or presence of natural flora, respectively. These microbial and pH changes modulated the ATR of L. monocytogenes at 35 degrees C. In filter-sterilized water washings, inoculated L. monocytogenes increased its ATR by at least 1.0 log CFU/ml from days 1 to 8, while in unfiltered water washings the pathogen was acid tolerant at day 1 (0.3 to 1.4 log CFU/ml reduction) and became acid sensitive (3.0 to >5.0 log CFU/ml reduction) at day 8. These results suggest that the predominant gram-negative flora of an aerobic fresh meat plant environment may sensitize bacterial pathogens to acid.     

Simple method to identify bacteriocin induction peptides and to auto-induce bacteriocin production at low cell density

The production of some bacteriocins by lactic acid bacteria is regulated by induction peptides (IPs) that are secreted by a dedicated secretion system. The IP gene cbaX, for carnobacteriocin A production by Carnobacterium piscicola LV17A, and a presumptive IP gene (orf6), associated with the genetic locus for enterocin B production in Enterococcus faecium BFE 900, were fused to the signal peptide of the bacteriocin divergicin A from Carnobacterium divergens LV13 to access the general secretory pathway. The culture supernatants of C. piscicola UAL26 and Lactococcus lactis MG1363 containing either of these constructs were used to induce bacteriocin production by Bac(-) cultures of C. piscicola LV17A or E. faecium CTC492. The cbaX fusion product induced bacteriocin production by Bac(-) C. piscicola LV17A, but the orf6 fusion product did not induce bacteriocin production by E. faecium CTC492. This represents a relatively simple method of confirming the role of presumptive IPs. The transformation of C. piscicola LV17A with the CbaX gene under expression of the P32 promoter from L. lactis resulted in constitutive production of bacteriocin by either the dedicated transport apparatus or the general secretory pathway.     

Pathogenicity of Listeria monocytogenes grown on crabmeat

The pathogenicity of Listeria monocytogenes as influenced by growth on crabmeat at 5 and 10 degrees C was studied. Crabmeat was inoculated with L. monocytogenes V7 (ca. 10(4) CFU/g) and incubated for up to 14 days at 5 and 10 degrees C. At selected incubation times, L. monocytogenes was removed from crabmeat by washing with 0.1 M potassium phosphate buffer (pH 7.0), and populations were determined by surface plating on LiCl-phenylethanol-moxalactam agar. Buffered suspensions were then centrifuged, and the resulting pellets were suspended in phosphate buffer containing 10% glycerol and stored at -18 degrees C. Thawed, diluted suspensions of cells were tested for pathogenicity by intraperitoneal injection into immunocompromised and nonimmunocompromised mice. L. monocytogenes cells recovered from crabmeat and then recultured in tryptose phosphate broth (TPB), as well as cells which had not been passed through crabmeat but had been cultured in TPB, were likewise harvested, suspended in buffered 10% glycerol, frozen, thawed, diluted, and tested for pathogenicity by intraperitoneal injection. Growth on crabmeat at 5 and 10 degrees C did not have a significant effect on pathogenicity. The population of L. monocytogenes necessary to kill about 50% of the immunocompromised mice in each test set within 7 days was about 10(4) CFU, and this result was not significantly affected by storage temperature of the crabmeat or type of substrate, i.e., crabmeat or TPB, on which it had grown.     

Pathogenicity of Listeria monocytogenes grown on crabmeat.

The pathogenicity of Listeria monocytogenes as influenced by growth on crabmeat at 5 and 10 degrees C was studied. Crabmeat was inoculated with L. monocytogenes V7 (ca. 10(4) CFU/g) and incubated for up to 14 days at 5 and 10 degrees C. At selected incubation times, L. monocytogenes was removed from crabmeat by washing with 0.1 M potassium phosphate buffer (pH 7.0), and populations were determined by surface plating on LiCl-phenylethanol-moxalactam agar. Buffered suspensions were then centrifuged, and the resulting pellets were suspended in phosphate buffer containing 10% glycerol and stored at -18 degrees C. Thawed, diluted suspensions of cells were tested for pathogenicity by intraperitoneal injection into immunocompromised and nonimmunocompromised mice. L. monocytogenes cells recovered from crabmeat and then recultured in tryptose phosphate broth (TPB), as well as cells which had not been passed through crabmeat but had been cultured in TPB, were likewise harvested, suspended in buffered 10% glycerol, frozen, thawed, diluted, and tested for pathogenicity by intraperitoneal injection. Growth on crabmeat at 5 and 10 degrees C did not have a significant effect on pathogenicity. The population of L. monocytogenes necessary to kill about 50% of the immunocompromised mice in each test set within 7 days was about 10(4) CFU, and this result was not significantly affected by storage temperature of the crabmeat or type of substrate, i.e., crabmeat or TPB, on which it had grown.