Ribosome Reconstruction during Recovery from High-Hydrostatic-Pressure-Induced Injury in Bacillus subtilis.

Vegetative cells of Bacillus subtilis can recover from injury after high-hydrostatic-pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that substantial numbers of ribosomal genes and translation-related genes (e.g., translation initiation factors) were upregulated during the growth arrest phase after HHP treatment. The transcript levels of cold shock-responsive genes, whose products play key roles in efficient translation, and heat shock-responsive genes, whose products mediate correct protein folding or degrade misfolded proteins, were also upregulated. In contrast, the transcript level of hpf, whose product (Hpf) is involved in ribosome inactivation through the dimerization of 70S ribosomes, was downregulated during the growth arrest phase. Sucrose density gradient sedimentation analysis revealed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed. We also found that cell growth after HHP-induced injury was apparently inhibited by the addition of Mn2+ or Zn2+ to the recovery medium. Ribosome reconstruction in the HHP-injured cells was also significantly delayed in the presence of Mn2+ or Zn2+ Moreover, Zn2+, but not Mn2+, promoted dimer formation of 70S ribosomes in the HHP-injured cells. Disruption of the hpf gene suppressed the Zn2+-dependent accumulation of ribosome dimers, partially relieving the inhibitory effect of Zn2+ on the growth recovery of HHP-treated cells. In contrast, it was likely that Mn2+ prevented ribosome reconstruction without stimulating ribosome dimerization. Our results suggested that both Mn2+ and Zn2+ can prevent ribosome reconstruction, thereby delaying the growth recovery of HHP-injured B. subtilis cells.IMPORTANCE HHP treatment is used as a nonthermal processing technology in the food industry to inactivate bacteria while retaining high quality of foods under suppressed chemical reactions. However, some populations of bacterial cells may survive the inactivation. Although the survivors are in a transient nongrowing state due to HHP-induced injury, they can recover from the injury and then start growing, depending on the postprocessing conditions. The recovery process in terms of cellular components after the injury remains unclear. Transcriptome analysis using vegetative cells of Bacillus subtilis revealed that the translational machinery can preferentially be reconstructed after HHP treatment. We found that both Mn2+ and Zn2+ prolonged the growth-arrested stage of HHP-injured cells by delaying ribosome reconstruction. It is likely that ribosome reconstruction is crucial for the recovery of growth ability in HHP-injured cells. This study provides further understanding of the recovery process in HHP-injured B. subtilis cells.

Optimization of broth recovery for repair of heat-injured Salmonella enterica serovar Typhimurium and Escherichia coli O157:H7.

AIMS: The purpose of this research was to determine optimum conditions for broth recovery of heat-injured Salmonella Typhimurium and Escherichia coli O157:H7. METHODS AND RESULTS: Exposure to 55°C for 15 and 25 min, respectively, induced cellular injury to those pathogens. Comparison was made with the commonly used overlay method using selective medium for recovering sublethally injured cells of S. Typhimurium. For E. coli O157:H7, phenol red agar base with 1% sorbitol was used. After cell suspensions were heated at 55°C for selected time intervals, microbes were 10-fold diluted with brain heart infusion (BHI), tryptic soy broth (TSB) and TSB with 0·6% yeast extract (TSBYE) and incubated at 37°C for up to 3 h. At hourly intervals, diluents were plated onto selective medium for recovery. Simultaneously, diluents were plated onto tryptic soy agar (TSA) for recovery of sublethally injured cells. For overlays, diluents were plated onto TSA and overlaid with selective agar after a resuscitation interval. Broth recovery conditions for S. Typhimurium and E. coli O157:H7 were determined to be 1 h in any of the following broth media: BHI, TSB or TSBYE. When liquid resuscitation was applied to sublethally injured cells in food samples (milk), 1 h was also sufficient time for recovery. CONCLUSIONS: The broth recovery method is a convenient alternative to conventional recovery methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Cells sublethally injured by control interventions might not grow on selective medium because they have no resistance to several selective compounds. However, injured cells can recuperate and multiply under conditions sufficient for recovery. To repair and detect heat-injured cells, the overlay method is commonly used but this method has some limitations. This study confirms the effectiveness of liquid resuscitation method on recovery of injured cells. The broth recovery can replace the overlay method due to greater convenience and timesaving.

Low-energy X-ray irradiation: A novel non-thermal microbial inactivation technology.

Over the last several decades, food irradiation technology has been proven neither to reduce the nutritional value of foods more than other preservation technologies, nor to make foods radioactive or dangerous to eat. Furthermore, food irradiation is a non-thermal food processing technology that helps preserve more heat sensitive nutrients than those found in thermally processed foods. Conventional food irradiation technologies, including γ-ray, electron beam and high energy X-ray, have certain limitations and drawbacks, such as involving radioactive isotopes, low penetration ability, and economical unfeasibility, respectively. Owing to the recent developments in instrumentation technology, more compact and cheaper tabletop low-energy X-ray sources have become available. The generation of low-energy X-ray, unlike γ-ray, does not involve radioactive isotopes and the cost is lower than high energy X-ray. Furthermore, low-energy X-ray possesses unique advantages, i.e., high linear energy transfer (LET) value and high relative biological effect (RBE) value. The advantages allow low-energy X-ray irradiation to provide a higher microbial inactivation efficacy than γ-ray and high energy X-ray irradiation. In the last few years, various applications reported in the literature indicate that low-energy X-ray irradiation has a great potential to become an alternative food preservation technique. This chapter discusses the technical advances of low-energy X-ray irradiation, microbial inactivation mechanism, factors influencing its efficiency, current applications, consumer acceptance, and limitations.

Effects of injured and dead cells of Escherichia coli on the colony-forming rate of live cells.

Osmotic stress-induced injured cells of Escherichia coli were prepared by sorting live cells onto tryptic soy agar (TSA) containing 10-50% sucrose. The time course of colony-forming rate (CFR%) was analyzed. A time delay in colony formation indicated a sublethal effect. The final CFR level at 24 h indicated the relative number of culturable cells irrespective of injury. A value of (100-CFR)% at 24 h indicated a lethal effect. When cells were grown on TSA containing 10% sucrose, the time delay was 4 h and the lethal effect was 4%. However, dead cells inhibited the growth of live cells. Physical contact with insoluble matter derived from dead cells or dead cells themselves might have caused growth inhibition. These findings highlight a novel perspective on colony count methods in practical situations, such as when sampling foods containing a high concentration of sucrose.

Evaluation of standard enrichment broths for recovery of healthy and chlorine-injured Escherichia coli O157:H7 cells in kimchi.

This study aimed to evaluate three standard enrichment broth preparations for the recovery of healthy and chlorine-injured E. coli O157:H7 cells in kimchi. The growth of healthy and chlorine-injured cells in kimchi was observed in three different broths for 24 h. Results showed that the three broths were equally effective for the growth of healthy cells, although the broth described by the International Organization for Standardization (ISO) showed better performance in terms of maximum growth rate when compared to the other two broths described by the Korea Food Code (KFC) and the Food and Drug Administration (FDA). In the case of chlorine-injured cells, similar growth patterns were observed in KFC and ISO broths, whereas inhibition or no growth was found in FDA broth. Thus, this study suggests that KFC and ISO broths were more suitable than FDA broth for the enrichment of E. coli O157:H7 cells in kimchi.

Inhibition of Initial Attachment of Injured Salmonella Typhimurium onto Abiotic Surfaces.

Following sanitation interventions in food processing facilities, sublethally injured bacterial cells can remain on food contact surfaces. We investigated whether injured Salmonella Typhimurium cells can attach onto abiotic surfaces, which is the initial stage for further biofilm development. We utilized heat, UV, hydrogen peroxide, and lactic acid treatments, which are widely utilized by the food industry. Our results showed that heat, UV, and hydrogen peroxide did not effectively change populations of attached Salmonella Typhimurium. Cells treated with hydrogen peroxide had a slightly higher tendency to adhere to abiotic surfaces, although there was no significant difference between the populations of control and hydrogen peroxide-treated cells. However, lactic acid effectively reduced the number of Salmonella Typhimurium cells attached to stainless steel. We also compared physicochemical changes of Salmonella Typhimurium after application of lactic acid and used hydrogen peroxide as a positive control because only lactic acid showed a decreased tendency for attachment and hydrogen peroxide induced slightly higher numbers of attached bacteria cells. Extracellular polymeric substance produced by Salmonella Typhimurium was not detected in any treatment. Significant differences in hydrophobicity were not observed. Surface charges of cell membranes did not show relevant correlation with numbers of attached cells, whereas autoaggregation showed a positive correlation with attachment to stainless steel. Our results highlight that when lactic acid is applied in a food processing facility, it can effectively interfere with adhesion of injured Salmonella Typhimurium cells onto food contact surfaces.

Neural cell injury microenvironment induces neural differentiation of human umbilical cord mesenchymal stem cells.

This study aimed to investigate the neural differentiation of human umbilical cord mesenchymal stem cells (hUCMSCs) under the induction of injured neural cells. After in vitro isolation and culture, passage 5 hUCMSCs were used for experimentation. hUCMSCs were co-cultured with normal or Aß1-40-injured PC12 cells, PC12 cell supernatant or PC12 cell lysate in a Transwell co-culture system. Western blot analysis and flow cytometry results showed that choline acetyltransferase and microtubule-associated protein 2, a specific marker for neural cells, were expressed in hUCMSCs under various culture conditions, and highest expression was observed in the hUCMSCs co-cultured with injured PC12 cells. Choline acetyltransferase and microtubule-associated protein 2 were not expressed in hUCMSCs cultured alone (no treatment). Cell Counting Kit-8 assay results showed that hUCMSCs under co-culture conditions promoted the proliferation of injured PC12 cells. These findings suggest that the microenvironment during neural tissue injury can effectively induce neural cell differentiation of hUCMSCs. These differentiated hUCMSCs likely accelerate the repair of injured neural cells.