last updated: 2017.7.13.

Study on isolation, characterization and application of bacteriophages active against foodborne pathogens

Isolation and bio-control of Extended Spectrum Beta-Lactamase (ESBL)-producing Escherichia coli contamination in raw chicken meat by using lytic bacteriophages

 

Extended Spectrum Beta-Lactamase-producing Escherichia coli (ESBLEC) were isolated from 12/13 (92.3%) raw chicken meat samples by using selective culture and PCR. Of the 27 ESBLEC analyzed, 33.3% (9/27) of isolates were positive for ESBL of CTX-M group 1 followed by TEM (22.2%), SHV (22.2%), CTX-M group 2 (11.1%), and CTX-M group 9 (11.1%). None of ESBLEC tested were positive for stx1, stx2, eae, ehxA, saa, and subAB genes of Shiga toxin-producing E. coli. Among 22 isolated phages, phages PBL66-CL1 and PBL116-CS6 infected 21 (77.7%) and 20 (74%) of 27 ESBLEC isolates examined, respectively. The remaining phages lysed less than 50% of the hosts tested. Compared to non-treatment, the treatment of isolate EBL116 in the broth medium with phage cocktail of PBL66-CL1 and PBL116-CS6 at 25 °C and 5 °C after 6 h significantly reduced bacterial viable counts by 5.06 and 1.33 log CFU/mL, respectively. When the treatment was performed on raw chicken meat samples, viable counts of EBL116 were also decreased by 2.02 and 1.67 log CFU/4 cm2 meat piece after 6 h at 25 °C and 5 °C, respectively. This study demonstrates a high prevalence of ESBLEC in raw chicken meat and possible use of lytic phages as bactericide for controlling ESBLEC.

 


 

Specific inhibition of cytotoxicity of Shiga-like toxin 1 of enterohaemorrhagic Escherichia coli by gallocatechin gallate and epigallocatechin gallate

 

Mechanism of inhibitory action of 8 catechins and teaflavin was investigated at low concentration against Shiga-like toxin (Stx). Viability of Vero cells largely decreased in the presence of Stx1 and Stx2 preparations. Cytotoxicity of Stx1 decreased after preincubation with gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) at 100 mg/L. However, the cytotoxicity of Stx2 was not inhibited by the preincubation with catechins and teaflavin tested. The inhibitory activity of GCg and EGCg at 15 mg/L (0.0327mM) was investigated against Stx preparations with various concentrations. The cytotoxicity of Stx1 at the concentration ranging from 1.6 to 50 ng/mL significantly reduced (p<0.01) by the preincubation of Stx1 with GCg at 15 mg/L. Similarly, the cytotoxicity of Stx1 at the concentration ranging from 3.1 to 25 ng/mL was significantly reduced (p<0.01) by the preincubation with EGCg at 15 mg/L. In contrast, GCg and EGCg did not inhibit cytotoxicity of Stx2 at any concentrations tested. On the other hand, EGC showed no significant effects on cytotoxicity of both Stx1 and Stx2 at the same concentrations tested. The pocket sizes formed at the center of the Stx1B and Stx2B pentamers were calculated to be 778Å3 and 475 Å3, respectively. Docking simulations were conducted with EGCg positioned in the center of the pore of StxB pentamers. The docking models showed that EGCg formed 7 hydrogen bonds with side chains of amino acids faced inside the pocket of the Stx1B pentarmer with the lowest intramolecular energy (strain energy + electrostatic energy) of -0.1 kcal/mol. In contrast, in the case of Stx2B pentamer, EGCg formed 6 hydrogen bonds with the lowest intramolecular energy of 5.2 kcal/mol. In silico study suggested that EGCg forms more stable structure with Stx1B pentamer than Stx2B pentamer. These results indicated that both GCg and EGCg specifically inhibited cytotoxicity of Stx1 but not of Stx2.

 


 

Studies on antibacterial action of catechin derived from green tea extract

The antibacterial activity of the green tea extracts(GTE) containing catechins was investigated on various kinds of bacteria at different pHa??s.A The antibacterial activity of GTE was determined after incubation in 50% L broth containing various concentrations of GTE for 48 h at 30 or 37C.A The tests were performed on 19 strains of gram-negative bacteria and 27 strains of gram-positive bacteria. At pH 6.5, GTE effectively inhibited the growth of Staphylococcus aureus, Bacillus brevis, B. cereus, B. subtilis, Vibrio alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnifivus at less than 250 ppm.A A On the other hand, E. coli O157:H7, Salmonella spp and Listeria monocytogenes, Lactobacillus spp. were resistant to GTE.A The antibacterial activity of GTE increased at pH 5.0 on B. cereus, L. monocytogenes, L.plantarum and V. parahaemolyticus.A In contrast, the activity increased more than 4 times at pH 8.0 on S. Enteritidis, E. coli O157:H7 and P. aeruginosa etc. compared with those at pH 6.5. The results of the antibacterial test using each of the main eight components of catechines suggest that galloyl and gallyl groups are important for the antibacterial action at acidic and alkaline pH, respectively. The antibacterial activity of GTE was the highest on S. Typhimurium DkatG mutant of all strains (wild type, DkatG, DahpC, and DahpF), suggesting H2O2 generated from catechins just outside the cell envelope is one of the important factors for the antibacterial action.

The effects of the food additives on the antibacterial activity of green tea extracts (GTE) containing catechins were investigated using various bacterial strains. In the presence of 4% NaCl, the antibacterial activity of GTE increased to 2 to 8 fold against almost of gram-positive and -negative bacteria tested. Antibacterial activity of GTE was determined against both Listeria monocytogenes and Escherichia coli O157:H7 in the presence of each of the 12 different food additives at pH 5.0, 6.5 and 8.0. The effects of combining various food additives with GTE were higher against L. monocytogenes than against E.coli O157:H7. Against L. monocytogenes, nisin A, sodium citrate, and chitosan in combination with GTE showed effects at all pHs examined and glycine, hexametaphosphate, EDTA, and ethanol enhanced the antibacterial activities of GTE at pH6.5 and 8.0. Against E.coli O157:H7, on the other hand, only sodium citrate showed combined effects with GTE at all pHs examined. At both pH 6.5 and 8.0, hexametaphosphate, EDTA, and ethanol enhanced the antibacterial activities of GTE. Combined effects of GTE and protamine were observed at pH 6.5 on both bacteria. This result suggests that the damage on cell surface is important in enhancing the antibacterial activity of GTE.
The antibacterial activity of Green tea extracts(GTE) containing catechins is generally weaker against Gram-negative bacteria than Gram-positive bacteria. The combined effects of the cell-surface damaging compounds on the antibacterial activity of GTE were investigated to enhance the antibacterial activity of GTE on Gram-negative bacteria(Pseudomonas aeruginosa). The combined effects of GTE and the compounds on P. aeruginosa were determined after incubation in 0.1M phosphate buffer containing various concentrations of GTE and the compounds for 24h at 30a??.Four kinds of surfactants (Sodium Laureth Sulfate, Lauramine Oxide, Benzalkonium Chloride, Glyceryl Laurate) showed the combined effects with GTE on P. aeruginosa. The nonionic surfactant enhanced the antibacterial activity of GTE at pH 5.0, 6.5,and 8.0. On the other hand, the amphoteric, cationic and anionic surfactants enhanced the antibacterial activity of GTE at both pH 5.0 and 8.0, but did not at pH 6.5. Two kinds ofA preservatives (methylparaben and phenoxyethanol)A with no electrical charge enhanced the antibacterial activity of GTE at pH 5.0, 6.5,and 8.0. These results suggested that the electrical charges of the compounds and catechines are important for the combined antibacterial activity.A Furthermore, the combined effect of GTE and glyceryl laurate , which exhibited the strong combined effect with GTE in phosphate buffer, was investigated on 5 kinds of bacteria in 50%L-broth for 24h under the condition of pH6.5 and 37 or 30C. The combined effect of glyceryl laurate and GTE was strong aginst B.cereus and B.subtillis, butA weak against L.monocytogenes and E.coli O157:H7. These results suggested the combined effect of GTE and glyceryl laurate was generally weaker against gram-negative bacteria than gram-positive bacteria, due to the interaction between glyceryl laurate and organic components in the medium.

Difference of EGCg adhesion on cell surface between Staphylococcusaureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride

We developed a novel method using indirect staining with cerium chloride for visualization of the catechin derivative epigallocatechin gallate (EGCg) on the surface of particles, i.e., polystyrene beads and bacterial cells, by electron microscopy. The staining method is based on the fact that in an alkaline environment, EGCg produces hydrogen peroxide, and then hydrogen peroxide reacts with cerium, resulting in a cerium hydroperoxide precipitate. This precipitate subsequently reacts with EGCg to produce larger deposits. The amount of precipitate is proportional to the amount of EGCg. Highly EGCg-sensitive Staphylococcus aureus and EGCg-resistant Escherichia coli were treated with EGCg under various pH conditions. Transmission electron microscopy observation showed that the amount of deposits on S. aureus increased with an increase in EGCg concentration. After treating bacterial cells with 0.5 mg/mL EGCg (pH 6.0), attachment of EGCg was significantly lower to E. coli than to S. aureus. This is the first report that shows differences in affinity of EGCg to the cell surfaces of Gram-positive and -negative bacteria by electron microscopy.

Clarification of the mechanism of epigallocatechin gallate (EGCg) antibacterial action against bacterial spores: EGCg inhibits growth, not germination, of bacterial spores

Spore-forming bacteria form highly stress-resistant spores in environmental conditions that are deleterious to growth, such as nutrient limitation, and are thus of particular concern in the food and beverage industry. Control of these bacteria is extremely important for food and beverage preservation. Catechins exhibit antibacterial action against the vegetative cells of a range of bacterial species, but the detailed mechanism of action of catechins on spores has not been clarified. Here, we examined the mechanism of antibacterial action of epigallocatechin gallate (EGCg) against spore-forming bacteria. The effects of catechins on the germination of spores and vegetative cell growth of the genus Bacillus were investigated by chronological measurements of viable counts and spore counts cultured in medium containing EGCg, using total cell count and heat resistance cell count as indices of spores. The spore count was independent of the presence of EGCg and decreased immediately after the start of culture, whereas vegetative growth was suppressed by EGCg. It is therefore likely that EGCg does not inhibit spores, but acts on vegetative cells. We also analyzed the amount of EGCg adsorbed by vegetative cells and spores with an electron microscopic observation technique using cerium. The amount of EGCg adsorbed by spores was extremely small in comparison to that adsorbed by vegetative cells. Our findings indicate that the reason why catechins do not suppress spore germination or inactivate spores is that catechins are not adsorbed by spores and thus cannot act on them.

A study of the antibacterial mechanism of catechins: Isolation and identification of Escherichia coli cell surface proteins that interact with epigallocatechin gallate

Catechins have high anti-bacterial activity against various microorganisms. In this study, the mechanism of anti-bacterial activity of catechins was investigated using Escherichia coli. Transmission electron microscope analysis revealed that deposits containing EGCg were found only on the outer membrane, which is the outermost layer of the cell surface, in E .coli cells treated with EGCg. Based on this observation, we focused on outer membrane proteins as targets of EGCg in E. coli. Two-dimensional electrophoresis identified 16 spots that had disappeared or showed markedly reduced intensity after treatment with EGCg compared to the control. Of these, an outer membrane porin protein, OmpG, acids suggested that the basic amino acids Lys, Arg, and His strongly interacted with EGCg. The docking simulation with EGCg and OmpG revealed that EGCg enters into the porin pore and binds to Arg residues present on the inner surface of the pore channel through hydrogen bonding, resulting in inhibition of the porin function. Furthermore, glucose uptake by E. coli was inhibited in cells treated with EGCg. Taken together, these results suggest that EGCg inhibits the major function of porin proteins, namely the passive transport of small hydrophilic molecules such as glucose, leading to growth inhibition of E. coli.


 

Study on mechanism for adhesion of foodborne pathogen on agricultural produce, and survival strategy of the pathogen in and on produce (lettuce, tomato, spinach)

 

Investigation of Routes of Salmonella Contamination Via Soils and the Use of Mulch for Contamination Control during Lettuce Cultivation

Foodborne illnesses associated with the consumption of fresh produce such as raw vegetables have become a major health concern worldwide in recent years. In the present study, we investigated the possible routes of Salmonella contamination in leafy lettuce via soil during cultivation. After 10-week cultivation of lettuce plants in soils inoculated with S. Enteritidis expressing green fluorescent protein (SE-EGFP), the bacterium was detected in soil inoculated with >104 cfu/g and from most lettuce leaves cultivated in soils inoculated with >4.4 × 107 cfu/g. As Salmonella was not detected in intact lettuce leaves or lettuce leaves with root injury cultivated in highly contaminated soils and after surface disinfection, the lettuce plants were not considered to internalize the bacterium. Overhead irrigation led to the contamination of one in 10 lettuce plants; however, all sets of three leaves of the plant were contaminated (>110 MPN/g). In an effort to prevent Salmonella contamination from soils, we investigated the effects of mulch on contamination levels during cultivation. Mulch effectively reduced Salmonella contamination levels of lettuce plants cultivated in highly contaminated soils.

 

 

 

 

 

 

Recently tomatoes have been the most implicated vehicles for produce-associated salmonellosis for some outbreaks.  Trace back reports suggested that pre-harvest contamination of Salmonella enterica might be the main reason of these outbreaks, however parts of tomato fruits to which the pathogen attaches remain unclear.  It is important to investigate mechanisms of contamination of fresh produce by Salmonella.  To trace the presence of Salmonella in the soil and plants, Salmonella Enteritidis transformed with a pEGFP plasmid vector (S. Enteritidis-EGFP) was used.  The soil was artificially contaminated with several levels of S. Enteritidis-EGFP (104, 106 or 108 CFU/g) followed by cultivation of tomato plants in the contaminated soil.  Samples of the soil and each organ of the tomato (fruit, stems/leaves, and root) were assayed for Salmonella by plating onto Tryptic Soy Agar.  Population of Salmonella in the soil was gradually decreased over the time and remained depending on the initial levels of inoculation.  Population of Salmonella was under detection limit (<100) in fruits and stems/leaves, regardless of contamination levels of soil.  Moreover, no S. Enteritidis-EGFP population was recovered from tomato fruits whose roots were injured. These results indicate that internalization of Salmonella to tomato fruitsmight not occur after cultivation in contaminated soil.  However,it is necessary to prevent the contaminated soil from attaching to fresh produce.

 


Development of Simple and Rapid Detection Method of Food Poisoning Bacteria

Salmonella spp., Escherichia coli, Staphylococcus aureus, Campylobacter spp., and Vibrio spp. are the major causes of food poisoning in the developed world. Conventional methods for the detection of these food poisoning bacteria in foods require 2 to 4 days to obtain presumptive results after initiation of sample analysis. The needs for more rapid and simple methods for the detection of food poisoning bacteria are growing in food industries and government agencies. In recent years, new methods have been developed to increase the efficiency and speed of food microorganism detection while reducing the labor. We have developed the rapid detection method for Salmonella by combination of a new selective enrichment and ELISA using monoclonal antibodies against dulcitol 1-phosphate dehydrogenase, a random amplified polymorphic DNA (RAPD) analysis for Salmonella. and fluorogenic assay for rapid detection of Vibrio parahaemolyticus in foods.

We have developed and are developing,

(1) Application of random amplified polymorphic DNA analysis for detection of Salmonella in foods.

(2) Rapid detection of Salmonella spp. by PCR amplification of Salmonella specific region in gat D gene.

(3) PCR assay for detecting Escherichia coli O157:H7 and O157:H-
(4) Rapid detection method of food poisoning bacteria by the combination of PCR and quartz crystal microbalance
(5) Rapid detection method for viable bacteria and food poisoning bacteria by using a photon- counting TV camera.
(6) Immunomagnetic-flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk
(7) Development of Rapid and Simple Method for Total Viable Bacterial Counts by Flow Cytometry
(8) Simple Identification of Some Bacillus Species and Related Genera in Food by RAPD Analysis Combined with Morphological Observation
(9) SNP typing and rapid identification of Listeria monocytogenes
 

Studies on inhibitors of adhesion of food poisoning bacteria

Inhibitors of Adhesion of Salmonella Enteritidis
The inhibitors of adhesion of Salmonella Enteritidis onto microplates were screened among 39 additives including natural additives and food additives with high safety. S. Enteritidis adhered strongly onto microplate after incubation in 0.1% Bacto-soytone for 24 hr.A The adhesion was inhibitied in the presence of pigments such as Annatto pigment, Gardenia yellow, and Monascus pigment, food color products such as Annatto AN, San-brown AC, San-yellow No. 2AU, San-red YM, San-red RCFU, San-beet L, and food additives such as protamine, Chili extract, sucrose fatty acid esters, monoglycerine fatty acid esters, and flavonoids such as Kaempferol.A Among them, Annatto AN, Gardenia yellow, Monascus pigment, and sugar fatty acid esters inhibited the adhesion at the concentration without antibacterial activity. In the case of sugar fatty acid esters with C8 to C18 tested, the longer the fatty acid chain, the stronger the inhibitory effects. Especially, Monascus pigment and sucrose monostearate most strongly inhibited the adhesion of S.Enteritidis even at 0.01% without antibacterial action.

Inhibition of adhesion of several bacteria onto microtiter plate by selected food additives

 Adhesion inhibitory effects of food additives, such as Polylysine (PL) and Whey protein (WP), as well as Sucrose fatty acid ester (SFE) with fatty acid of C8 to C18, Monoglycerin fatty acid ester (MFE) with fatty acid of C8 to C18, Gardenia yellow pigment (GY), Monascus pigment (MP), and Protamine (PT) that had been shown to inhibit adhesion of Salmonella Enteritidis onto microtiter plate, were determined on several bacteria. Among SFE tested, adhesion of S. Typhimurium onto microtiter plate was decreased to less than 50% of the control by SFE with fatty acid of C10, C12, C14, and C16 at 0.05% and that of C18 at 0.01%.  MFE with fatty acid of C8, C10, C12, C16, C18 also inhibited the adhesion to less than 50% of the control. The adhesion of S. Typhimurium was also almost completely inhibited by PT and PL at 0.01%, 0.1% MP, 0.025%WP, and 1% GY. Adhesion of Pseudomonas aeruginosawasdecreased to less than 50% of the control by SFE with C8 and C16 fatty acid at 0.05%, C12 and C14 at 0.01%, 0.1% PT, 0.01% PL, and 0.025% WP, but not by 0.05% MFE tested, 1% GY, and 1% MP.  Adhesion of P. fluorescens was almost completely inhibited by SFE with fatty acid of C14, C16, and C18 at 0.05, 0.005, and 0.005%, respectively, and 1% GY, 1% MP, 0.1% PT, and 1% PL, but not by MFE and WP even at 0.05 and 0.25%, respectively. Adhesion of Listeria monocytogenes wasdecreased to less than 50% of the control by SFE with fatty acid of C10, C12, C16, and C18 at 0.05% and by MFE with fatty acid of C10, C14, C16, and C18 at 0.05, 0.05, 0.005, and 0.005, respectively.  The adhesion decreased to less than 50% of the control by 0.1% MP, 1% PT, and 1% PL, but not by 1% GY and 0.25% WP.  Adhesion of Staphylococcus aureusdecreased to less than 50% of the control by SFE with fatty acid of C10, C12, C14, C16, and C18 at 0.05, 0.01, 0.005, 0.005, and 0.005%, respectively. The adhesion was inhibited by more than 50% by 0.05% MFE with C8 fatty acid and by 0.005% MFE with C10 to C18 fatty acid.  GY, MP, PT, PL, and WP decreased the adhesion by more than 50% at 0.1, 0.01, 0.01, 0.01, and 0.025%, respectively.  It seems to be important to select suitable substances for inhibition of adhesion of each of the bacterial species.

Combined effects of selected food additives on adhesion of various foodborne pathogens onto microtiter plate and cabbage leaves

The study was conducted to examine the adhesion inhibition and antibacterial activities by combined use of some selected food additives such as Sucrose Fatty Acid Ester (SE) C18, Gurdania Yellow (GY), Monascus Pigment (MP), Protamine (PT), ε-polylysine (PL) and Milk Serum Protein (MSP), against Salmonella Enteritidis, S. Typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus. The adhesion of those pathogenic bacteria was reduced by several combination of food additives compared to that of each of the single use. The combinations decreased the relative adhesion more than 10% compared to that of each of the single use were taken into consideration. The following combinations such as SE18 & GY, PT & MSP, and PL & MSP were effective in inhibiting the attachment of S. Enteritidis onto microtiter plate compared to that of each of the single use. In case of S. Typhimurium, the combination of MSP & MC, SE18  & GY, SE18 & PT, SE18  & PL, SE18  & MC, SE18  & MSP, MC  & PT, MSP & PL, GY & PL, and MSP & PT were effective in adhesion inhibition. The combination of SE18 & GY, GY & MSP, and PL & MSP were effective in inhibiting the attachment of P. aeruginosa onto microtiter plate compared to that of each of the single use. The combination of GY & MSP, MSP & PT or PL and MC & PL were effective to inhibit the attachment of L. monocytogenes. The adhesion of S. aureus was reduced by combined use of SE18 & PT, GY & PT, GY & PL, MSP & GY, MSP & PL, SE18 & GY, SE18 & MC, and MSP & PT compared to that of each of the single use. On the other hand, there were no such significant changes in viable counts of all pathogenic bacteria tested by using combination of food additives compared to that of each of the single use. However, the viable counts of S. Enteritidis and P. aeruginosa decreased drastically in the presence of PL (0.01%) and PT (1%), respectively and reached to the lower detection limit. In addition, the viable counts of L. monocytogenes decreased around 4 Log in the presence of PL. These results clearly showed that the combination of MSP and PT or PL effectively reduced adhesion of all the Gram-positive and negative pathogens tested on the microtiter plate. The pretreatment of cabbage leaves with combination of PL & MSP was also effective to reduce the viable counts of secondary-contaminated SE-EGFP on the cabbage leaves by washing with water compared to that without the pretreatment.

 


Sterilization of fresh produce by the combined use of slightly acidic hypochlorous water and sucrose fatty acid ester

Treatment by slightly acidic hypochlorous water (SAHW) in combination of pretreatment with sucrose fatty acid ester under microbubble generation was effective for sterilization of lettuce. Sufficient contact time of SAHW containing 30 ppm of available chlorine on reduction of viable counts of lettuce was determined to be 5 min. For 5 min at 18-20°C, treatment with 30 ppm of available chlorine in SAHW appeared more effective in the reduction of bacteria on lettuce compared with 15ppm. The treatment of lettuce at 50°C with SAHW at 30ppm of available chlorine showed a considerable reduction of bacterial counts by 2 log without injury in the tissue. The treatment at 50°C, SAHW also delayed browning on cut lettuce for the first 5 to 6 days of subsequent storage at 6°C. Among 2 sucrose fatty acid esters tested, sucrose monopalmitate at 100 ppm had a higher efficacy for pretreatment under microbubble generation. After pretreatment for 5 min with 100 ppm sucrose monopalmitate under microbubble generation and subsequent treatment with SAHW at 50°C for 5 min, viable counts of lettuce were decreased by about 3-4 logs. After the same treatment, Pseudomonas sp. predominant on lettuce decreased drastically. These results indicate the effectiveness of the combined treatments of sucrose fatty acid ester under microbubble generation and SAHW at 50°C for sterilization of fresh produce.

 

 


Sporulation of Bacillus cereus

Bacillus cereus is one of the food poisoning bacteria which produces heat resistant enterotoxin and spores. The presence of its spore in food is one of the biggest problems in food industry because the spore is resistant to heating, ultraviolet light and drugs. It is very difficult to sterilize these foods contaminated with spores of B. cereus.
The formation of an asymmetrically sited division septum is an early event of sporulation of Bacillus. The asymmetric division results in two distinct cells, that have radically different developmental fates. This sporulation division contrasts with division during vegetative growth of Bacillus, in which the septum is symmetrically situated with respect to the ends of the dividing cell.
The penicillin-binding proteins (PBPs) are membrane bound enzymes required for peptidoglycan synthesis and septum formation. At least seven PBPs are reported in Escherichia coli. There are various beta-lactam antibiotics and the target sites of them have been studied in detail on E. coli. Some of these antibiotics have specific target PBPs, for example, cefaloridine specifically binds to and inhibits PBP 1, mecillinam is specific for PBP 2 and cephalexin for PBP 3 of E. coli. The studies on the mechanism of action of penicillin on B. subtilis have shown the presence of PBPs that increase or decrease during sporulation. However, the mechanism of the gene expression for these PBPs at the initial stage of sporulation is not clear.
We have studied the mechanism of sporulation of B. cereus. We previously showed the presence of chromosomal proteins that seem to be involved in the initiation of sporulation of B. cereus. We identified one of the proteins as IMP dehydrogenase. In B. cereus, the enzyme activity was the largest in the cells that were induced to sporulate by nutritional shift down at 40 min from the initiation of chromosome replication, the sensitive stage for sporulation. The result suggests that high IMP dehydrogenase activity is necessary for the induction of sporulation of B. cereus. We are studying the involvement of IMP dehydrogenase in induction of sporulation of B. cereus. We have also shown that cephalexin inhibits the sporulation of B. cereus which has been induced to sporulate by nutrient downshift and that the presence of PBPs that has high affinity to cephalexin. We are also studying the mechanism of expression of the PBPs sensitive to cephalexin in B. cereus.

Injury and recovery of Salmonella

Salmonella enteritidis is currently the main cause of human salmonellosis in most industrial countries [1]. Heat treatments and freezing kill a fraction of a bacterial population, but they also generate damaged cells. The ability of these stressed cells to survive depends on the recovery conditions. Under favorable environmental conditions such as in nonselective medium, injured cells usually undergo repair and become functionally normal. Chemicals with selective properties often are added to media to facilitate detection of specific bacteria in foods. Some of these agents inhibit repair of injured cells. Successful enrichment is the decisive step in the detection of salmonellas. Several studies have been done to improve techniques for recovering sublethaly injured Salmonella [2, 3, 4]. In order to develop an effective methods or media for the enrichment of damaged S. enteritidis in food, it is necessary to know the mechanism and the level of the damage on the bacterium. In this study, we have investigated the effects of pH of medium on viability of S. enteritidis after heating and freezing. Effects of some reagents on the recovery of the injured S. enteritidis were also investigated.

Changes in transcription during recovery from Heat-Injury in SalmonellaTyphimurium and effects of BCAA on recovery

Mechanisms of recovery from heat-injury in Salmonella Typhimurium were elucidated.  Recovery of the heat-injured S. Typhimurium cells in TSB resulted in full recovery after 3 h of incubation at 37°C.  The DNA microarray analysis of 30-min and 60-min recovering cells resulted in an increase in transcription of 89 and 141 genes, respectively.  Among them, 15 genes, with known function, seemed to be somewhat involved in recovery.  They encoded proteins involved in branched-chain amino acid (BCAA) transport (livJ, livH), cell envelope integrity (ddg), heat-shock response (cpxP, rrmJ), phage shock protein (pspA), ribosome modulation factor (rmf), virulence (sseB) transcriptional regulation ( rpoE, rpoH, rseA, rseB, rseC) and ArcB signal transduction (sixA) and cytoplasmic membrane protein (fxsA). It seems that BCAA promoted the recovery by promoting protein synthesis either directly through their use in translation or indirectly through stimulation of protein synthesis by activation of the Lrp protein.

 


Subtyping of Listeria monocytogenes Based on Nucleotide Polymorphism in clpC, inlA, hlyA, and plcA Genes and Rapid Detection of L. monocytogenes Genetically Similar to Clinical Isolates

The Listeria species are widely distributed in the environment. They are found in soil and in mammals, thus they are highly detected in various types of food, mainly meats and dairy products. L. monocytogenes is a significant food-borne pathogen and causes an infectious disease known as listeriosis. In the food industry, contamination of food with the bacterium may lead to serious problems since it can grow even at low temperatures and high salt concentrations during preservation of ready-to-eat foods such as unsterilized dairy products and raw vegetables. In Japan, although sporadic cases of listeriosis have been reported, no serious epidemics have occurred. However, L. monocytogenes is often detected in foodstuffs, which may lead to a potential outbreak of listeriosis. Listeriosis may result in mortality for pregnant women, infants, immunocompromised persons, and the elderly. For identification and subtyping of L. monocytogenes, several techniques, such as phenotypic typing (serological typing, phage typing, and multilocus enzyme electrophoresis) and molecular typing techniques (ribotyping, restriction enzyme analysis, PCR-based typing, and DNA sequencing), have been used in epidemiology.


Enhancement of Freezing Tolerance in Plants

Some plant species develop freezing tolerance when exposed to low, non-freezing temperatures. If freezing tolerance could be provided to cold-sensitive plants, the contribution to stable production of crops would be significant. In our laboratory, we found that hardened cells of Chlorella vulgaris C-27 survive slow freezing to -196°C. Freezing tolerance of the cells were developed by expression of hardening-induced Chlorella (hiC) genes. We have isolated 17 cDNA clones corresponding to hiC genes. Out of them, hiC6 accounted for 75% of hiC clones and was shown to be siginificantly involved in the development of freezing tolerance.
In order to investigate whether the HIC6 protein will confer freezing tolerance to plants, the coding region of the hiC6 gene was introduced into tobacco plant. However, the aquired level of freezing tolerance of the transgenic tobacco leaves was relatively low and not satisfactory. Now, we are trying to enhance the level of freezing tolerance by increasing the expression level of the HIC6 protein and by introducing additional genes encoding stress-inducible proteins such as fatty acid desaturase and scavenging enzymes, superoxidase dismutase and ascorbate peroxidase etc..