Acid Tolerance Response Research Articles

Acid tolerance responses and their mechanisms in Lactiplantibacillus plantarum LM1001.

The online version contains supplementary material available at 10.1007/s10068-024-01582-4.

Methods for studying microbial acid stress responses: from molecules to populations.

The study of how micro-organisms detect and respond to different stresses has a long history of producing fundamental biological insights while being simultaneously of significance in many applied microbiological fields including infection, food and drink manufacture, and industrial and environmental biotechnology. This is well-illustrated by the large body of work on acid stress. Numerous different methods have been used to understand the impacts of low pH on growth and survival of micro-organisms, ranging from studies of single cells to large and heterogeneous populations, from the molecular or biophysical to the computational, and from well-understood model organisms to poorly defined and complex microbial consortia. Much is to be gained from an increased general awareness of these methods, and so the present review looks at examples of the different methods that have been used to study acid resistance, acid tolerance, and acid stress responses, and the insights they can lead to, as well as some of the problems involved in using them. We hope this will be of interest both within and well beyond the acid stress research community.

Acid tolerance response of Salmonella during the squid storage and its amine production capacity analysis.

Salmonella exhibits a strong inducible acid tolerance response (ATR) under weak acid conditions, and can also induce high-risk strains that are highly toxic, acid resistant, and osmotic pressure resistant to aquatic products. However, the induction mechanism is not yet clear. Therefore, this study aims to simulate the slightly acidic, low-temperature, and high-protein environment during squid processing and storage. Through λRed gene knockout, exploring the effects of low-acid induction, long-term low-temperature storage, and two-component regulation on Salmonella ATR. In this study, we found the two-component system, PhoP/PhoQ and PmrA/PmrB in Salmonella regulates the amino acid metabolism system and improves bacterial acid tolerance by controlling arginine and lysine. Compared with the two indicators of total biogenic amine and diamine content, biogenic amine index and quality index were more suitable for evaluating the quality of aquatic products. The result showed that low-temperature treatment could inhibit Salmonella-induced ATR, which further explained the ATR mechanism from the amino acid metabolism.

Determination of Probiotic Viability in Yoghurts Produced with Acid Adapted Lactobacillus acidophilus ATCC 4356 and Bifidobacterium bifidum ATCC 11863 During Refrigerated Storage

Microorganisms have various stress response systems to maintain their viability when exposed to different stress conditions. In this study, Lactobacillus acidophilus ATCC 4356 and Bifidobacterium bifidum ATCC 11863 strains, used in probiotic yoghurt production, were subjected to acid (lactic and hydrochloric acid) stress to induce acid tolerance response (ATR). Yoghurts produced with both acid-adapted and non-adapted strains were stored at +4°C for 21 days. During the storage period, the pH and titratable acidity values of the yoghurts were measured, and the viability levels of the probiotic strains in the yoghurts were determined. In all yoghurt groups, a decrease in pH values and an increase in titratable acidity were observed during storage. The highest viability levels of the probiotic strains were detected on the first day of storage. Lactic acid-adapted Lb. acidophilus ATCC 4356 and B. bifidum ATCC 11863 in yoghurt showed growth at a level of 8.08 ± 0.12 and 8.08 ± 0.09 log10 Cfu/g at the first day of storage, respectively. Additionally, hydrochloric acid-adapted Lb. acidophilus ATCC 4356 and B. bifidum ATCC 11863 in yoghurt exhibited growth at levels of 7.90 ± 0.08 and 5.99 ± 0.03 log10 Cfu/g, respectively. The viability of acid-adapted Lb. acidophilus ATCC 4356 and B. bifidum ATCC 11863 showed a decrease similar way to that of the control group (non-acid adapted) during the storage period.

Acid adaptation alters Streptococcus mutans drug susceptibility profile

The ability of Streptococcus mutans, the primary causative agent of dental caries, to generate and tolerate an acidic environment is a leading factor in its capacity to outcompete other organisms within the oral cavity. The acid tolerance response (ATR) involves coordination of multiple pathways to upregulate macromolecule repair, maintenance of cytosolic pH, and restructuring the membrane. In this study, we asked if the changes associated with adaptation to a low pH environment would result in detectable changes to drug susceptibility. Accordingly, we screened using the Selleck library of 853 FDA approved drugs for agents that exhibit antimicrobial activity toward planktonic cultures of S. mutans or disrupt biofilm formation in growth medium titrated to pH 5 or in the presence of hydrogen peroxide. Results revealed that a total of 182 compounds displayed antimicrobial properties toward the test strain in at least one screening condition. Our findings provide evidence that the S. mutans ATR is characterized by a concerted shift in drug susceptibility. The presence of an intact ATR, associated with growth in continuous culture at pH 5, leads to increased sensitivity to 22 compounds and decreased sensitivity to a separate set of 20 compounds. Disruption of the ATR in a mutant lacking the fabM gene resulted in a susceptibility profile that was distinct from the result of our results. This suggests that loss of unsaturated fatty acids produces a physiological change that is not only measurable, but perhaps more significant than the process of ATR. Additional tests revealed that S. mutans exhibits a distinct drug susceptibility profile compared to a closely related, non-cariogenic bacterium, S. gordonii. Overall, shifts in S. mutans susceptibility patterns based on pH of growth medium or type of culture (batch vs. continuous culture), indicate that the ATR can be tracked using changes in drug susceptibility profile. Furthermore, our results provide clues that compounds could be useful in treatment of dental caries as well as providing molecular tools to study shifts in physiology of disease-associated bacteria.

Magnetic chitin beads (MCB) coated with Vibrio cholerae reveals transcriptome dynamics in adult mice with a complex gut microbiota

ABSTRACT Vibrio cholerae adapts to the host environment by altering gene expression. Because of the complexity of the gut microbiome, current in vivo V. cholerae transcriptome studies have focused on microbiota-undeveloped conditions, neglecting the interaction between the host’s commensal gut microbiota and V. cholerae. In this study, we analyzed the transcriptome of fully colonized adult mice in vivo using V. cholerae coated-magnetic chitin beads (vcMCB). This provides a simple yet powerful method for obtaining high-quality RNA from V. cholerae during colonization in mice. The transcriptome of V. cholerae recovered from adult mice infected with vcMCB shows differential expression of several genes when compared to V. cholerae recovered from the infant mouse and infant rabbit model. Some of these genes were also observed to be differentially expressed in previous studies of V. cholerae recovered from human infection when compared to V. cholerae grown in vitro. In particular, we confirmed that V. cholerae resists the inhibitory effects of low pH and formic acid from gut microbiota, such as Anaerostipes caccae and Dorea formicigenerans, by downregulating vc1080. We propose that the vc1080 product may protect V. cholerae from formic acid stress through a novel acid tolerance response mechanism. Transcriptomic data obtained using the vcMCB system provide new perspectives on the interaction between V. cholerae and the gut microbiota, and this approach can also be applied to studies of other pathogenic bacteria.

BglG Regulates the Heterogeneity Driven by the Acid Tolerance Response in Lacticaseibacillus paracasei L9.

The acid tolerance of lactic acid bacteria is crucial for their fermentation and probiotic functions. Acid adaption significantly enhances the acid tolerance of strains, and the phenotypic heterogeneity driven by the acid tolerance response (ATR) contributes to this process by providing a selective advantage in harsh environments. The mechanism of heterogeneity under the ATR is not yet clear, but individual gene expression differences are recognized as the cause. In this study, we observed four heterogeneous subpopulations (viable, injured, dead, and unstained) of Lacticaseibacillus paracasei L9 (L9) induced by acid adaption (pH 5.0, 40 min) using flow cytometry. The viable subpopulation represented a significantly superior acid tolerance to the injured subpopulation or total population. Different subpopulations were sorted and transcriptomic analysis was performed. Five genes were found to be upregulated in the viable subpopulation and downregulated in the injured subpopulation, and bglG (LPL9_RS14735) was identified as having a key role in this process. Using salicin (glucoside)-inducing gene expression and gene insertion mutagenesis, we verified that bglG regulated the heterogeneity of the acid stress response and that the relevant mechanisms might be related to activating hsp20. This study provides new evidence for the mechanism of the ATR and may contribute to the theoretical basis of improving the acid tolerance of Lacticaseibacillus paracasei L9.

A novel RofA-family transcriptional regulator, GadR, controls the development of acid resistance in Listeria monocytogenes.

The ability to survive the acidic conditions found in the stomach is crucial for the food-borne pathogen Listeria monocytogenes to gain access to the mammalian gastrointestinal tract. Little is currently known about how acid resistance is regulated in this pathogen and why this trait is highly variable between strains. Here, we used comparative genomics to identify a novel RofA-family transcriptional regulator, GadR, that controls the development of acid resistance. The RofA family of regulators was previously found only in a small group of bacterial pathogens, including streptococci, where they regulate virulence properties. We show that gadR encodes the dominant regulator of acid resistance in L. monocytogenes and that its sequence variability accounts for previously observed differences between strains in this trait. Together, these findings significantly advance our understanding of how this important pathogen copes with acid stress and suggest a potential molecular target to aid its control in the food chain.

An underlying mechanism for MleR activating the malolactic enzyme pathway to enhance acid tolerance in Lacticaseibacillus paracasei L9.

Tolerance to acid stress is a crucial property of probiotics against gastric acids. The malolactic enzyme pathway is one of the most important acid resistance systems in lactic acid bacteria. It has been reported that the malolactic enzyme pathway was regulated by the transcriptional regulator, MleR. However, regulatory mechanisms underlying malolactic enzyme pathway to cope with acid stress remain unknown. In this study, the acid tolerance ability of the ΔmleR deletion strain was significantly lower than that of the wild-type strain, and the complementation of the mleR gene into the ΔmleR strain restored the acid tolerance of the ΔmleR strain, indicating that MleR was involved in acid tolerance response of Lacticaseibacillus paracasei L9. Real-time quantitative PCR and transcriptional fusion experiments confirmed MleR-activated transcription of the mleST gene cluster. Furthermore, MleR was confirmed to directly bind to the promoter region of the mleST operon using ChIP assays and EMSAs. The transcription start site G of the mleST operon was located at position -198 relative to the start codon of the mleS gene. The region from -80 to -61 upstream of the transcription start site was determined to be essential for MleR binding. Moreover, L-malic acid acted as an effector for MleR to activate the transcription of the mleST operon in a dose-dependent manner. These results revealed the regulatory mechanism behind MleR-mediated activation of the malolactic enzyme pathway to enhance acid tolerance in Lc. paracasei L9. IMPORTANCE Lacticaseibacillus paracasei is extensively used as probiotics in human health and fermented dairy production. Following consumption, Lc. paracasei is exposed to a variety of physico-chemical stresses, such as low pH in the stomach and bile salts in the intestines. The high acidity of the stomach severely inhibits bacterial metabolism and growth. Therefore, the acid tolerance response is critical for Lc. paracasei to survive. It has been reported that the malolactic enzyme (MLE) pathway plays an important role for LAB to resist acid stress. However, the regulatory mechanism has not yet been investigated. In this study, we determined that the LysR-type regulator MleR positively regulated the MLE pathway to enhance acid tolerance by binding -80 to -61 upstream of the transcription start site of the mleST operon. Further, L-malic acid acts as a co-inducer for MleR transcriptional regulation. Our study provides novel insights into acid tolerance mechanisms in LAB.

Limosilactobacillus reuteri inhibits the acid tolerance response in oral bacteria

Probiotic bacteria show promising results in prevention of the biofilm-mediated disease caries, but the mechanisms are not fully understood. The acid tolerance response (ATR) allows biofilm bacteria to survive and metabolize at low pH resulting from microbial carbohydrate fermentation. We have studied the effect of probiotic strains: Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus on ATR induction in common oral bacteria. Communities of L. reuteri ATCC PTA5289 and Streptoccus gordonii, Streptococcus oralis, Streptococcus mutans or Actinomyces naeslundii in the initial stages of biofilm formation were exposed to pH 5.5 to allow ATR induction, followed by a low pH challenge. Acid tolerance was evaluated as viable cells after staining with LIVE/DEAD®BacLight™. The presence of L. reuteri ATCC PTA5289 caused a significant reduction in acid tolerance in all strains except S. oralis. When S. mutans was used as a model organism to study the effects of additional probiotic strains (L. reuteri SD2112, L. reuteri DSM17938 or L. rhamnosus GG) as well as L. reuteri ATCC PTA5289 supernatant on ATR development, neither the other probiotic strains nor supernatants showed any effect. The presence of L. reuteri ATCC PTA5289 during ATR induction led to down-regulation of three key genes involved in tolerance of acid stress (luxS, brpA and ldh) in Streptococci. These data suggest that live cells of probiotic L. reuteri ATCC PTA5289 can interfere with ATR development in common oral bacteria and specific strains of L. reuteri may thus have a role in caries prevention by inhibiting development of an acid-tolerant biofilm microbiota.

The role of PhoP/PhoQ system in regulating stress adaptation response in Escherichia coli O157:H7.

The development of acid tolerance response (ATR) as a result of low pH in Escherichia coli O157:H7 (E. coli O157:H7) contaminating beef during processing is considered a major food safety concern. Thus, in order to explore the formation and molecular mechanisms of the tolerance response of E. coli O157:H7 in a simulated beef processing environment, the resistance of a wild-type (WT) strain and its corresponding ΔphoP mutant to acid, heat, and osmotic pressure was evaluated. Strains were pre-adapted under different conditions of pH (5.4 and 7.0), temperature (37°C and 10°C), and culture medium (meat extract and Luria-Bertani broth media). In addition, the expression of genes related to stress response and virulence was also investigated among WT and ΔphoP strains under the tested conditions. Pre-acid adaptation increased the resistance of E. coli O157:H7 to acid and heat treatment while resistance to osmotic pressure decreased. Moreover, acid adaptation in meat extract medium simulating slaughter environment increased ATR, whereas pre-adaptation at 10°C reduced the ATR. Furthermore, it was shown that mildly acidic conditions (pH=5.4) and the PhoP/PhoQ two-component system (TCS) acted synergistically to enhance acid and heat tolerance in E. coli O157:H7. Additionally, the expression of genes related to arginine and lysine metabolism, heat shock, and invasiveness was up-regulated, which revealed that the mechanism of acid resistance and cross-protection under mildly acidic conditions was mediated by the PhoP/PhoQ TCS. Both acid adaptation and phoP gene knockout reduced the relative expression of stx1 and stx2 genes which were considered as critical pathogenic factors. Collectively, the current findings indicated that ATR could occur in E. coli O157:H7 during beef processing. Thus, there is an increased food safety risk due to the persistence of tolerance response in the following processing conditions. The present study provides a more comprehensive basis for the effective application of hurdle technology in beef processing.

LssR plays a positive regulatory role in acid and nisin tolerance response of Lactococcus lactis

In Lactococcus lactis, different regulation mechanisms can be activated to overcome the effects of adverse environmental stresses. Here, a TetR family regulator LssR was demonstrated as a positive regulator in the activation of the mechanisms involved in acid and nisin tolerance of L. lactis. The deletion of lssR led to the reduction of tolerance of L. lactis NZ9000 to nisin and acid stress, and the survival rates of NZ9000 under nisin and acid stress were roughly 20-fold, 10-fold (pH 3.0, hydrochloric acid), and 8.9-fold (pH 4.0, lactic acid) of the lssR mutant NZΔlssR, respectively. Moreover, the lssR mutant NZΔlssR also displayed a lower intracellular pH stability and a changed cell surface morphology. Subsequently, transcriptome analysis revealed that genes related to the arginine deiminase pathway, the surface polysaccharides biosynthesis, carbohydrates transport and metabolism, multidrug resistance, cell repair proteins and chaperones were predominantly down transcribed in NZΔlssR. The transcript levels of the arginine deiminase pathway and the surface polysaccharides biosynthesis-associated genes under acid and nisin stresses were compared between the wild type NZ9000 and NZΔlssR using real-time fluorescence quantitative PCR. It revealed that the arginine deiminase pathway genes (arcD1C1C2T) and the surface polysaccharides biosynthesis genes (cgT, gmhB, gmhA, hddA, tagH and tarS) were proposed to be the main regulatory mechanisms of LssR in response to the acid and nisin stresses. Overall, the important role of LssR in the acid and nisin stresses response was demonstrated and the putative regulation mechanism of LssR was revealed.

Effect of Lipoteichoic Acid Synthesis-Related Gene dltD on Acid Tolerance of Highly Cariogenic Strains of Streptococcus mutans

To study the role and possible mechanism of dltD in the acid tolerance of Streptococcus mutans 593 (SM593), and to provide a theoretical basis for the ecological prevention and control of dental caries by constructing the dltD gene deletion strain of SM593 (SM593-ΔdltD). 1) SM593-Δ dltD was constructed by homologous recombination. 2) The growth curve of SM593 dltD and SM593-Δ dltD under different pH culture conditions was drawn by the automatic growth curve analyzer to compare their acid tolerance. Colony forming unit (CFU) at different time points was used to calculate the survival rate and to compare the acid tolerance response (ATR) of SM593 and SM593-Δ dltD. 3) Under different pH conditions, glycolysis experiments, proton permeability test and H +-ATPase activity test were conducted to make preliminary exploration into the mechanisms of how dltD gene deletion may affect acid tolerance. 1) PCR and sequencing results showed that the SM593-Δ dltD was constructed successfully. 2) With decreasing pH value of the culture medium, the growth of SM593-Δ dltD slowed down. When the pH value of the culture medium was 5.0, SM593-Δ dltD was not allowed to grow, and its acid tolerance was lower than that of SM593. Compared with SM593, the ATR capability of SM593-Δ dltD was decreased. 3) SM593 dltD and SM593-Δ dltD did not show obvious difference in their glycolysis ability under different pH conditions. Compared with SM593 dltD, the proton permeability of SM593-Δ dltD under different pH conditions was increased significantly (P<0.05), and H +-ATPase activity decreased significantly (P<0.05). Compared with SM593 dltD, SM593-Δ dltD showed obvious decrease in acid tolerance, which may be caused by the significant increase in proton permeability and significant decrease in the H +-ATPase activity induced by the deletion of the dltD gene, hence reducing its ability to maintain intracellular pH homeostasis.

The acid tolerance responses of the Salmonella strains isolated from beef processing plants

The development of the stationary-phase, low-pH-inducible acid tolerance response (ATR) in the Salmonella contaminant of beef during the processing arises food safety concerns, because it may evoke bacterial coping mechanisms against bactericidal insults and alter gene expression that contribute to pathogen virulence. However, information on the development of the ATR and the stability (defined as the capacity to maintain the acquired acid tolerance after induction) in the Salmonella during the production and distribution of beef is limited. After adaptation overnight, ATRs in the 79 strains of Salmonella isolated from beef processing plants were investigated by comparing the log reduction in the 2-h acid challenge trials at pH 3.0. Six representative strains were selected to further estimate the effect of three factors in the incubation period on the development of the ATR, including adapted pH values (5.0, 5.4, 6.0, and 7.0), temperatures (10 °C and 37 °C), and the adaptation media (meat extract and brain heart infusion media). The stability of acid tolerance during the long-time chilled storage (4 °C for 13 days) was also observed on two strains of serotypes S. Derby and S. Meleagridis. All the strains isolated from beef processing plants exhibited an enhanced acid tolerance indicating the widespread existence of ATR. The results also revealed that strain variability was present in the development of ATR. Significant tolerance to lethal acidic environments (pH 3.0) was found when the Salmonella strains had been acid-adapted in meat extract at pH 5.0, pH 5.4, or pH 6.0, which indicated the possible induction of ATR during beef production. After the acid adaptations, the population reduction after the acid challenge (BHI, pH = 3) in the strains was significantly lower than the non-induced at the 1d, 7 day and 13 day's storage in meat extract media at 4 °C, which revealed the persistence of ATR during beef distribution. Compared to 37 °C, adaptation in lower temperature (10 °C) significantly reduced the ATR and no ATR was developed when adapted in 4 °C. This emphasizes the importance of keeping a low temperature of beef throughout the supply chains of beef industry.

Pyruvate Oxidase as a Key Determinant of Pneumococcal Viability during Transcytosis across Brain Endothelium.

Streptococcus pneumoniae invades a myriad of host tissues following efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for evasion of host intracellular defenses governing successful transcytosis across host cellular barriers remain elusive. In this study, using brain endothelium as a model host barrier, we observed that pneumococcus containing endocytic vacuoles (PCVs), formed following S. pneumoniae internalization into brain microvascular endothelial cells (BMECs), undergo early maturation and acidification, with a major subset acquiring lysosome-like characteristics. Exploration of measures that would preserve pneumococcal viability in the lethal acidic pH of these lysosome-like vacuoles revealed a critical role of the two-component system response regulator, CiaR, which was previously implicated in induction of acid tolerance response. Pyruvate oxidase (SpxB), a key sugar-metabolizing enzyme that catalyzes oxidative decarboxylation of pyruvate to acetyl phosphate, was found to contribute to acid stress tolerance, presumably via acetyl phosphate-mediated phosphorylation and activation of CiaR, independent of its cognate kinase CiaH. Hydrogen peroxide, the by-product of an SpxB-catalyzed reaction, was also found to improve pneumococcal intracellular survival by oxidative inactivation of lysosomal cysteine cathepsins, thus compromising the degradative capacity of the host lysosomes. As expected, a ΔspxB mutant was found to be significantly attenuated in its ability to survive inside the BMEC endocytic vacuoles, reflecting its reduced transcytosis ability. Collectively, our studies establish SpxB as an important virulence determinant facilitating pneumococcal survival inside host cells, ensuring successful trafficking across host cellular barriers. IMPORTANCE Host cellular barriers have innate immune defenses to restrict microbial passage into sterile compartments. Here, by focusing on the blood-brain barrier endothelium, we investigated mechanisms that enable Streptococcus pneumoniae to traverse through host barriers. Pyruvate oxidase, a pneumococcal sugar-metabolizing enzyme, was found to play a crucial role in this via generation of acetyl phosphate and hydrogen peroxide. A two-pronged approach consisting of acetyl phosphate-mediated activation of acid tolerance response and hydrogen peroxide-mediated inactivation of lysosomal enzymes enabled pneumococci to maintain viability inside the degradative vacuoles of the brain endothelium for successful transcytosis across the barrier. Thus, pyruvate oxidase is a key virulence determinant and can potentially serve as a viable candidate for therapeutic interventions for better management of invasive pneumococcal diseases.

Pharmacophore-based screening and modification of amiloride analogs for targeting the NhaP-type cation-proton antiporter in Vibrio cholerae.

The genome of Vibrio cholerae contains three structural genes for the NhaP-type cation-proton antiporter paralogues, Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3, mediating exchange of K+ and or Na+ for protons across the membrane. Based on phenotypic analysis of chromosomal Vc-NhaP1, Vc-NhaP2, and Vc-NhaP3 triple deletion mutants, we suggest that Vc-NhaP paralogues are primarily K+/H+ antiporters and might play a role in the acid tolerance response of V.cholerae as it passes through the gastric acid barrier of the stomach. Comparison of the biochemical properties of Vc-NhaP isoforms revealed that Vc-NhaP2 was the most active among all three paralogues. Therefore, the Vc-NhaP2 antiporter is a plausible therapeutic target for developing novel inhibitors targeting these ion exchangers. Our structural and mutational analysis of Vc-NhaP2 identified a putative cation-binding pocket formed by antiparallel extended regions of two transmembrane segments (TMSs V and XII) along with TMS VI. Molecular dynamics simulations suggested that the flexibility of TMSs V and XII is crucial for intramolecular conformational events in Vc-NhaP2. In this study, we developed putative Vc-NhaP2 inhibitors from amiloride analogs. Molecular docking of the modified amiloride analogs revealed promising binding properties. The four selected drugs potentially interacted with functionally important amino acid residues located on the cytoplasmic side of TMS VI, the extended chain region of TMSs V and XII, and the loop region between TMSs VIIII and IX. Molecular dynamics simulations revealed that binding of the selected drugs can potentially destabilize Vc-NhaP2 and alter the flexibility of functionally important TMS VI. This work presents the utility of in silico approaches for the rational identification of potential targets and drugs that could target NhaP2 cation proton antiporters to control V. cholerae. The goal was to identify potential drugs that could be validated in future experiments.

A pH-Dependent Gene Expression Enables Bacillus amyloliquefaciens MBNC to Adapt to Acid Stress.

Acid tolerance response (ATR), a process by which bacteria optimize their growth conditions for cellular functions, is a well-characterized bacterial stress response. A bacterial isolate identified, as Bacillus amyloliquefaciens MBNC, was isolated from acidic soil and studied for its acid tolerance response under several range of acidic stress conditions imposed through inorganic acid, organic acid, acetate buffer, and soil extract. The ability of the B. amyloliquefaciens MBNC to tolerate extreme acidic conditions (pH 4.5) increased when exposed to moderate-acidic pH (pH 5.5). Along with ATR, the bacterial cell density was also critical to its ability to tolerate low pH as the cells of late log phase were more tolerant to low pH stress compared to the early log phase cells. A comparative expression study of 28 genes of B. amyloliquefaciens MBNC was assessed in cells grown in neutral (pH 7.0) and acidic condition (pH 4.5) through qRT-PCR. Among the 28 genes analyzed, 24 genes showed increased expression whereas the expression of 4 genes was downregulated under acid stress indicating to the involvement of the genes in acid stress response.

Effect οf οxygen availability and pH οn adaptive acid tolerance response of immobilized Listeria monocytogenes in structured growth media

The aim of the present study was to evaluate the effect of oxygen availability (aerobic, hypoxic and anoxic conditions) and sub-optimal pH (6.2 and 5.5) in a structured medium (10% w/V gelatin) on the growth of two immobilized L. monocytogenes strains (C5, 6179) at 10 °C and their subsequent acid resistance (pH 2.0, e.g., gastric acidity). Anaerobic conditions resulted in lower bacterial population (P < 0.05) (7.8–8.2 log CFU/mL) at the end of storage than aerobic and hypoxic environment (8.5–9.0 log CFU/mL), a phenomenon that was intensified at lower pH (5.5), where no significant growth was observed for anaerobically grown cultures. Prolonged habituation of L. monocytogenes (15 days) at both pH increased its acid tolerance resulting in max. 10 times higher t4D (appx. 60 min). The combined effect though of oxygen availability and suboptimal pH on L. monocytogenes acid resistance was found to vary with the strain. Anoxically grown cultures at pH 5.5 exhibited the lowest tolerance towards lethal acid stress, with countable survivors occurring only until 20 min of exposure at pH 2.0. Elucidating the role of oxygen limiting conditions, often encountered in structured foods, on acid resistance of L. monocytogenes, would assist in assessing the capacity of L. monocytogenes originated from different food-related niches to withstand gastric acidity and possibly initiate infection.

Vibrio parahaemolyticus CadC regulates acid tolerance response to enhance bacterial motility and cytotoxicity.

Pathogens adapted to sub‐lethal acidic conditions could increase the virulence and survival ability under lethal conditions. In the aquaculture industry, feed acidifiers have been used to increase the growth of aquatic animals. However, there is limited study on the effects of acidic condition on the virulence and survival of pathogens in aquaculture. In this study, we investigated the survival ability of Vibrio parahaemolyticus at lethal acidic pH (4.0) after adapted the bacteria to sub‐lethal acidic pH (5.5) for 1 hr. Our results indicated that the adapted strain increased the survival ability at lethal acidic pH invoked by an inorganic (HCl) or organic (citric) acid. RNA‐sequencing (RNA‐seq) results revealed that 321 genes were differentially expressed at the sub‐lethal acidic pH including cadC, cadBA and groES/groEL relating to acid tolerance response (ATR), as well as genes relating to outer membrane, heat‐shock proteins, phosphotransferase system and flagella system. Quantitative real‐time polymerase chain reaction (qRT‐PCR) confirmed that cadC and cadBA were upregulated under sub‐lethal acidic conditions. The CadC protein could directly regulate the expression of cadBA to modulate the ATR in V. parahaemolyticus. RNA‐seq data also indicated that 113 genes in the CadC‐dependent way and 208 genes in the CadC‐independent way were differentially expressed, which were related to the regulation of ATR. Finally, the motility and cytotoxicity of the sub‐lethal acidic adapted wild type (WT) were significantly increased compared with the unadapted strain. Our results demonstrated that the dietary acidifiers may increase the virulence and survival of V. parahaemolyticus in aquaculture.

The responses of Salmonella enterica serovar Typhimurium to vanillin in apple juice through global transcriptomics

Salmonella enterica serovar Typhimurium can survive some extreme environment in food processing, and vanillin generally recognized as safe is bactericidal to pathogens. Thus, we need to explore the responses of S. Typhimurium to vanillin in order to apply this antimicrobial agent in food processing. In this study, we exposed S. Typhimurium to commercial apple juice with/without vanillin (3.2 mg/mL) at 45 °C for 75 min to determine the survival rate. Subsequently, the 10-min cultures were selected for transcriptomic analysis. Using high-throughput RNA sequencing, genes related to vanillin resistance and their expression changes of S. Typhimurium were identified. The survival curve showed that S. Typhimurium treated with vanillin were inactivated by 5.5 log after 75 min, while the control group only decreased by 2.3 log. Such a discrepancy showed the significant antibacterial effect of vanillin on S. Typhimurium. As a result, 265 differentially expressed genes (DEGs) were found when coping with vanillin, among which, 225 showed up-regulation and 40 DEGs were down-regulated. Treated with vanillin, S. Typhimurium significantly up-regulated genes involved in cell membrane, acid tolerance response (ATR) and oxidative stress response, cold shock cross-protection, DNA repair, virulence factors and some key regulators. Firstly, membrane-related genes, including outer membrane (bamE, mepS, ygdI, lolB), inner membrane (yaiY, yicS) and other proteins (yciC, yjcH), were significantly up-regulated because of the damaged cell membrane. Then, up-regulated proteins associated with arginine synthesis (ArgABCDIG) and inward transportation (ArtI, ArtJ, ArtP and HisP), participated in ATR to pump out the protons inside the cell in this scenario. Next, superoxide stress response triggered by vanillin was found to have a significant up-regulation as well, which was controlled by SoxRS regulon. Besides, NADH-associated (nuoA, nuoB, nuoK, nadE, fre and STM3021), thioredoxin (trxA, trxC, tpx and bcp) and glutaredoxin (grxC and grxD) DEGs led to the increase of the oxidative stress response. Cold shock proteins such as CspA and CspC showed an up-regulation, suggesting it might play a role in cross-protecting S. Typhimurium from vanillin stress. Furthermore, DEGs in DNA repair and virulence factors, including flagellar assembly, adhesins and type III secretion system were up-regulated. Some regulators like fur, rpoE and csrA played a pivotal role in response to the stress caused by vanillin. Therefore, this study sounds an alarm for the risks caused by stress tolerance of S. Typhimurium in food industry.