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A chromosomal gtrB homolog and dam differentially contribute to dry-heat and high hydrostatic pressure resistance in Salmonella enterica.

Salmonella enterica can persist in low-moisture foods and shows enhanced dry-heat resistance under low water activity, posing significant food safety challenges. However, the genetic basis of extreme dry-heat resistance and its relationship with other processing stresses remain unclear. In this study, twelve S. enterica strains were screened for dry-heat treatment at 60 °C and 80 °C, with S. Infantis CICC21649 identified as the most resistant strain. Comparative genomics and transcriptional analysis identified candidate genes related to envelope integrity and regulation, including gtrB and dam. Deletion of the chromosomal gtrB homolog reduced dry-heat resistance, producing an additional 0.91-log10 reduction relative to the parent strain at 80 °C. Deletion of dam caused broader stress sensitivity, reducing resistance to both dry heat and high hydrostatic pressure, with the stronger phenotype observed under high hydrostatic pressure. Proteomic analysis of the chromosomal gtrB homolog mutant revealed broad alterations in envelope-associated proteins, transport functions, oxidative stress pathways, and central metabolism under dry-heat stress. These findings indicate that the chromosomal gtrB homolog is an important contributor to extreme dry-heat resistance, whereas dam contributes to resistance against both dry-heat and high hydrostatic pressure, likely through a broader regulatory role in stress adaptation. These results reveal distinct structural and regulatory layers underlying stress adaptation in S. enterica and provide practical guidance for low-moisture food processing by highlighting the need to account for strain-dependent and stress-specific resistance during process validation.

Hydrostatic Pressure↗

Bacterial inactivation by high-pressure homogenisation and high hydrostatic pressure.

The resistance of five gram-positive bacteria, Enterococcus faecalis, Staphylococcus aureus, Lactobacillus plantarum, Listeria innocua and Leuconostoc dextranicum, and six gram-negative bacteria, Salmonella enterica serovar typhimurium, Shigella flexneri, Yersinia enterocolitica, Pseudomonas fluorescens and two strains of Escherichia coli, to high-pressure homogenisation (100-300 MPa) and to high hydrostatic pressure (200-400 MPa) was compared in this study. Within the group of gram-positive bacteria and within the group of gram-negative bacteria, large differences were observed in resistance to high hydrostatic pressure, but not to high-pressure homogenisation. All gram-positive bacteria were more resistant than any of the gram-negative bacteria to high-pressure homogenisation, while in relative to high hydrostatic pressure resistance both groups overlapped. Within the group of gram-negative bacteria, there also existed another order in resistance to high-pressure homogenisation than to high hydrostatic pressure. Further it appears that the mutant E. coli LMM1010, which is resistant to high hydrostatic pressure is not more resistant to high-pressure homogenisation than its parental strain MG1655. The preceding observations indicate a different response of the test bacteria to high-pressure homogenisation compared to high hydrostatic pressure treatment, which suggests that the underlying inactivation mechanisms for both techniques are different. Further, no sublethal injury could be observed upon high-pressure homogenisation of Y. enterocolitica and S. aureus cell population by using low pH (5.5 7), NaCl (0 6%) or SDS (0-100 mg/l) as selective components in the plating medium. Finally, it was observed that successive rounds of high-pressure homogenisation have an additive effect on viability reduction of Y. enterocolitica and S. aureus.

Colony Count, Microbial↗

Variation in resistance to high hydrostatic pressure and rpoS heterogeneity in natural isolates of Escherichia coli O157:H7.

Several natural isolates of Escherichia coli O157:H7 have previously been shown to exhibit stationary-phase-dependent variation in their resistance to inactivation by high hydrostatic pressure. In this report we demonstrate that loss of the stationary-phase-inducible sigma factor RpoS resulted in decreased resistance to pressure in E. coli O157:H7 and in a commensal strain. Furthermore, variation in the RpoS activity of the natural isolates of O157:H7 correlated with the pressure resistance of those strains. Heterogeneity was noted in the rpoS alleles of the natural isolates that may explain the differences in RpoS activity. These results are consistent with a role for rpoS in mediating resistance to high hydrostatic pressure in E. coli O157:H7.

Animals↗

Heat shock protein-mediated resistance to high hydrostatic pressure in Escherichia coli.

A random library of Escherichia coli MG1655 genomic fragments fused to a promoterless green fluorescent protein (GFP) gene was constructed and screened by differential fluorescence induction for promoters that are induced after exposure to a sublethal high hydrostatic pressure stress. This screening yielded three promoters of genes belonging to the heat shock regulon (dnaK, lon, clpPX), suggesting a role for heat shock proteins in protection against, and/or repair of, damage caused by high pressure. Several further observations provide additional support for this hypothesis: (i). the expression of rpoH, encoding the heat shock-specific sigma factor sigma(32), was also induced by high pressure; (ii). heat shock rendered E. coli significantly more resistant to subsequent high-pressure inactivation, and this heat shock-induced pressure resistance followed the same time course as the induction of heat shock genes; (iii). basal expression levels of GFP from heat shock promoters, and expression of several heat shock proteins as determined by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins extracted from pulse-labeled cells, was increased in three previously isolated pressure-resistant mutants of E. coli compared to wild-type levels.

Culture Media↗

Inactivation of Listeria innocua inoculated in liquid whole egg by high hydrostatic pressure.

The resistance of Listeria innocua, as a model microorganism for Listeria monocytogenes, to high hydrostatic pressure in liquid whole egg was studied at several pressures (300, 350, 400, and 450 MPa),temperatures (- 15, 2, and 20 degrees C), and times (5, 10, and 15 min). Listeria innocus was added to liquid whole egg at approximately 10(6) CFU/ml. Listeria innocua was not totally inactivated in any of the treatments. In general, reduction was better at 2 degrees than at room temperature, but the greatest inactivation was obtained at 450 MPa at 20 degrees C for 15 min (over 5 log of reduction), The results indicate that microbial inactivation was increased with prolonged exposure to pressure. D values for Listeria innocua were obtained at 400 MPa for two temperatures (2 and 20 degrees C), and different times (0 to 20 min). The microbial inactivation followed apparent first-order kinetics, exhibiting a decimal reduction time of 7.35 min at 2 degrees C and 8.23 min at 20 degrees C.

Animals↗

Evidence for contribution of neutral trehalase in barotolerance of Saccharomyces cerevisiae.

In yeast, trehalose accumulation and its hydrolysis, which is catalyzed by neutral trehalase, are believed to be important for thermotolerance. We have shown that trehalose is one of the important factors for barotolerance (resistance to hydrostatic pressure); however, nothing is known about the role of neutral trehalase in barotolerance. To estimate the contribution of neutral trehalase in resisting high hydrostatic pressure, we measured the barotolerance of neutral trehalase I and/or neutral trehalase II deletion strains. Under 180 MPa of pressure for 2 h, the neutral trehalase I deletion strain showed higher barotolerance in logarithmic-phase cells and lower barotolerance in stationary-phase cells than the wild-type strain. Introduction of the neutral trehalase I gene (NTH1) into the deletion mutant restored barotolerance defects in stationary-phase cells. Furthermore, we assessed the contribution of neutral trehalase during pressure and recovery conditions by varying the expression of NTH1 or neutral trehalase activity with a galactose-inducible GAL1 promoter with either glucose or galactose. The low barotolerance observed with glucose repression of neutral trehalase from the GAL1 promoter was restored during recovery with galactose induction. Our results suggest that neutral trehalase contributes to barotolerance, especially during recovery.

Galactose↗

Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses.

Strains of Escherichia coli O157 isolated from patients with clinical cases of food-borne illness and other sources exhibited wide differences in resistance to high hydrostatic pressure. The most pressure-resistant strains were also more resistant to mild heat than other strains. Strain C9490, a representative pressure-resistant strain, was also more resistant to acid, oxidative, and osmotic stresses than the pressure-sensitive strain NCTC 12079. Most of these differences in resistance were observed only in stationary-phase cells, the only exception being acid resistance, where differences were also apparent in the exponential phase. Membrane damage in pressure-treated cells was revealed by increased uptake of the fluorescent dyes ethidium bromide and propidium iodide. When strains were exposed to the same pressure for different lengths of time, the pressure-sensitive strains took up stain sooner than the more resistant strain, which suggested that the differences in resistance may be related to susceptibility to membrane damage. Our results emphasize the importance of including stress-resistant strains of E. coli O157 when the efficacy of a novel or mild food preservation treatment is tested.

Cell Membrane↗

Escherichia coli mutants resistant to inactivation by high hydrostatic pressure.

Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing.

Cell Membrane Permeability↗

Interactions of high hydrostatic pressure, pressurization temperature and pH on death and injury of pressure-resistant and pressure-sensitive strains of foodborne pathogens.

The objective of this study is to determine the interactions between high hydrostatic pressure, pressurization temperature, time and pH during pressurization on death and injury of pressure-resistant and pressure-sensitive strains of four foodborne pathogens: Staphylococcus aureus 485 and 765, Listeria ,monocytogenes CA and OH2, Escherichia coli O157:H7 933 and 931, Salmonella enteritidis FDA and Salmonella typhimurium E21274. Among these strains S. aureus 485, L. monocytogenes CA, E. coli O157:H7 933 and S. enteritidis FDA were reported to be more pressure-resistant than the respective strain of the same species (Alpas et al., 1999). In general, viability loss of all pathogens was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). All the strains except S. aureus 485 demonstrated more than 8 log cycle reduction when pressurized at 345 MPa at 50 degrees C for 5 min. This strain seemed to be the most pressure-resistant strain within the conditions of the study. Pressurization in the presence of either citric or lactic acid increased the viability loss by an additional 1.2-3.9 log cycles at pH 4.5 for both acids at 345 MPa. This study has indicated that high hydrostatic pressure applied in conjunction with mild heat and acidity can be an effective method for inactivating pressure-resistant and pressure-sensitive strains of four foodborne pathogens in organic acid solutions. This combination treatment indicates possible pressure pasteurization applications to liquid foods that have low pH. reserved.

Analysis of Variance↗

Evaluation of high hydrostatic pressure sensitivity of Staphylococcus aureus and Escherichia coli O157:H7 by differential scanning calorimetry.

Differential scanning calorimetry (DSC) was used to evaluate the relative high hydrostatic pressure (HHP) resistances of bacterial strains from Staphylococcus aureus and Escherichia coli O157:H7 in vivo. The total apparent enthalpy change and thermal stability were two DSC parameters used to compare bacterial strains of untreated control and pressure-treated bacteria. DSC thermograms indicated that ribosomal denaturation appears to be a major factor in cell death by both thermal and high pressure treatments. However, the analysis of calorimetric data for control samples as well as pressure-treated samples clearly showed that the sensitivities of bacteria to various physical stresses can be different. While S. aureus 765 had a relatively higher resistance to thermal treatment in comparison to S. aureus 485, S. aureus 485 was determined to be more resistant to pressure than S. aureus 765. This information can be utilized in the design of processes specific to targeting certain cellular components by using different physical stresses.

Calorimetry, Differential Scanning↗

Sensitisation of Escherichia coli to antibacterial peptides and enzymes by high-pressure homogenisation.

The sensitisation of Escherichia coli towards lysozyme, nisin and lactoperoxidase by high-pressure homogenisation (HPH) at pressures ranging from 100 to 300 MPa was studied. At above 150 MPa, E. coli became sensitive to lysozyme and nisin when these compounds were added before the HPH treatment, but cells treated by HPH remained insensitive for lysozyme and nisin added after that treatment. No sensitisation to the lactoperoxidase enzyme system was observed, and no evidence of cytoplasmic membrane damage could be found in cells surviving HPH treatment using SYTO 9/propidium iodide staining. A previously isolated spontaneous E. coli mutant that is resistant to lysozyme under high hydrostatic pressure, was also found to be resistant to lysozyme upon HPH treatment. Further, lysozyme resistance in this mutant was not due to enhanced levels of periplasmic lysozyme inhibitor (Ivy), since knock-out of the ivy gene did not increase the sensitivity of the mutant to lysozyme upon HPH treatment. These results indicate that HPH treatment at pressures up to 300 MPa, unlike treatment with high hydrostatic pressure, does not cause sublethal injury, and thus does not sensitise bacteria to antimicrobial compounds that disturb cellular homeostasis such as might be the case for the lactoperoxidase system. However, like high hydrostatic pressure treatment, HPH may sensitise E. coli to lysozyme and nisin by inducing a transient permeabilisation of the outer membrane that does not involve a physical disruption and that is immediately repaired after the process.

Adaptation, Physiological↗

Inactivation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides.

We studied the inactivation in milk of four Escherichia coli strains (MG1655 and three pressure-resistant mutants isolated from MG1655) by high hydrostatic pressure, alone or in combination with the natural antimicrobial peptides lysozyme and nisin and at different temperatures (10 to 50 degrees C). Compared with that of phosphate buffer, the complex physicochemical environment of milk exerted a strong protective effect on E. coli MG1655 against high-hydrostatic-pressure inactivation, reducing inactivation from 7 logs at 400 MPa to only 3 logs at 700 MPa in 15 min at 20 degrees C. An increase in lethality was achieved by addition of high concentrations of lysozyme (400 microg/ml) and nisin (400 IU/ml) to the milk before pressure treatment. The additional reduction amounted maximally to 3 logs in skim milk at 550 MPa but was strain dependent and significantly reduced in 1.55% fat and whole milk. An increase of the process temperature to 50 degrees C also enhanced inactivation, particularly for the parental strain, but even in the presence of lysozyme and nisin, a 15-min treatment at 550 MPa and 50 degrees C in skim milk allowed decimal reductions of only 4.5 to 6.9 for the pressure-resistant mutants. A substantial improvement of inactivation efficiency at ambient temperature was achieved by application of consecutive, short pressure treatments interrupted by brief decompressions. Interestingly, this pulsed-pressure treatment enhanced the sensitivity of the cells not only to high pressure but also to the action of lysozyme and nisin.

Animals↗

Effects of minerals on resistance of Bacillus subtilis spores to heat and hydrostatic pressure.

Among Bacillus subtilis IFO13722 spores sporulated at 30, 37, and 44 degrees C, those sporulated at 30 degrees C had the highest resistance to treatments with high hydrostatic pressure (100 to 300 MPa, 55 degrees C, 30 min). Pressure resistance increased after demineralization of the spores and decreased after remineralization of the spores with Ca(2+) or Mg(2+), whereas the resistance did not change when spores were remineralized with Mn(2+) or K(+), suggesting that former two divalent ions were involved in the activation of cortex-lytic enzymes during germination.

Bacillus subtilis↗

Ultrastructural organization of contractile and cytoskeletal proteins in glomerular podocytes of chicken, rat, and man.

By immunoelectron microscopy the podocyte foot processes of the rat and human kidney have been shown to contain three major proteins of the contractile apparatus in muscle, i.e., actin, myosin, and the Z-line protein, alpha-actinin. Gel electrophoresis and immunoblot analysis of isolated glomeruli suggests that these proteins constitute an important part of the total glomerular protein contents. In the chicken kidney, the plasmalemmal portion of the foot processes that abuts the glomerular basement membrane was specifically labeled with antibodies against chicken gizzard vinculin and talin, two proteins thought to be important for the linkage of actin filaments to the lipid bilayer and to the receptor for fibronectin and laminin. Such a linkage may not only be important for the attachment of actin filaments to the plasma membrane, but could also be of functional significance for restricting the fibronectin-laminin receptor in its lateral diffusion in the plane of the lipid bilayer and to localize it at the basis of the podocytic foot processes. Assuming that actin, myosin, and alpha-actinin are arranged in a way that would allow the foot processes to generate contractile force this filament system might help the glomerular capillaries to resist the high intraluminal hydrostatic pressure as well as to actively modify the surface area for filtration. Vimentin and tubulin, the main protein subunits of intermediate filaments and microtubules, respectively, were confined to the podocyte cell body and the major processes but were virtually absent from the foot processes. This suggests that both proteins and their polymers are not important for the structure and function of the foot processes.

Actinin↗

The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence.

A spontaneous high hydrostatic pressure (HHP)-tolerant mutant of Listeria monocytogenes ScottA, named AK01, was isolated previously. This mutant was immotile and showed increased resistance to heat, acid and H2O2 compared with the wild type (wt) (Karatzas, K.A.G. and Bennik, M.H.J. 2002 Appl Environ Microbiol 68: 3183-3189). In this study, we conclusively linked the increased HHP and stress tolerance of strain AK01 to a single codon deletion in ctsR (class three stress gene repressor) in a region encoding a highly conserved glycine repeat. CtsR negatively regulates the expression of the clp genes, including clpP, clpE and the clpC operon (encompassing ctsR itself), which belong to the class III heat shock genes. Allelic replacement of the ctsR gene in the wt background with the mutant ctsR gene, designated ctsRDeltaGly, rendered mutants with phenotypes and protein expression profiles identical to those of strain AK01. The expression levels of CtsR, ClpC and ClpP proteins were significantly higher in ctsRDeltaGly mutants than in the wt strain, indicative of the CtsRDeltaGly protein being inactive. Further evidence that the CtsRDeltaGly protein lacks its repressor function came from the finding that the Clp proteins in the mutant were not further induced upon heat shock, and that HHP tolerance of a ctsR deletion strain was as high as that of a ctsRDeltaGly mutant. The high HHP tolerance possibly results from the increased expression of the clp genes in the absence of (active) CtsR repressor. Importantly, the strains expressing CtsRDeltaGly show significantly attenuated virulence compared with the wt strain; however, no indication of disregulation of PrfA in the mutant strains was found. Our data highlight an important regulatory role of the glycine-rich region of CtsR in stress resistance and virulence.

Adenosine Triphosphatases↗

Periplasmic lysozyme inhibitor contributes to lysozyme resistance in Escherichia coli.

The product of the Escherichia coli ORFan gene ykfE was recently shown to be a strong inhibitor of C-type lysozyme in vitro. The gene was correspondingly renamed ivy (inhibitor of vertebrate lysozyme), but its biological function in E. coli remains unknown. In this work, we investigated the role of Ivy in the resistance of E. coli to the bactericidal effect of lysozyme in the presence of outer-membrane-permeabilizing treatments. Both in the presence of lactoferrin (3.0 mg/ml) and under high hydrostatic pressure (250 MPa), the lysozyme resistance of E. coli MG1655 was decreased by knock-out of Ivy, and increased by overexpression of Ivy. However, knock-out of Ivy did not increase the lysozyme sensitivity of an E. coli MG1655 mutant previously described to be resistant to lysozyme under high pressure. These results indicate that Ivy is one of several factors that affect lysozyme resistance in E. coli, and suggest a possible function for Ivy as a host interaction factor in commensal and pathogenic E. coli.

Carrier Proteins↗

Pathogenetic mechanisms of venous congestion after the Fontan procedure.

BACKGROUND: The hemodynamic status after a Fontan type procedure for definitive palliation of functionally univentricular hearts is dominated by a high central venous pressure, which seems to be one of several factors responsible for venous congestion appearing as a frequent complication in the early and late postoperative course. The purpose of our study was to find other hemodynamic parameters correlating with the presence of venous congestion and effusions in these patients. METHODS: We compared the hemodynamic data of 18 patients who had an uneventful long-term course after a Fontan type procedure with the respective data of 10 patients who developed symptoms of venous congestion in the immediate postoperative period. Based on a theoretical model, we developed an algorithm to calculate mean hydrostatic capillary pressure from mean arterial pressure, systemic vascular resistance index and central venous pressure. RESULTS: Pulmonary vascular resistance index (2.1 +/- 1.0 mmHg L-1 min m2), mean left atrial pressure (9.7+/-4.0 mmHg) and cardiac index (3.6+/-0.6 l/min/m2) are mainly normal in patients with venous congestion in the immediate postoperative period, but mean hydrostatic capillary pressure is significantly higher compared to patients without venous congestion (24.3+/-3.1 vs 18.3+/-4.0 mmHg). Lower mean hydrostatic capillary pressures in these patients are due to a highly significant increase of systemic vascular resistance index (18.6+/-4.2 versus 33.6+/-6.6 mmHg L-1 min m2) and a concomitant decrease of cardiac index to 2.4+/-0.3 l/min/m2. CONCLUSIONS: The increase of mean hydrostatic capillary pressure, caused by high central venous pressures but also by relatively low systemic vascular resistance indexes, seems to be the hemodynamic key parameter responsible for venous congestion and effusions in patients after a Fontan type procedure in the immediate postoperative period.

Adolescent↗