Microorganisms under high pressure — Adaptation,growth and biotechnological potential

Hydrostatic pressure is a well-known physical parameter which is now considered an important variable of life, since organisms have the ability to adapt to pressure changes, by the development of resistance against this variable. In the past decades a huge interest in high hydrostatic pressure (HHP) technology is increasingly emerging among food and biosciences researchers. Microbial specific stress responses to HHP are currently being investigated, through the evaluation of pressure effects on biomolecules, cell structure, metabolic behavior, growth and viability. The knowledge development in this field allows a better comprehension of pressure resistance mechanisms acquired at sub-lethal pressures. In addition, new applications of HHP could arise from these studies, particularly in what concerns to biotechnology. For instance, the modulation of microbial metabolic pathways, as a response to different pressure conditions, may lead to the production of novel compounds with potential biotechnological and industrial applications. Considering pressure as an extreme life condition, this review intends to present the main findings so far reported in the scientific literature, focusing on microorganisms with the ability to withstand and to grow in high pressure conditions, whether they have innated or acquired resistance, and show the potential of the application of HHP technology for microbial biotechnology.     

The development of high hydrostatic pressure processes as an alternative to other pathogen reduction methods

In biology, scientist's interest for high hydrostatic pressure (HHP) has increased over the last 20 years, for both research and industrial developments, mainly because of the low energy associated with its application in liquid phase and its capacity to inactivate pathogens. It is now considered as an interesting alternative to heat treatments for the inactivation of contaminants in many products, from foods to pharmaceutical preparations. This last statement implies different objectives according to the type of product. The therapeutic properties of pharmaceutical preparations or other biological media of physiological importance are in general associated with specific and well-defined molecules such as proteins. Their activity mainly depends on their spatial conformation, maintained by weak chemical bonds that are often pressure sensitive. In this case, the optimization of a HHP process can be more complex than for foods, for which the organoleptic molecules are less pressure sensitive, and the evaluation of their preservation is more subjective and highly dependent on the consumers acceptance. The objective of this review is therefore to underline how, even if the basic concept for the optimization of a pathogen reduction process using HHP is the same whatever the product, major differences arise from the product itself and its final use.     

Synthetic reconstruction of extreme high hydrostatic pressure resistance in Escherichia coli

Although high hydrostatic pressure (HHP) is an interesting parameter to be applied in bioprocessing, its potential is currently limited by the lack of bacterial chassis capable of surviving and maintaining homeostasis under pressure. While several efforts have been made to genetically engineer microorganisms able to grow at sublethal pressures, there is little information for designing backgrounds that survive more extreme pressures. In this investigation, we analyzed the genome of an extreme HHP-resistant mutant of E. coli MG1655 (designated as DVL1), from which we identified four mutations (in the cra, cyaA, aceA and rpoD loci) causally linked to increased HHP resistance. Analysing the functional effect of these mutations we found that the coupled effect of downregulation of cAMP/CRP, Cra and the glyoxylate shunt activity, together with the upregulation of RpoH and RpoS activity, could mechanistically explain the increased HHP resistance of the mutant. Using combinations of three mutations, we could synthetically engineer E. coli strains able to comfortably survive pressures of 600–800 MPa, which could serve as genetic backgrounds for HHP-based biotechnological applications.     

Modeling conductive heat transfer and process uniformity during batch high-pressure processing of foods

A numerical model for predicting conductive heat transfer during batch high hydrostatic pressure (HHP) processing of foods was developed and tested for a food simulator (agar gel). For a comprehensive evaluation of the proposed method, both "conventional" HHP processes, HHP processes with gradual, step-by-step pressure buildup and pressure release, and pressure cycling HHP processes were included. In all cases, good agreement between experimental and predicted temperature profiles was observed. The model provides a very useful tool to evaluate batch HHP processes in terms of uniformity of any heat- and/or pressure-related effect. This is illustrated for inactivation of Bacillus subtilis alpha-amylase, an enzymatic model system with known pressure-temperature degradation kinetics.     

超高压对植物乳杆菌能量代谢影响的研究

以植物乳杆菌ATCC8014为试材,研究超高压对其能量代谢的影响。建立了用氯化碘硝基四唑紫测定ATCC8014的INT代谢还原活性的比色法。用比色法测定了超高压对ATCC8014的INT代谢还原活性与葡萄糖利用的影响。试验结果表明,150~250MPa作用15min在MRS琼脂培养基上随着压力的增大菌落数显著降低,INT代谢还原活性降低显著,葡萄糖的利用变化不明显;超过300MPa后,葡萄糖的利用才显著降低;400MPa处理15min,尽管在MRS琼脂培养基上菌落数低于检测限,INT代谢还原活性为0%,而葡萄糖的利用能力仍为对照组的56.1%,超高压作用下ATCC8014的灭活与INT代谢还原活性的降低的相关性较好。说明ATCC8014的细胞膜上参与葡萄糖的吸收和运输的酶、糖酵解的酶与调节系统比三羧酸循环的酶与调节系统较耐压。三羧酸循环比糖酵解对超高压敏感,三羧酸循环的抑制是超高压灭活其的重要原因,这为了探讨超高压杀灭植物乳杆菌的机制提供了一定的理论依据。     

Application of Detergents or High Hydrostatic Pressure as Decellularization Processes in Uterine Tissues and Their Subsequent Effects on In Vivo Uterine Regeneration in Murine Models

Infertility caused by ovarian or tubal problems can be treated using In Vitro Fertilization and Embryo Transfer (IVF-ET); however, this is not possible for women with uterine loss and malformations that require uterine reconstruction for the treatment of their infertility. In this study, we are the first to report the usefulness of decellularized matrices as a scaffold for uterine reconstruction. Uterine tissues were extracted from Sprague Dawley (SD) rats and decellularized using either sodium dodecyl sulfate (SDS) or high hydrostatic pressure (HHP) at optimized conditions. Histological staining and quantitative analysis showed that both SDS and HHP methods effectively removed cells from the tissues with, specifically, a significant reduction of DNA contents for HHP constructs. HHP constructs highly retained the collagen content, the main component of extracellular matrices in uterine tissue, compared to SDS constructs and had similar content levels of collagen to the native tissue. The mechanical strength of the HHP constructs was similar to that of the native tissue, while that of the SDS constructs was significantly elevated. Transmission electron microscopy (TEM) revealed no apparent denaturation of collagen fibers in the HHP constructs compared to the SDS constructs. Transplantation of the decellularized tissues into rat uteri revealed the successful regeneration of the uterine tissues with a 3-layer structure 30 days after the transplantation. Moreover, a lot of epithelial gland tissue and Ki67 positive cells were detected. Immunohistochemical analyses showed that the regenerated tissues have a normal response to ovarian hormone for pregnancy. The subsequent pregnancy test after 30 days transplantation revealed successful pregnancy for both the SDS and HHP groups. These findings indicate that the decellularized matrix from the uterine tissue can be a potential scaffold for uterine regeneration.     

Inactivation and injury of pressure-resistant strains of Escherichia coli O157 and Listeria monocytogenes in fruit juices

AIMS: The aim of the study was to investigate the combined antimicrobial action of the plant-derived volatile carvacrol and high hydrostatic pressure (HHP). METHODS AND RESULTS: Combined treatments of carvacrol and HHP have been studied at different temperatures, using exponentially growing cells of Listeria monocytogenes, and showed a synergistic action. The antimicrobial effects were higher at 1 degrees C than at 8 or 20 degrees C. Furthermore, addition of carvacrol to cells exposed to sublethal HHP treatment caused similar reductions in viable numbers as simultaneous treatment with carvacrol and HHP. Synergism was also observed between carvacrol and HHP in semi-skimmed milk that was artificially contaminated with L. monocytogenes. CONCLUSION: Carvacrol and HHP act synergistically and the antimicrobial effects of the combined treatment are greater at lower temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study demonstrates the synergistic antimicrobial effect of essential oils in combination with HHP and indicates the potential of these combined treatments in food processing.     

Molecular Basis of the Behavior of Hepatitis A Virus Exposed to High Hydrostatic Pressure

Food-borne hepatitis A outbreaks may be prevented by subjecting foods at risk of virus contamination to moderate treatments of high hydrostatic pressure (HHP). A pretreatment promoting hepatitis A virus (HAV) capsid-folding changes enhances the virucidal effect of HHP, indicating that its efficacy depends on capsid conformation. HAV populations enriched in immature capsids (125S provirions) are more resistant to HHP, suggesting that mature capsids (150S virions) are more susceptible to this treatment. In addition, the monoclonal antibody (MAb) K24F2 epitope contained in the immunodominant site is a key factor for the resistance to HHP. Changes in capsid folding inducing a loss of recognition by MAb K24F2 render more susceptible conformations independently of the origin of such changes. Accordingly, codon usage-associated folding changes and changes stimulated by pH-dependent breathings, provided they confer a loss of recognition by MAb K24F2, induce a higher susceptibility to HHP. In conclusion, the resistance of HAV to HHP treatments may be explained by a low proportion of 150S particles combined with a good accessibility of the epitope contained in the immunodominant site close to the 5-fold axis.     

Comparative proteome approach to characterize the high-pressure stress response of Lactobacillus sanfranciscensis DSM 20451(T)

High hydrostatic pressure (HHP) exerts diverse effects on microorganisms, leading to stress response and cell death. While inactivation of microorganisms by lethal HHP is well investigated in the context of food preservation and the hygienic safety of minimal food processes, sublethal HHP stress response and its effect on adaptation and cross-protection is less understood. In this study, the HHP stress response of Lactobacillus sanfranciscensis was characterized and compared with cold, heat, salt, acid and starvation stress at the proteome level by using 2-DE so as to provide insight into general versus specific stress responses. Sixteen proteins were found to be affected by HHP and were identified by using N-terminal amino acid sequencing and MS. Only one slightly increased protein was specific to the HHP response and showed homology to a clp protease. The other proteins were influenced by most of the investigated stresses in a similar way as HHP. The highest similarity in the HHP proteome was found to be with cold- and NaCl-stressed cells, with 11 overlapping proteins. At the proteome level, L. sanfranciscensis appears to use overlapping subsets of stress-inducible proteins rather than stereotype responses. Our data suggest that a specific pressure response does not exist in this bacteria.     

High hydrostatic pressure treatment of porcine oocytes before handmade cloning improves developmental competence and cryosurvival

An innovative technique, called the high hydrostatic pressure (HHP) treatment, has been recently reported to improve the cryosurvival of gametes or embryos in certain mammalian species. The aim of the present study was to investigate the in vitro and in vivo developmental competence and cryotolerance of embryos produced by handmade cloning (HMC) after pressure treatment of recipient oocytes. In vitro-matured porcine oocytes were treated with a sublethal hydrostatic pressure of 20 MPa (200 times greater than atmospheric pressure) and recovered for either 1 or 2 h (HHP1 and HHP2 groups, respectively) before they were used for HMC. After 7 days of in vitro culture, blastocyst rates and mean cell numbers were determined. Randomly selected blastocysts were vitrified with the Cryotop method based on minimum volume cooling procedure. The blastocyst rate was higher in the HHP2 group than in the control group (68.2 +/- 4.1% vs. 46.4 +/- 4.2%; p < 0.01), while there was no difference between HHP1 and control group (52.1 +/- 1.2% vs. 49.0 +/- 2.7%; p > 0.05). Similar mean cell numbers of produced blastocysts were obtained in HHP2 and control groups (56 +/- 4 vs. 49 +/- 5; p > 0.05). Subsequent blastocyst vitrification with the Cryotop method resulted in significantly higher survival rate after thawing in the HHP2 group than in the control group (61.6 +/- 4.0% vs. 30.2 +/- 30.9%; p < 0.01). Fifty-six and 57 day 5 to day 7 fresh blastocysts in HHP1 group were transferred into two recipient sows on day 5 of the estrous cycle. One recipient was diagnosed pregnant and gave birth to two healthy piglets by naturally delivery on day 122 of gestation. This pilot study proved that the sublethal HHP treatment of porcine oocytes before HMC results in improved in vitro developmental competence and cryotolerance, and supports embryonic and fetal development as well as pregnancy establishment and maintenance up to the birth of healthy piglets.     

Preparation of the β-cyclodextrin-vitamin C (β-CD-Vc) inclusion complex under high hydrostatic pressure (HHP)

In this study, a novel high hydrostatic pressure (HHP) technique was used to prepare the β-cyclodextrin-vitamin C (β-CD-Vc) inclusion complex. The inclusion ratio was positively correlated with the pressure under 300MPa and remained at above 50.0% when the pressure was more than 300MPa. Fourier-transform infrared spectroscopy (FI-IR) and UV-visible spectroscopy (UV-vis) analysis showed that characteristic absorption bands and the absorption peak of Vc disappeared in the spectra of the β-CD-Vc inclusion complex. Furthermore, differential scanning calorimetry (DSC) data revealed that only one endothermic peak appeared at about 138°C in the DSC curve of the β-CD-Vc inclusion complex. These results indicate that the HHP treatment effectively induces the formation of β-CD-Vc inclusion complex.     

Emergence and stability of high-pressure resistance in different food-borne pathogens

High hydrostatic pressure (HHP) processing is becoming a valuable nonthermal food pasteurization technique, although there is reasonable concern that bacterial HHP resistance could compromise the safety and stability of HHP-processed foods. While the degree of natural HHP resistance has already been shown to vary greatly among and within bacterial species, a still unresolved question remains as to what extent different food-borne pathogens can actually develop HHP resistance. In this study, we therefore examined and compared the intrinsic potentials for HHP resistance development among strains of Escherichia coli, Shigella flexneri, Salmonella enterica serovars Typhimurium and Enteritidis, Yersinia enterocolitica, Aeromonas hydrophila, Pseudomonas aeruginosa, and Listeria innocua using a selective enrichment approach. Interestingly, of all strains examined, the acquisition of extreme HHP resistance could be detected in only some of the E. coli strains, indicating that a specific genetic predisposition might be required for resistance development. Furthermore, once acquired, HHP resistance proved to be a very stable trait that was maintained for >80 generations in the absence of HHP exposure. Finally, at the mechanistic level, HHP resistance was not necessarily linked to derepression of the heat shock genes and was not related to the phenomenon of persistence.     

Pressure response in the yeast Saccharomyces cerevisiae: from cellular to molecular approaches.

Yeasts are unicellular organisms that are exposed to a highly variable environment, concerning the availability of nutrients, temperature, pH, radiation, access to oxygen and, specially, water activity. Evolution has selected yeasts to tolerate, to a certain extent, these environmental stresses. High hydrostatic pressure (HHP) exerts a broad effect upon yeast cells, interfering with the cell membranes, cellular architecture and in processes ofpolymerisation and denaturation of proteins. Gene expression patterns in response to HHP revealed a stress response profile. The majority of the upregulated genes were involved in stress defence and carbohydrate metabolism while most of the repressed ones were in cell cycle progression and protein synthesis categories. In addition, in the present work it was seen that mild pressure induced cell cycle arrest and protection against severe stresses, such as high temperature, high pressure and ultra cold shock. Nevertheless, this protection was only significant if the cells were incubated at atmospheric pressure after the HHP treatment. Expression of genes that were upregulated by HHP and are related to resistance to this stresses were also analyzed, and, for the majority of them, higher induction was attained after 15 min post-pressurization. Taken together, the results imply an interconnection among stresses.     

High hydrostatic pressure inactivated human tumour cells preserve their immunogenicity.

High hydrostatic pressure (HHP) is an established method to inactivate biomolecules and microoganisms. It is routinely used for the sterilization of foodstuff. Recently, new applications as inactivation of microorganisms and tumour cells for bone transplants or for cancer vaccines have emerged. Characterization of the HHP-induced cellular responses are a prerequisite for its clinical use. To this end, we investigated the fate of human cells after HHP by cytofluorometry. We observed that the induction by HHP of cell death is time- and pressure-dependent. Surprisingly, an HHP-treatment of 100 MPa did not reduce viability at any time point. Pressures from 150 to 250 MPa-induced programmed cell death in most cells. However, survivors were observed in long term culture experiments under these conditions. Pressures above 300 MPa immediately induced cell death by necrosis and completely inactivated the cells. In contrast to inactivation by other necrosis inducing treatments like heat, freeze/thaw, or chemical agents, HHP avoids generation of Maillard products and disintegration and lysis of the cells. Instead HHP generates a gelatinised mixture of antigens captured in a distinct and robust particle and maintains their humoral immunogenicity. The high viscosity of the internal matrix of a pressurised cell is reflected by the slow penetration of the low molecular compound propidium iodide and limits the bleeding of antigen before uptake by antigen presenting cells. Taken together, HHP is an alternative method for the inactivation of mammalian cells in clinical settings.     

In Process Citation

Abstract Background: Allogeneic bone transplantation is at risk of infection, and established disinfection methods typically compromise bone quality. High hydrostatic pressure (HHP) is well established for disinfection in food technology, and also it does protect biomechanical and biological properties of bone. This study is the first investigation of HHP regarding disinfection of bone biopsies. Materials and methods: Bone biopsies of 34 patients with chronic infections were subjected to HHP and assessed for persisting bacterial growth. In series 1, bone biopsies were proceeded directly to HHP (10 min; maximal pressure P(max) 600 MPa). In series 2, HHP was applied after 5-day incubation in growth media (10 min or 2x30 min; P(max) 600 MPa). Furthermore, HHP-induced changes of bacterial morphology on artificially infected bone samples were evaluated by scanning electron microscopy (SEM). Results: For series 1, 71% of the bone samples were sterilised by HHP (n=17), compared to 38% of the untreated control samples, which were obtained during the same surgery (n=8). For series 2, after prior incubation, HHP disinfected 7% of the bone specimens (n=55), all control samples showed bacterial growth (n=33). Destruction of cell wall integrity of Gram-negative strains was observed by SEM. Conclusion: The effectiveness of HHP for bone disinfection should be improved by optimising treatment parameters. Infections with barosensitive Gram-negative bacteria or yeast might represent possible clinical indications.     

New insights into conformational and functional stability of human alpha-thrombin probed by high hydrostatic pressure.

The effects of high hydrostatic pressure (HHP) and urea on conformational transitions of human alpha-thrombin structure were studied by fluorescence spectroscopy and by measuring the catalytic activity of the enzyme. Treatment of thrombin with urea produced a progressive red shift in the center of mass of the intrinsic fluorescence emission spectrum, with a maximum displacement of 650 cm(-1). HHP (270 MPa) shifted the centre of mass by only 370 cm(-1). HHP combined with a subdenaturing urea concentration (1.5 m) displaced the centre of mass by approximately 750 cm(-1). The binding of the fluorescent probe bis(8-anilinonaphthalene-1-sulfonate) to thrombin was increased by 1.8-, 4.0-, and 2.7-fold after treatment with high urea concentration, HHP or HHP combined with urea, respectively, thus suggesting that all treatments convert the enzyme to partially folded intermediates with exposed hydrophobic regions. On the other hand, treatment of thrombin with urea (but not HHP) combined with dithiothreitol progressively displaced the fluorescent probe, thus suggesting that this condition converts the enzyme to a completely unfolded state. Urea and HHP also led to different conformations when changes in the thrombin catalytic site environment were assessed using the fluorescence emission of fluorescein-d-Phe-Pro-Arg-cloromethylketone-alpha-thrombin: addition of urea up to 2 m gradually decreased the fluorescence emission of the probe to 65% of the initial intensity, whereas HHP caused a progressive increase in fluorescence. Hydrolysis of the synthetic substrate S-2238 was enhanced (35%) in 2 m urea and gradually abolished at higher concentrations, while HHP (270 MPa) inhibited the enzyme's catalytic activity by 45% and abolished it when 1.5 m urea was also present. Altogether, analysis of urea and HHP effects on thrombin structure and activity indicates the formation of dissimilar intermediate states during denaturation by these agents.     

Hydrostatic pressure induced death of mammalian cells engages pathways related to apoptosis or necrosis.

We investigated the response to high hydrostatic pressure (HHP) of mammalian cells, since HHP is proposed to be suitable to inactivate mammalian cells in biopharmaceutics and patient's material. We observed that cells were not restricted in their viability by pressures up to 100 MPa. Mammalian cells die when treated with pressures of 200 MPa or more. But the effects of 200, 300 or 400 MPa do not follow the same pattem. At 200 MPa, cells die in a way that is related to apoptosis. Some apoptotic characteristics like phosphatidylserine (PS) exposure and morphological alterations appear very fast. Other features like a higher exposure of intracellular NPn ligands and pronounced degradation of DNA and lectin ligands are unique features of HHP induced apoptosis. Cells treated with 300 and 400 MPa die immediately following a unique necrotic pathway, since treated cells harbour high DNA and glycoprotein degrading activities.     

Food processing by high hydrostatic pressure

High hydrostatic pressure (HHP) process, as a nonthermal process, can be used to inactivate microbes while minimizing chemical reactions in food. In this regard, a HHP level of 100 MPa (986.9 atm/1019.7 kgf/cm²) and more is applied to food. Conventional thermal process damages food components relating color, flavor, and nutrition via enhanced chemical reactions. However, HHP process minimizes the damages and inactivates microbes toward processing high quality safe foods. The first commercial HHP-processed foods were launched in 1990 as fruit products such as jams, and then some other products have been commercialized: retort rice products (enhanced water impregnation), cooked hams and sausages (shelf life extension), soy sauce with minimized salt (short-time fermentation owing to enhanced enzymatic reactions), and beverages (shelf life extension). The characteristics of HHP food processing are reviewed from viewpoints of nonthermal process, history, research and development, physical and biochemical changes, and processing equipment.     

Effect of high hydrostatic pressure on murine norovirus in Manila clams

Aims: Eating raw or insufficiently cooked bivalve molluscs contaminated with human noroviruses (NVs) can result in acute cases of gastroenteritis in humans. Manila clams (Ruditapes philippinarum) are particularly prone to exposure to NVs due to the brackish environment in which they are farmed which is known to be susceptible to human faecal contamination. High hydrostatic pressure processing (HHP) is a food treatment technique that has been shown to inactivate NV. Methods and results: In this study we investigated the ability of HHP to inactivate murine norovirus (MNV‐1), a recognised surrogate for NV, in experimentally contaminated manila clams. Pools of contaminated live clams were subjected to hydrostatic pressure ranging from 300 to 500 MPa for different time intervals of between one and 10 min. The trial was repeated three times, at monthly intervals. Conclusions: Virus vitality post‐treatment was assessed and the data obtained indicates that the use of high hydrostatic pressures of at least 500 MPa for 1 min was effective in inactivating MNV‐1. Significance and Impact of the Study: HHP results to be an effective technique that could be applied to industrial process to obtain safe Manila clams ready to eat.