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.     

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.     

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.     

Acid adaptation and survival of Listeria monocytogenes in Italian-style soft cheeses

AIMS: The ability of Listeria monocytogenes to survive and grow at high salt concentrations and low pH makes it a potential hazard after the consumption of milk and dairy products, often implicated in severe outbreaks of listeriosis. This study was designed to evaluate the behaviour of L. monocytogenes in traditional acid and salted Italian-style soft cheeses and to investigate whether Listeria occurrence and growth in these environments may represent a potential increase of hazard. METHODS AND RESULTS: A first approach was addressed to in vitro evaluate survival, acid tolerance response, ability to produce biofilm, and capability to invade intestinal-like cells of a L. monocytogenes strain grown under experimental conditions mimicking environmental features that this pathogen encounters in soft cheeses (such as acid pH and high NaCl content). A second set of experiments was performed to monitor, during the storage at 4 degrees C, the survival of acid-adapted and nonadapted Listeriae in artificially contaminated soft cheeses. Both acid tolerance response and invasion efficiency of acid-adapted bacteria resulted in an increase, even when bacteria were simultaneously pre-exposed to increasing salt stress. The contamination of cheeses with acid-adapted and nonadapted bacteria evidenced in all products a good survival. A significant increased survival, the recovery of bacterial cells highly resistant to lethal pH exposure, and the prevalence of filamentous structures were observed in crescenza cheese during the storage. CONCLUSIONS: The Listeria survival and acid pH tolerance observed during refrigerated storage are probably related to the intrinsic acid and saline features of soft cheeses analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Italian soft cheeses tested may represent a potential hazard for the recovery of acid-adapted L. monocytogenes cells with enhanced ability to adhere to inert surfaces and/or to penetrate host cells.     

Physiological and biochemical aspects of the acid survival of Listeria monocytogenes

The physiological aspects of the response to acidic conditions and the correlated protein synthesis were studied by using Listeria monocytogenes grown in a chemically defined synthetic medium. This growth was greatly affected by pH of the medium. It decreased when pH declined and was arrested at pH 4. When pH went under 4, the bacteria began to die. If the bacteria had been adapted to an intermediary sublethal pH before imposition of lethal pH stress, they would have resisted better lethal pH. A prolonged treatment at intermediary pH, however, rendered the bacteria more sensitive to subsequent lethal pH. Organic volatile acids exerted a more deleterious effect on L. monocytogenes than inorganic acids at the same stressing pH. The acquired acid tolerance was conserved after several weeks of storage of the adapted bacteria at 4 degrees C. Acid stress and acid adaptation (tolerance) affected the synthesis patterns of bacterial proteins: Many proteins were repressed and several others increased in expression level. These acid-induced proteins were separated by two-dimensional (2D-) electrophoresis and analyzed by a computer-aided 2D-gel analysis system. The results obtained suggested that acid tolerance and acid stress responses require the synthesis of a certain number of shared proteins and that additional acid-induced proteins are needed when the bacteria must face more severe acidic pH.     

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.     

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   

A small heat shock protein from Leuconostoc oenos induced by multiple stresses and during stationary growth phase

In Leuconostoc oenos , a malolactic bacterium, the synthesis of a stress protein called LO18 with an apparent molecular mass of 18 kDa was greatly induced after heat (42°C), acid (pH 3) or ethanolic (12% (v/v)) shocks. Moreover, the LO18 protein synthesis was induced in stationary growth phase and was detected for a long time (30 h) during this growth phase. Significant identity was found between the N-terminal parts of the LO18 protein and the Hsp18 from Clostridium acetobutylicum suggesting that LO18 protein belongs to the family of small heat shock proteins conserved in prokaryotic and eukaryotic cells.     

Characterization of the protein-synthesis dependent adaptive acid tolerance response in Lactobacillus acidophilus

Exposure of L. acidophilus CRL 639 cells to sublethal adaptive acid conditions (pH 5.0 for 60 min) was found to confer protection against subsequent exposure to lethal pH (pH 3.0). Adaptation, which only occurred in complex media, was dependent on de novo protein synthesis and was inhibited by amino acid analogues. There was no modification in the protein synthesis rate during adaptation, but the protein degradation rate decreased. Synthesis of acid stress proteins may increase the stability of pre-existing proteins. By 2D-PAGE, induction of nine acid stress proteins and repression of several housekeeping proteins was observed. Putative heat shock proteins DnaK, DnaJ, GrpE, GroES and GroEL (70, 43, 24, 10 and 55 kDa, respectively) were among the proteins whose synthesis was induced in response to acid adaptation.     

The adaptive acid response in Mesorhizobium sp.

Mesorhizobium huakuii strain LL56 and Mesorhizobium sp. strain LL22, which nodulate Lotus glaber, developed an adaptive acid response during exponential growth upon exposure to sublethal acid conditions. The adaptive acid response was found to be dependent on the sublethal pH and the strain intrinsic acid tolerance: the lowest adaptation pH was 4.0 for strain LL56 and 5.7 for strain LL22, and the lowest pH values tolerated after adaptation were 3.0 and 4.0, respectively. Both complex and minimal medium allowed the development of the adaptive acid response, although in complex medium this response was more effective. Three low molecular weight polypeptides (LMWPs) showed increased expression in strain LL56 during the adaptation to pH 4.0. However, the adaptive acid tolerance was only partially dependent on de novo protein synthesis, and constitutive systems may play a significant role on the acid tolerance of Mesorhizobium huakuii strain LL56.     

The Lactic Acid Stress Response of Lactococcus lactis subsp. lactis

The lactic acid tolerance response (LATR) of the lactic acid bacterium Lactococcus lactis subsp. lactis has been studied. A dramatic increase in survival to a severe acid stress (pH 3.9) was obtained by preexposing the cells for 30 min to a mildly acid shock at pH 5.5. Whole-cell protein extract analysis revealed that during the acid tolerance response 33 polypeptides are induced over the level of naive cells. Among these are the major heat shock proteins DnaK and GroEL. In conjunction with a previous report (Hartke et al. 1994), the results establish that L. lactis can adapt to lactic acid exposure in two different ways: a logarithmic phase LATR, which may be activated by protons, and a stationary-phase LATR, which needs no activation by protons. Both systems are independent of de novo protein synthesis. Received: 8 February 1996 / Accepted: 11 March 1996     

Characterization of a highly hop-resistant Lactobacillus brevis strain lacking hop transport

Resistance to hops is a prerequisite for lactic acid bacteria to spoil beer. In this study we analyzed mechanisms of hop resistance of Lactobacillus brevis at the metabolism, membrane physiology, and cell wall composition levels. The beer-spoiling organism L. brevis TMW 1.465 was adapted to high concentrations of hop compounds and compared to a nonadapted strain. Upon adaptation to hops the metabolism changed to minimize ethanol stress. Fructose was used predominantly as a carbon source by the nonadapted strain but served as an electron acceptor upon adaptation to hops, with concomitant formation of acetate instead of ethanol. Furthermore, hop adaptation resulted in higher levels of lipoteichoic acids (LTA) incorporated into the cell wall and altered composition and fluidity of the cytoplasmic membrane. The putative transport protein HitA and enzymes of the arginine deiminase pathway were overexpressed upon hop adaptation. HorA was not expressed, and the transport of hop compounds from the membrane to the extracellular space did not account for increased resistance to hops upon adaptation. Accordingly, hop resistance is a multifactorial dynamic property, which can develop during adaptation. During hop adaptation, arginine catabolism contributes to energy and generation of the proton motive force until a small fraction of the population has established structural improvements. This acquired hop resistance is energy independent and involves an altered cell wall composition. LTA shields the organism from accompanying stresses and provides a reservoir of divalent cations, which are otherwise scarce as a result of their complexation by hop acids. Some of the mechanisms involved in hop resistance overlap with mechanisms of pH resistance and ethanol tolerance and as a result enable beer spoilage by L. brevis.     

Role of Listeria monocytogenes sigma(B) in survival of lethal acidic conditions and in the acquired acid tolerance response

The food-borne pathogen Listeria monocytogenes can acquire enhanced resistance to lethal acid conditions through multiple mechanisms. We investigated contributions of the stress-responsive alternative sigma factor, sigma(B), which is encoded by sigB, to growth phase-dependent acid resistance (AR) and to the adaptive acid tolerance response in L. monocytogenes. At various points throughout growth, we compared the relative survival of L. monocytogenes wild-type and DeltasigB strains that had been exposed to either brain heart infusion (pH 2.5) or synthetic gastric fluid (pH 2.5) with and without prior acid adaptation. Under these conditions, survival of the DeltasigB strain was consistently lower than that of the wild-type strain throughout all phases of growth, ranging from 4 orders of magnitude less in mid-log phase to 2 orders of magnitude less in stationary phase. Survival of both DeltasigB and wild-type L. monocytogenes strains increased by 6 orders of magnitude upon entry into stationary phase, demonstrating that the L. monocytogenes growth phase-dependent AR mechanism is sigma(B) independent. sigma(B)-mediated contributions to acquired acid tolerance appear to be greatest in early logarithmic growth. Loss of a functional sigma(B) reduced the survival of L. monocytogenes at pH 2.5 to a greater extent in the presence of organic acid (100 mM acetic acid) than in the presence of inorganic acid alone (HCl), suggesting that L. monocytogenes protection against organic and inorganic acid may be mediated through different mechanisms. sigma(B) does not appear to contribute to pH(i) homeostasis through regulation of net proton movement across the cell membrane or by regulation of pH(i) buffering by the GAD system under the conditions examined in this study. In summary, a functional sigma(B) protein is necessary for full resistance of L. monocytogenes to lethal acid treatments.     

Type I IFN are host modulators of strain-specific Listeria monocytogenes virulence

Type I IFN (IFN-I) increase the sensitivity of cells and mice to lethal infection with Listeria monocytogenes . Therefore the amount of IFN-I produced during infection might be an important factor determining Listeria virulence. Two commonly used strains of L. monocytogenes , EGD and LO28, were identified as, respectively, low and high inducers of IFN-I synthesis in infected macrophages. Increased IFN-I production resulted from the stronger ability of the LO28 strain to trigger the IRF3 signalling pathway and correlated with an increased sensitization of macrophages to lethal infection. In contrast, stimulation of NFκB, MAPK, or inflammasome signalling by the LO28 and EGD strains did not differ significantly. The LO28 strain was more virulent in wild-type (wt) C57/BL6 mice than the EGD strain whereas both strains were similarly virulent in IFN-I receptor-deficient C57/BL6 mice. Together our data suggest that isolates of wt L. monocytogenes differ in their ability to trigger the IRF3 signalling pathway and IFN-I production, and that the amount of IFN-I produced during infection is an important determinant of Listeria virulence.     

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.     

Evaluation of housekeeping genes in Listeria monocytogenes as potential internal control references for normalizing mRNA expression levels in stress adaptation models using real-time PCR

Listeria monocytogenes is an important food-borne pathogen that can tolerate a wide range of stress conditions. However, its stress adaptation processes are still poorly understood. Real-time-based quantitative RT-PCR (qRT-PCR) provides a tool to probe gene expression changes underlying stress adaptation. But, a limitation to study mRNA levels by real-time qRT-PCR is that validated reference genes are required for normalization. Such genes are currently lacking for experimental models that may be applied to evaluate stress-related gene expression changes in L. monocytogenes. Therefore, five housekeeping genes (HKG) were studied as potential reference genes. Their expression stability was evaluated across 16 L. monocytogenes strains. Three experimental models designed to assess gene expression changes induced by cold, acid and high NaCl concentration stress adaptation were applied. The 16S rRNA gene was consistently the most stably expressed HKG across the different L. monocytogenes strains under all the experimental conditions. While the expressions of beta-glucosidase (bglA), Glyceraldehyde-3P-dehydrogenase (gap), RNA polymerase beta subunit (rpoB) and Ribosomal protein L4 (rplD) was stable amongst the different L. monocytogenes strains, they were prone to significant variations under the different stress adaptation models.