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.     

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.     

Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation.

We have previously shown that tolerance to severe acid stress (pH 3.5) can be induced in Listeria monocytogenes following a 1-h adaptation to mild acid (pH 5.5), a phenomenon termed the acid tolerance response (ATR) (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1966). In an attempt to determine the industrial significance of the ATR, we have examined the survival of adapted and nonadapted cells in a variety of acidic foods. Acid adaptation enhanced the survival of L. monocytogenes in acidified dairy products, including cottage cheese, yogurt, and whole-fat cheddar cheese. Acid-adapted L. monocytogenes cultures also demonstrated increased survival during active milk fermentation by a lactic acid culture. Similarly, acid-adapted cells showed greatly improved survival in low-pH foods (orange juice and salad dressing) containing acids other than lactic acid. However, in foods with a marginally higher pH, such as mozzarella cheese, a commercial cottage cheese, or low-fat cheddar cheese, acid adaptation did not appear to enhance survival. We have previously isolated mutants of L. monocytogenes that are constitutively acid tolerant in the absence of an induction step (O'Driscoll et al., Appl. Environ. Microbiol. 62:1693-1698, 1996). In the present study, one such mutant, ATM56, demonstrated an increased ability to survive in low-pH foods and during milk fermentation when compared with the wild-type strain. Significant numbers of ATM56 could be recovered even after 70 days in both whole-fat and low-fat cheddar cheese. Collectively, the data suggest that ATR mechanisms, whether constitutive or induced, can greatly influence the survival of L. monocytogenes in low-pH food environments.     

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.     

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.     

Simple method to observe the adaptive response of Listeria monocytogenes in food

A simple, novel method for determining stress-adaptive response of Listeria monocytogenes in food systems is presented. The method involves plating samples on Listeria-selective agar (LSA) acidified to pH 5.25 with incubation at 36 degrees C for 60 h to detect acid adaptation and plating on LSA with 70 gl-1 NaCl and incubation at 7 degrees C for 7 d to detect cold-osmotic adaptation. Adapted cells produced larger colonies (> 1 mm) under these conditions than unadapted cells. Scot A (97%) and Brie-1 (100%) cells incubated in milk at pH 5 for 3 h manifested the acid-adapted colony type compared with 6% and 21% of viable cells in the unstressed control population. After a 5-d adaptation period at 4 degrees C in milk with 80 gl-1 salt, 29% of Scot A and 91% of Brie-1 viable cells exhibited the adapted colony type compared with < 1% of the unstressed control population. Stress-adapted L. monocytogenes were isolated from soft cheese held for 42 d at 10 C.     

Modelling the behaviour of Listeria monocytogenes in ground pork as a function of pH, water activity, nature and concentration of organic acid salts

AIMS: to study and model the effect of sodium acetate, sodium lactate, potassium sorbate and combination of acid salts on the behaviour of Listeria monocytogenes in ground pork. METHODS AND RESULTS: Water activity (a(w)), pH and concentration of acid salt of the meat were adjusted. The behaviour of inoculated L. monocytogenes was studied and modelled according to physicochemical parameters values. Whatever the acid salt concentration used, we observed an inhibition of the growth of L. monocytogenes at pH 5.6 and a(w) 0.95. At pH 6.2 and a(w) 0.97, addition of 402 mmol l(-1) of sodium lactate or 60 mmol l(-1) of potassium sorbate was required to observe a slower growth. CONCLUSIONS: The inhibitory effect of acid salts was a function of pH, a(w), as well as of the nature and concentration of acid salts added. When one acid salt was added, the Augustin's model (Augustin et al. 2005) yielded generally correct predictions of either the survival or growth of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The suggested model can be used for risk assessment concerning L. monocytogenes in pork products.     

Factors controlling acid tolerance of Listeria monocytogenes: effects of nisin and other ionophores.

The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.     

Sigma B在单核细胞增生李斯特菌耐受环境压力胁迫中的作用

摘要：Sigma B（σB）因子在单核细胞增生李斯特菌（Listeria monocytogenes）的压力应答和毒力调控中起着重要的作用。研究发现单核细胞增生李斯特菌的致病力与耐受环境条件的胁迫息息相关。在压力存在的情况下能启动一些基因的表达,以增强细菌对环境的耐受性,这些基因包括与耐受渗透压、酸碱性环境、氧化、极端温度和宿主体内胆碱等环境压力相关的基因。本文综述了σB因子在上述几种环境压力胁迫中的作用,为深入了解该菌的生理特征、探讨食品的最佳生产和保藏条件、防止细菌的感染等方面提供新的理论依据。     

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.     

Growth, morphology and surface properties of Listeria monocytogenes Scott A and LO28 under saline and acid environments

AIMS: The effect of salt and acid on the growth and surface properties of two strains of Listeria monocytogenes was investigated. METHODS AND RESULTS: Medium acidification and NaCl supplementation induced a marked increase in the lag and growth times (up to fivefold higher) and a decrease in the maximal optical density. Due to a strong synergic effect of pH and NaCl, growth was only detected after 280 h incubation for Scott A and not detected after 600 h for LO28 at pH 5.0 and 10% NaCl. Furthermore, the addition of NaCl in acidic conditions gave rise to cell filamentation and cell surfaces became strongly hydrophilic. CONCLUSIONS: Some L. monocytogenes strains subjected to high NaCl concentrations in acidic conditions are able to grow but may present altered adhesion properties due to modification of their surface properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted that L. monocytogenes do represent a hazard in acid and salted foods, such as soft cheese.     

Comparative acid stress response of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium after habituation at different pH conditions

AIMS: The aim of the study was to evaluate the effect of habituation at different pH conditions on the acid resistance of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium, and to identify potential differences between the adaptive responses of the three pathogens. METHODS: Stationary phase cells of L. monocytogenes, E. coli O157:H7 and S. Typhimurium, grown in glucose-free media, were exposed to pH 3.5 broth directly or after habituation for 90 min at various pH conditions from 4.0 to 6.0. Survivors at pH 3.5 were determined by plating on tryptic soy agar and incubating at 30 degrees C for 48 h. The kinetics (death rate) of the pathogens at pH 3.5 was calculated by fitting the data to an exponential model. RESULTS: Habituation to acidic environments provided protection of the pathogens against lethal acid conditions. This acid protection, however, was found to be pH dependent. For example, for E. coli O157:H7 an increased acid resistance was observed after habituation at a pH range from 4.0 to 5.5, while the maximum acid tolerance was induced at pH 5.0. Furthermore, the effect of low pH habituation was different among pathogens. For L. monocytogenes, E. coli O157:H7 and S. Typhimurium, the pH range within which habituation resulted to increased acid resistance was 5.0-6.0, 4.0-5.5 and 4.0-5.0, respectively, while the maximum acid tolerance was induced after habituation at pH 5.5, 5.0 and 4.5, respectively. SIGNIFICANCE: Acid stress conditions are common within current food processing technologies. The information on adaptive responses of L. monocytogenes, E. coli O157:H7 and S. Typhimurium after habituation to different pH environments provided in the present study, could lead to a more realistic evaluation of food safety concerns and to a better selection of processes in order to avoid adaptation phenomena and to minimize the potential for food safety risks.     

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.     

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.     

Fate of low temperature and acid-adapted Yersinia enterocolitica and Listeria monocytogenes that contaminate lactic acid decontaminated meat during chill storage

Pathogens found in the environment of abattoirs may become adapted to lactic acid used to decontaminate meat. Such organisms are more acid tolerant than non-adapted parents and can contaminate meat after lactic acid decontamination (LAD). The fate of acid-adapted Yersinia enterocolitica and Listeria monocytogenes, inoculated on skin surface of pork bellies 2 h after LAD, was examined during chilled storage. LAD included dipping in 1%, 2% or 5% lactic acid solutions at 55°C for 120 s. LAD brought about sharp reductions in meat surface pH, but these recovered with time after LAD at ≈1–1·5 pH units below that of water-treated controls. Growth permitting pH at 4·8–5·2 was reached after 1% LAD in less than 0·5 d (pH 4·8–5·0), 2% LAD within 1·5 d (pH 4·9–5·1) and after 5% LAD (pH 5·0–5·2) within 4 d. During the lag on 2% LAD meat Y. enterocolitica counts decreased by 0·9 log₁₀ cfu per cm² and on 5% LAD the reduction was more than 1·4 log₁₀ cfu per cm². The reductions in L. monocytogenes were about a third of those in Y. enterocolitica . On 1% LAD the counts of both pathogens did not decrease significantly. The generation times of Y. enterocolitica and L. monocytogenes on 2–5% LAD meats were by up to twofold longer than on water-treated controls and on 1% LAD-treated meat they were similar to those on water-treated controls. Low temperature and acid-adapted L. monocytogenes and Y. enterocolitica that contaminate skin surface after hot 2–5% LAD did not cause an increased health hazard, although the number of Gram-negative spoilage organisms were drastically reduced by hot 2–5% LAD and intrinsic (lactic acid content, pH) conditions were created that may benefit the survival and the growth of acid-adapted organisms.     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

Acid adaptation sensitizes Salmonella typhimurium to hypochlorous acid.

Acid adaptation of Salmonella typhimurium at a pH of 5.0 to 5.8 for one to two cell doublings resulted in marked sensitization of the pathogen to halogen-based sanitizers including chlorine (hypochlorous acid) and iodine. Acid-adapted S. typhimurium was more resistant to an anionic acid sanitizer than was its nonadapted counterpart. A nonselective plating medium of tryptose phosphate agar plus 1% pyruvate was used throughout the study to help recover chemically stressed cells. Mechanisms of HOCl-mediated inactivation of acid-adapted and nonadapted salmonellae were investigated. Hypochlorous acid oxidized a higher percentage of cell surface sulfhydryl groups in acid-adapted cells than in nonadapted cells, and sulfhydryl oxidation was correlated with cell inactivation. HOCl caused severe metabolic disruptions in acid-adapted and nonadapted S. typhimurium, such as respiratory loss and inability to restore the adenylate energy charge from a nutrient-starved state. Sensitization of S. typhimurium to hypochlorous acid by acid adaptation also involved increased permeability of the cell surface because nonadapted cells treated with EDTA became sensitized. The results of this study establish that acid-adapted S. typhimurium cells are highly sensitized to HOCl oxidation and that inactivation by HOCl involves changes in membrane permeability, inability to maintain or restore energy charge, and probably oxidation of essential cellular components. This study provides a basis for improved practical technologies to inactivate Salmonella and implies that acid pretreatment of food plant environments may increase the efficacy of halogen sanitizers.     

Detection of Listeria monocytogenes in Italian-style soft cheeses

AIMS: A rapid detection system specific for Listeria monocytogenes and based on the polymerase chain reaction (PCR) was developed. METHODS AND RESULTS: Primers annealing to the coding region of the actA gene, critically involved in virulence and capable of discrimination between two different alleles naturally occurring in L. monocytogenes, have been utilized. The procedure was applied to recover L. monocytogenes cells in artificially contaminated fresh Italian soft cheeses (mozzarella, crescenza and ricotta). Low levels of L. monocytogenes were detected in mozzarella and crescenza homogenates (0.04-0.4 and 4 CFU g(-1), respectively) whereas in ricotta the detection limit was higher (40 CFU g(-1)). CONCLUSIONS: This PCR-based assay is highly specific as primers used recognize the DNA from different L. monocytogenes strains of clinical and food origin, while no amplification products result with any other Listeria spp. strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted a low-cost and rapid procedure that can be appropriated for the detection in real time of low L. monocytogenes levels in soft cheese.     

Effect of acid-adaptation on Listeria monocytogenes survival and translocation in a murine intragastric infection model

Acid tolerance response mechanisms can greatly influence Listeria monocytogenes survival in low pH foods. In the present paper, the effect of acid-adaptation together with control of gastric pH level on L. monocytogenes survival and translocation was analyzed after intragastric inoculation in the BALB/c mouse model. Our results showed that acid-adaptation led to an increase in resistance to the first barrier constituted by the low gastric pH and that inoculation at alkaline pH had a synergistic effect. It resulted in a higher live bacterial load reaching the next intestinal compartments and was correlated with increased translocation rates to the mesenteric lymph nodes, both at the frequency and quantitative levels. Our results in this murine model suggest that acid-adaptation of L. monocytogenes in low pH foods, together with control of gastric pH level through dietary practices, or use of inhibitors of gastric acid secretion, may be potential aggravating risk factors to food-borne listeriosis.     

Inhibition of Listeria monocytogenes by food-borne yeasts

Many bacteria are known to inhibit food pathogens, such as Listeria monocytogenes, by secreting a variety of bactericidal and bacteriostatic substances. In sharp contrast, it is unknown whether yeast has an inhibitory potential for the growth of pathogenic bacteria in food. A total of 404 yeasts were screened for inhibitory activity against five Listeria monocytogenes strains. Three hundred and four of these yeasts were isolated from smear-ripened cheeses. Most of the yeasts were identified by Fourier transform infrared spectroscopy. Using an agar-membrane screening assay, a fraction of approximately 4% of the 304 red smear cheese isolates clearly inhibited growth of L. monocytogenes. Furthermore, 14 out of these 304 cheese yeasts were cocultivated with L. monocytogenes WSLC 1364 on solid medium to test the antilisterial activity of yeast in direct cell contact with Listeria. All yeasts inhibited L. monocytogenes to a low degree, which is most probably due to competition for nutrients. However, one Candida intermedia strain was able to reduce the listerial cell count by 4 log units. Another four yeasts, assigned to C. intermedia (three strains) and Kluyveromyces marxianus (one strain), repressed growth of L. monocytogenes by 3 log units. Inhibition of L. monocytogenes was clearly pronounced in the cocultivation assay, which simulates the conditions and contamination rates present on smear cheese surfaces. We found no evidence that the unknown inhibitory molecule is able to diffuse through soft agar.