Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of the Acid Tolerance Response in Listeria monocytogenes LO28

Listeria monocytogenes is capable of withstanding low pH after initial exposure to sublethal acidic conditions, a phenomenon termed the acid tolerance response (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1996). Treatment of L. monocytogenes LO28 with chloramphenicol during acid adaptation abrogated the protective effect, suggesting that de novo protein synthesis is required for the acid tolerance response. Analysis of protein expression during acid adaptation by two-dimensional gel electrophoresis revealed changes in the levels of 53 proteins. Significant protein differences were also evident between nonadapted L. monocytogenes LO28 and a constitutively acid-tolerant mutant, ATM56. In addition, the analysis[S_TABC] revealed differences in protein expression between cells induced with a weak acid (lactic acid) and those induced with a strong acid (HCl). Comparison of both acid-adapted LO28 and ATM56 revealed that both are capable of maintaining their internal pH (pH(infi)) at higher levels than nonadapted control cells during severe acid stress. Collectively, the data demonstrate the profound alterations in protein synthesis which take place during acid adaptation in L. monocytogenes and ultimately lead to an increased ability to survive severe stress conditions.     

The survival of Listeria monocytogenes in cottage cheese

Because of the difficulty of ensuring that cottage cheese is produced in conditions that prevent contamination with Listeria monocytogenes , the ability of this bacterium to survive in cottage cheese from three sources was investigated (a) during shelf-life at chill temperature and (b) in conditions of temperature abuse. Three batches of creamed cottage cheese, from three sources, received within 24 h of production, were inoculated with L. monocytogenes strain F6861 and stored at 4, 8 or 12°C for 14 d. The three batches differed in their initial pH, titratable acidity and content of lactic acid and of lactic acid bacteria. No increase in numbers of L. monocytogenes occurred in the cottage cheeses during storage in these conditions. The numbers of listeria decreased; the rate of decrease differed in products from the three sources and was least in the product with the highest pH and lowest content of lactic acid. Acid formation by lactic acid bacteria during storage of the products probably contributed to the inhibition of listeria.     

The survival of Listeria monocytogenes in cottage cheese

Because of the difficulty of ensuring that cottage cheese is produced in conditions that prevent contamination with Listeria monocytogenes, the ability of this bacterium to survive in cottage cheese from three sources was investigated (a) during shelf-life at chill temperature and (b) in conditions of temperature abuse. Three batches of creamed cottage cheese, from three sources, received within 24 h of production, were inoculated with L. monocytogenes strain F6861 and stored at 4, 8 or 12 degrees C for 14 d. The three batches differed in their initial pH, titratable acidity and content of lactic acid and of lactic acid bacteria. No increase in numbers of L. monocytogenes occurred in the cottage cheeses during storage in these conditions. The numbers of listeria decreased; the rate of decrease differed in products from the three sources and was least in the product with the highest pH and lowest content of lactic acid. Acid formation by lactic acid bacteria during storage of the products probably contributed to the inhibition of listeria.     

Evaluation of the pH-dependent,stationary-phase acid tolerance in Listeria monocytogenes and Salmonella Typhimurium DT104 induced by culturing in media with 1% glucose: a comparative study with Escherichia coli O157:H7

AIMS: To comparatively evaluate the adaptive stationary-phase acid tolerance response (ATR) in food-borne pathogens induced by culturing in glucose-containing media, as affected by strain variability and antibiotic resistance, growth temperature, challenge pH and type of acidulant. METHODS AND RESULTS: Antibiotic resistant or sensitive strains of Listeria monocytogenes, Salmonella including S. Typhimurium DT104, and Escherichia coli O157:H7 were cultured (30 degrees C for 24 h; 10 degrees C for up to 14 days) in trypticase soya broth with yeast extract (TSBYE) with 1% or without glucose to induce or prevent acid adaptation, respectively. Cultures were subsequently exposed to pH 3.5 or 3.7 with lactic or acetic acid at 25 degrees C for 120 min. Acid-adapted cultures were more acid tolerant than nonadapted cultures, particularly those of L. monocytogenes and Salmonella. No consistent, positive or negative, influence of antibiotic resistance on the pH-inducible ATR or acid resistance (AR) was observed. Compared with 30 degrees C cultures, growth and acid adaptation of L. monocytogenes and S. Typhimurium DT104 at 10 degrees C markedly reduced their ATR and AR in stationary phase. E. coli O157:H7 had the greatest AR, relying less on acid adaptation. A 0.2 unit difference in challenge pH (3.5-3.7) caused great variations in survival of acid-adapted and nonadapted cells. CONCLUSIONS: Culturing L. monocytogenes and Salmonella to stationary phase in media with 1% glucose induces a pH-dependent ATR and enhances their survival to organic acids; thus, this method is suitable for producing acid-adapted cultures for use in food challenge studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial pathogens may become acid-adapted in foods containing glucose or other fermentable carbohydrates. Low storage temperatures may substantially decrease the stationary-phase ATR of L. monocytogenes and S. Typhimurium DT104, but their effect on ATR of E. coli O157:H7 appears to be far less dramatic.     

Acid-tolerant Listeria monocytogenes persist in a model food system fermented with nisin-producing bacteria

AIMS: To investigate the induction of the acid tolerance response (ATR) in Listeria monocytogenes and to assess the persistence of the pathogen in broth fermented using a nisin-producing starter culture. METHODS AND RESULTS: Lactic, acetic and hydrochloric acids were used to induce the ATR in L. monocytogenes growing at early exponential phase. Cells were then challenged in medium acidified to pH 3.5 with the same acid. Only lactic acid induced a detectable ATR. ATR+ cells maintained their initial numbers after 1 h exposure while ATR- were reduced by c. 4 log10 CFU. ATR+ or ATR- cells were also inoculated in M17G broth fermented with nisin-producing (nis+) or control (nis-) Lactococcus lactis. When exposed to nisin, the numbers of ATR+ cells were c. 2 log10 CFU higher than non detectable ATR- cells at day 3. In the absence of nisin (nis- culture), L. monocytogenes was recovered from all ATR+ and ATR- samples after 30 days. In contrast, no L. monocytogenes were recovered from any nis+ATR- samples but four of five nis+ATR+ samples were positive for L. monocytogenes after 30 days. CONCLUSIONS: The ATR confers cross-resistance to nisin for at least 30 days in a system fermented by nisin-producing bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The cross-resistance induced by the ATR should be considered for the safety of foods fermented with bacteriocin-producing cultures.     

Acid adaptation promotes survival of Salmonella spp. in cheese.

Salmonella typhimurium was adapted to acid by exposure to hydrochloric acid at pH 5.8 for one to two doublings. Acid-adapted cells had increased resistance to inactivation by organic acids commonly present in cheese, including lactic, propionic, and acetic acids. Recovery of cells during the treatment with organic acids was increased 1,000-fold by inclusion of 0.1% sodium pyruvate in the recovery medium. Acid-adapted S. typhimurium cells survived better than nonadapted cells during a milk fermentation by a lactic acid culture. Acid-adapted cells also showed enhanced survival over a period of two months in cheddar, Swiss, and mozzarella cheeses kept at 5 degrees C. Acid adaptation was found in Salmonella spp., including Salmonella enteritidis, Salmonella choleraesuis subsp. choleraesuis serotype heidelberg, and Salmonella choleraesuis subsp. choleraesuis serotype javiana, associated with food poisoning. These observations support the theory that acid adaptation is an important survival mechanism enabling Salmonella spp. to persist in fermented dairy products and possibly other acidic food products.     

Acid adaptation promotes survival of Salmonella spp. in cheese.

Salmonella typhimurium was adapted to acid by exposure to hydrochloric acid at pH 5.8 for one to two doublings. Acid-adapted cells had increased resistance to inactivation by organic acids commonly present in cheese, including lactic, propionic, and acetic acids. Recovery of cells during the treatment with organic acids was increased 1,000-fold by inclusion of 0.1% sodium pyruvate in the recovery medium. Acid-adapted S. typhimurium cells survived better than nonadapted cells during a milk fermentation by a lactic acid culture. Acid-adapted cells also showed enhanced survival over a period of two months in cheddar, Swiss, and mozzarella cheeses kept at 5 degrees C. Acid adaptation was found in Salmonella spp., including Salmonella enteritidis, Salmonella choleraesuis subsp. choleraesuis serotype heidelberg, and Salmonella choleraesuis subsp. choleraesuis serotype javiana, associated with food poisoning. These observations support the theory that acid adaptation is an important survival mechanism enabling Salmonella spp. to persist in fermented dairy products and possibly other acidic food products.     

The effect of nisin on Listeria monocytogenes in culture medium and long-life cottage cheese

M.A.S.S. FERREIRA AND B.M. LUND. 1996. The sensitivity to nisin of 27 strains of Listeria monocytogenes , four of L. innocua and one of L. ivanovii was estimated at pH 6.8 and pH 5.5. Strains of L. monocytogenes showed differences in sensitivity which were not correlated with serotype. Strains of L. innocua were as resistant as the most resistant strains of L. monocytogenes , whereas the strain of L. ivanovii was relatively sensitive. Two of the most resistant strains of L. monocytogenes multiplied in aerated liquid medium adjusted to pH 5.0 with HCl, incubated at 20°C; nisin, 500 IU ml^-1, prevented multiplication and caused death. Following inoculation of a resistant strain into long-life cottage cheese, pH 4.6–4.7, the number of viable L. monocytogenes decreased approximately 10-fold during storage at 20°C for 7 d; addition of nisin, 2000 IU g^-1, to the cottage cheese increased the rate of inactivation to approximately a 1000-fold decrease in 3 d.     

Salmonella enterica serovar Typhimurium and Listeria monocytogenes acid tolerance response induced by organic acids at 20 degrees C: optimization and modeling

An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20 degrees C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (t(s)), the log-linear decline (k(max)), and the magnitude of a critical component (C). The magnitude of k(max) gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.     

Adaptive acid tolerance response in Listeria monocytogenes: isolation of an acid-tolerant mutant which demonstrates increased virulence.

The ability of Listeria monocytogenes to tolerate low-pH environments is of particular importance because the pathogen encounters such environments in vivo, both during passage through the stomach and within the macrophage phagosome. In our study, L. monocytogenes was shown to exhibit a significant adaptive acid tolerance response following a 1-h exposure to mild acid (pH 5.5), which is capable of protecting cells from severe acid stress (pH 3.5). Susceptibility to pH 3.5 acid is growth phase dependent. Stationary-phase Listeria cultures are naturally resistant to the challenge pH (pH 3.5), while exponential-phase cultures require adaptation at pH 5.5 to induce acid tolerance. Adaptation requires protein synthesis, since treatment with chloramphenicol prevents the development of acid tolerance. Induction of the acid tolerance response also protects L. monocytogenes against the effect of other environmental stresses. Acid-adapted cells demonstrate increased tolerance toward thermal stress, osmotic stress, crystal violet, and ethanol. Following prolonged exposure of L. monocytogenes to pH 3.5, we isolated mutants which constitutively demonstrate increased acid tolerance at all stages of the growth cycle. These mutants do not display full acid tolerance, but their resistance to low pH can be further increased following adaptation to mild-acid conditions. The mutants demonstrated increased lethality for mice relative to that of the wild type when inoculated by the intraperitoneal route. When administered as lower inocula, the mutants reached higher levels in the spleens of infected mice than did the wild type. The data suggest that low-pH conditions may have the potential to select for L. monocytogenes mutants with increased natural acid tolerance and increased virulence.     

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.     

Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0

The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.     

Inhibition of Listeria monocytogenes by using bacteriocin PA-1 produced by Pediococcus acidilactici PAC 1.0.

The bacteriocin produced by Pediococcus acidilactici PAC 1.0, previously designated PA-1 bacteriocin, was found to be inhibitory and bactericidal for Listeria monocytogenes. A dried powder prepared from PAC 1.0 culture supernatant fortified with 10% milk powder was found to contain bacteriocin activity. An MIC against L. monocytogenes and lytic effects in broth cultures were determined. Inhibition by PA-1 powder occurred over the pH range 5.5 to 7.0 and at both 4 and 32 degrees C. In addition, inhibition of L. monocytogenes was demonstrated in several food systems including dressed cottage cheese, half-and-half cream, and cheese sauce.     

Rosmarinus officinalis L. essential oil and the related compound 1,8-cineole do not induce direct or cross-protection in Listeria monocytogenes ATCC 7644 cultivated in meat broth

Listeria monocytogenes has the capability of adapting to 1 or more antimicrobial compounds or procedures applied by the food industry to control the growth and survival of microorganisms in foods. In this study, the effects of Rosmarinus officinalis essential oil (EO) and the related compound 1,8-cineole on the inhibition of the growth and survival of L.?monocytogenes ATCC 7644 were determined. The ability of the R.?officinalis EO and 1,8-cineole to induce direct and cross-protection of bacteria against various stresses (lactic acid, pH?5.2; NaCl, 3?g/100?mL; high temperature, 45?°C) was also determined. At all concentrations tested (minimum inhibitory concentration (MIC), ? MIC, and ? MIC), both compounds inhibited the cell viability of L.?monocytogenes over 120?min of exposure. Overnight exposure of L.?monocytogenes to sublethal amounts of either the R.?officinalis EO or 1,8-cineole in meat broth revealed no induction of direct or cross-protection against lactic acid, NaCl, or high temperature. Similarly, cells subjected to 24?h cycles of adaptation with increasing amounts (? MIC to 2× MIC) of the EO and 1,8-cineole showed no increase in direct tolerance, as they were able to survive in growth medium containing up to ? MIC of either substance. These results show the antimicrobial efficacy of R.?officinalis EO and 1,8-cineole for use in systems, particularly as anti-L.?monocytogenes compounds.     

Presence of GadD1 glutamate decarboxylase in selected Listeria monocytogenes strains is associated with an ability to grow at low pH

The glutamate decarboxylase (GAD) system is critical to the survival of Listeria monocytogenes LO28 at low-pH stress (相似文献   

Growth limits of Listeria monocytogenes as a function of temperature, pH, NaCl, and lactic acid

Models describing the limits of growth of pathogens under multiple constraints will aid management of the safety of foods which are sporadically contaminated with pathogens and for which subsequent growth of the pathogen would significantly increase the risk of food-borne illness. We modeled the effects of temperature, water activity, pH, and lactic acid levels on the growth of two strains of Listeria monocytogenes in tryptone soya yeast extract broth. The results could be divided unambiguously into "growth is possible" or "growth is not possible" classes. We observed minor differences in growth characteristics of the two L. monocytogenes strains. The data follow a binomial probability distribution and may be modeled using logistic regression. The model used is derived from a growth rate model in a manner similar to that described in a previously published work (K. A. Presser, T. Ross, and D. A. Ratkowsky, Appl. Environ. Microbiol. 64:1773-1779, 1998). We used "nonlinear logistic regression" to estimate the model parameters and developed a relatively simple model that describes our experimental data well. The fitted equations also described well the growth limits of all strains of L. monocytogenes reported in the literature, except at temperatures beyond the limits of the experimental data used to develop the model (3 to 35 degrees C). The models developed will improve the rigor of microbial food safety risk assessment and provide quantitative data in a concise form for the development of safer food products and processes.     

A mutant of Listeria monocytogenes LO28 unable to induce an acid tolerance response displays diminished virulence in a murine model.

Exposing Listeria monocytogenes LO28 to sublethal pH induces protection against normally lethal pH conditions, a phenomenon known as the acid tolerance response. We identified a mutant, L. monocytogenes ATR1, which is incapable of inducing such tolerance, either against low pH or against any other stress tested. The virulence of this mutant was considerably decreased, suggesting that the acid tolerance response contributes to in vivo survival of L. monocytogenes.     

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.     

Evaluation of a spray-dried lacticin 3147 powder for the control of Listeria monocytogenes and Bacillus cereus in a range of food systems

AIMS: The potential of a powdered preparation of the bacteriocin, lacticin 3147, was investigated for the inhibition of Listeria monocytogenes and Bacillus cereus. METHODS AND RESULTS: A 10% solution of reconstituted demineralized whey powder was fermented with Lactococcus lactis DPC3147 for the generation of a lacticin 3147 containing powdered product. A 99.9% reduction in L. monocytogenes numbers occurred in the presence of the lacticin 3147 powder within 2 h in natural yogurt, and an 85% reduction was observed in cottage cheese within the same time frame. Counts of B. cereus were reduced by 80% in soup, in the presence of 1% (w/w) lacticin 3147 powder, within 3 h. CONCLUSIONS: A powdered preparation of lacticin 3147 was effective for the control of Listeria and Bacillus in natural yogurt, cottage cheese and soup. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioactive lacticin 3147 powder may find broad applications for control of Gram-positive pathogens/spoilage bacteria in a range of foods.