Abstract: Eight isolates of Salmonella typhimurium were contaminated different sources of meat, poultry products and caused deadly diseases in humans. SDS-polyacrylamide gel electrophoretic technique showed that the protein banding patterns of Salmonella typhimurium. Isolates were more similar in the upper part of the gel. While difference were clearly detected in the lower part of the gel. The protein band of 289 KDa was a common protein band in all isolater of Salmonella typhimurium isolates. The similarity coefficient among Salmonella typhimurium protein profiles ranged from 0.43 to 0.63. By studying plasmid profiles to all isolates, it showed that the intensity of the extracted plasmids from Salmonella typhimurium No. 4. is much denser than others which indicate that there are more copie number of plasmids which could be related to the resistance of this strains. Further study on the isolate No. 4. The effect of irradiation on the viability of bacterial cell which occur drops in the viable count of bacteria in response to increasing doses of gamma irradiation from (0-1.0 KGY) and the D10 value was calculated which equal to 0.561 KGY. Decrease in viable cell number when heating times were extended to 2 min at 65°C. Antibiotic profiles of isolate No. 4 was detected against ten different antibiotic (Imipenem, ampicilin/sulbactum, amoxicillin, nitrofurantion, gentamycin, tobramycin, ofloxacin, cefotaxime cefaclor and chloromphenicol). Detection of MICs by E-test Imipenem. The most effective essential oils were garlic, carvacrol, thyme and thymol. Heating without irradiation caused destabitization of the cytoplasmic membrane allowing penetration of hydrophobic dye. Further, the lethality of heating followed by irradiation for S. typhimurium was additive, reflecting irradiation induced DNA-damage and heat induced membrane destabilization, but when irradiation preceded heating, more cell were inactivated because of heat- inactivating radiation damage DNA. The transmission electron microscope indicate the variation on the effect of radiation heat treatment and combined radiation and heat. The amino acid contents detected that glutamic acid was the dominant.
INTRODUCTION
Polyacrylamide gel electrophoresis technique is used extensively for the identification of many bacteria. The proteins separate as bands across the gel and the pattern is characteristic for a given species. The sodium dodecyle sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a low cost, reproducible and rapid method for quantifying, comparing and characterizing proteins (Laemmli, 1970). This form of PAGE has been used to classify and identify different species of bacteria (Plikaytis et al., 1986). The numerical analysis of PAGE of whole cell protein extracts was used for the classification and rapid identification of pathogenic species (Vanzyl and Syten, 1990).
The analysis of the electrophoretic profile of proteins secreted by the bacteria were used in order to determine a precise and quick identification technique for pseudomonas and another species (Lacroix et al., 1995). The use of ionizing irradiation has been suggested as a method for eliminating or reducing contamination of food by pathogens such as Salmonella and E. coli from meat and poultry products. Both direct and indirect reactions between ionizing radiation and cellular components occur in direct proportion to the amount of energy that is absorbed. Since 50 to 70% of the bacterial cell mass is water, it absorbs much of the radiation. As a result, hydroxyl radicals and hydrated electrons which are important in radiation-induced cell inactivation are produced. Irradiation damage of DNA is considered a major cause of cell inactivation, while those of protein, lipid and RNA contribute less (Von Sonntag, 1987). Reaction of DNA with hydroxyl radicals may result in single and/or double-strand breaks, protein DNA cross-linkage and base attractions, leading to cellular inactivation (Von Sonntag, 1987).
Survival of bacterial cells following irradiation depends upon intrinsic factors, such as the physiological conditions of individual cells and their potential for repair. The process of thermal control of food -borne pathogens in various food systems is well established. Typically the bacterial population decreases exponentially, while a portion is heat resistant (Gould, 1989; Hurst, 1977). Summarized some of the mechanisms by which heat may cause cellular inactivation. (i) damage of DNA, (ii) inhibition of protein synthesis (iii) damage of cell membrane and (iv) inactivation of critical metabolic enzymes. Because multiple changes may occur during the inactivation of bacteria by heat, thermal inactivation is not linear (Gould, 1989). Some or all of these mechanisms may also be applicable during inactivation of bacterial cell by irradiation. Simultaneous applications of ionizing irradiation and heating to control food -borne pathogens have been proposed to maximize food safety, while preserving food quality by reducing the detrimental effects of heating and irradiation on the food (Radomysko et al., 1994). However, current regulations require irradiation of poultry at -4°C during processing. An enhanced (synergistic) heat sensitivity of irradiated Salmonella typhimurium has been reported by Thayer et al. (1991) suggesting that, if a few Salmonella cells should survive, the irradiation treatment, they would be very unlikely to survive cooking, assuming that the irradiated product has been properly refrigerated, thus preventing recovery and multiplication of the cells.
Antibiotic resistance in Salmonella typhimurium, one of the most frequent etiologic pathogens of food borne bacterial gastroenteritis in humans, is a serous health problem world wide (Wu et al., 2005). Essential oil extracts of various plants have been reported to have inhibitory effects against diverse types of microorganisms including Gram positive and Gram negative (Chao et al., 2000). Garlic exhibits abroad antibacterial spectrum against both G +ve and G -ve bacteria (Sivam, 2001). Further, the effect of irradiation increase in the total amounts of the amino acids contents by either individual or combined treated with heat and irradiation (Khaled, 2004). Tawfik et al. (1992) reported that the ultra structure study for S. typhimurium isolate irradiated at different dose levels showed that the effect of radiation was concentrated on the cellular material rather than on the cellular membrane.
The objective of this study was to identify mechanisms by which irradiation and heat act synergistically to produce greater in activation of S. typhimurium than would be predicted from the effects of heating and irradiation alone. The relative amounts of DNA and cellular membrane damage were determined.
MATERIALS AND METHODS
Bacterial isolate: Eight Salmonella typhimurium were isolated from different sources (meat and poultry products). The isolates were enriched in selenit F- broth, isolated on selective medium; Salmonella shigella (ss) agar and finally transferred to triple sugar agar slants.
SDS-PAGE method: Cellular proteins of 8 isolate of Salmonella typhimurium were analyzed by SDS-PAGE method (Laommli, 1970).
Agarose Gel Electrophoresis: Agarose gel electrophoresis technique was employed for detection and preliminary characterization of plasmid DNA present in the most resistant isolates of S. typhimurium according to Millesimo et al. (1996).
Antibiotic susceptibility test: Antimicrobial susceptibility of the isolate was done by the disc diffusion technique using commercially available antibiotic disc as recommended by Bauer et al. (1966).
Determination of minimum inhibitory (MICs): The Minimum Bacteriodical Concentrations (MBCs) was detected by Et-strip.
Effect of essential oils on the growth of the isolates: Antibacterial activity of ten essential oils were tested by the filter paper disc diffusion method.
Radiation treatment: Source of radiation used was cobalt-60 Gamma cell 220/located at National center for Radiation Research and Technology (Nasr city, Cairo). The dose rate of this source was 2.85 KGy at the time of experiment. Cells (24 h age) suspended in nutrient broth medium was exposed to increasing doses of gamma radiation (0.0, 0.2, 0.4, 0.6, 0.8, 1.0 and 2.0 KGy).
The viable cells mL-1 were recorded prior and after irradiation. The log of surviving fractions were calculated as log N/No. (n = No. of cell after treatment and No = No. of cells before treatment) and D10 values (radiation dose which reduced the microbial counts by one log cycle) were detected.
Heat treatment: Bacterial cell suspensions each of 4 mL in test tubes were immersed into a water bath at 65°C. Temperature of cell suspensions were monitored by a thermometer. The tubes were incubated at 65°C from 1 to 5 min and then rapidly cooled to 0°C.
Combination treatment: To determine the relationship between radiation and heating, bacterial cell suspensions were heated at 65°C for 2 min either before or after irradiation with 1.0 KGY. The total viable counts were determined for each treatment.
Studying the ultra structure of bacterial cell (S. typhimurium): Bacterial strain S. typhimurium was subcultured in nutrient broth and incubated for 24 h at 30°C. After incubation, bacterial culture was centrifuged, get rid of the supernatant and the bacterial precipitate was prepared for electron microscopy by the methods recommended by Phillip (1981).
Amino acid analyzer: The amino acid composition of Salmonella typhimirum before and after irradiation was carried out using LC 3000 amino acid alanyzer at flow rate 0.2 mL min-1 pressure of buffer from 0-50 bar, pressure of reagent from 0 -150 bar and reaction temperature 123°C.
RESULTS
About Eight isolates of Salmonella typhimurium were isolated and identified according to Bergy's manual Holt et al. (1994) and (Murray et al., 1999).
| Table 1: | The number of the bands of protein and molecular weight of
highest concentration of protein band in relation to "SDS.PAGE" |
| Table 2: | Antibiotic susceptibility of Salmonella thyphimurium |
| Fig. 1: | Electrophoretic patterns of total cellular protein of S.
typhimurium isolates (i.e., S1, S2 , S3
, S4 , S5 , S6, S7 and S8).
Protein bands are identified from top to bottom |
| Table 3: | Effect of volatile oils on selected isolate S. typhimrium
No. 4 using paper disc diffusion method and detected the MIC |
| Table 4: | Effect of different temperature/for 2 min on the surviving of S. typhimirum |
The result in Fig. 1 and Table 1 show in the electrophoregram (scans of protein profiles or fingerprints) of total cellular proteins of S. typhimurium isolates revealed from 12 to 22 discrete protein bands. The protein profiles of isolates (8 S. typhimurium.) contains 19, 21, 21, 18, 22, 15, 14 and 12 bands, respectively. Comparison of protein patterns from Salmonella typhimurium isolates revealed that the patterns of these isolates were similar especially in the high molecular weight parts of the gels. Most of the bands in each isolate could be matched with bands in each of the others.
The dendogram tree Fig. 2 was divided among the isolates of S. typhimurium into three main groups representing S1, S7, S5 on one side and S2, S3, S4 on the one side and S8, S6 on the other side, the average marker similarity was 0.43 across all isolates. The highest similarity was scored between isolates S7 and S5 (0.83) followed by that between S3 and S2 (0.60). On the other hand, the least similarity was scored between S8, S6 and all other isolates (0.43). The markers used in the present study proved to be sufficient that can be used in distinguishing among different bacterial isolates with similar genetic background.
| Fig. 2: | Phenogram demonstrating the relationship among 8 isolates
of S. typhimurium based on a complied data set recorded from protein
polymorphism |
Furthermore, the electrophoretic patterns of S. typhimurium isolates were nearly identical and contain the protein band of 289 KDa. as a common band in all isolates, corresponding with the close relationship found between morphological and cultural characters of S. typhimurium isolates which can reflect genetic relatedness (Sacks et al., 1969 and Abo-El-Khair et al., 1986).
By studying Fig. 3, it showed that the intensity of the extracted plasmids from Salmonells typhimurium No. 4 is much denser than others isolates which indicate that there are more copie number of the plasmids (marked at RF of size 23, 2.2 and 2.0 kpb) which could be related to the resistance to this isolate. These results are in agreement with (Son and Lin, 2001) which detected that plasmid analysis of Salmonella species showed only three plasmid profiles.
The result revealed in Table 2 variation in their response of the isolate No. 4 to antibiotic (Imipenem, Ampicilin/sulbactum, Amoxicilin, Gentamycin, Tobramycin, Ofloxacin, Cefotoxime, Cefaclor and Chloromphenicol). These results indicated that S. typhimurium No. 4 was resistant to most previously useful antibiotic resistant. These results are in agreement with (Graham, 2002) concluded that S. typhimurium was resistant to most antibiotics.
In the present study Fig. 4 revealed the minimum concentrations (MIC) of imipenem (IPM) by E-test was 1.25 mg mL-1 against S. typhimurium No. 4. The data obtained indicated that there was a good correlation between disc diffusion test and E-test against different multidrug resistant isolates. These results are in agreement with the results of Hanberger et al. (1999) found that MIC of imipenem against E. coli, Klebsiellai, Salmonella typhiumium and P. aeruginosa were 100/100/98, 100/100/95, 100/100/100 and 84/84/84 for antimicrobial chemotherapy.
| Fig. 3: | Plasmid profile of S. typhimrium (1, 2, 3, 4, 5, 6, 7 and 8) strains. M1 λ Hind III, M2 1 KB, M3 100 bp |
| Fig. 4: | MIC of imipinem (IPM) against S. typhimrium using E-test was 1.25 ug mL-1 |
The result in Table 3 indicated the antimicrobaial potential of Garlic, thymol, G. Fennel, Carvacrol and thyme was evaluated by determining the MIC of 1.60, 1.56, 0.04, 1.56 and 2.75 respectively were observed for Salmonella typhi. No. 4. These results are in agreement with the Chiasson et al. (2004) which indicated the antimicrobaial polential of carvacrol, thymol and thyme were evaluated by determining the MIC of 1.2, 1.60 and 2.75, respectively were observed for Salmonella typhimurium.
From the Table 4 showed the average values of the viable counts for irradiated as well as non-irradiated Salmonella typhimurium No. 4. The dose response curve was obtained by plotting log N/No. The results revealed that generally, a significant drop in the viable counts of bacteria in response to increasing doses by Gamma irradiation and formation a simple exponential relationship of cells over the whole dose range (type A) from the plotted surviving curve D10 value was calculated which equal to 0.561 KGY on the other hand the results obtained by Twafik and Abu-Shady (1992) which reported the D10 value of S. typhi. was found to 0.24 KGY.
| Fig. 5: | Electron micrograph of Salmonella typhimirium (a) control,
(b) effect of heat for 2 min at 65°C, (c) effect of gamma irradiation,
(d) combined effect of heat and irradiation |
| Table 5: | Response of Salmonella typhimurium to increasing doses of gamma radiation |
| Table 6: | Irradiation dose was 1 KGY followed by heat treatment at 65°C for 2 min |
| Table 7: | D10 values for S. typhimurium following irradiation and/or heating |
| Cell suspensions were heated at 65°C for 2 min either before or after radiation | |
The results of heat sensitivity of S. typhi. as represented in Table 5 showed the greatest decrease in viable cells occurred during the come-up period with a small additional decrease in viable cell number when heating times were extend to 2 min at 65°C. Heat can damage protein, lipid, nucleic acids and destabilize membranes (Gould, 1989). On the other hand, the combined effects of irradiation and heating in S. typhimurium. detected in Table 6 showed that the combined irradiation followed heat decreased the number of survived cell. It is possible that heat and irradiation act on different sites but also some targets such as DNA to cause additive or synergistic effects on cells (Yatvin and Grummer, 1987). Alternatively, heated cells could be more sensitive to irradiation either because the heat-labile recovery capacity of bacterial cells is affected (Bridges, 1969) or because recovery from heating-induced damage by irradiation is abolished, preventing protein synthesis as a consequence of DNA damage in vegetative bacterial cells (Mukherjee and Bhatta Charjee, 1970). The dose-independent irradiation enhanced heat sensitivity of S. typhimurium may be explained by the degree of association between DNA and cytoplasmic membrane as has been suggested by Yatvin and Grummer (1987). Irradication may induce functional changes in the cytoplsmic membrane in additional to the heating induce destabilization such as oxidation of sulfydryl groups of the membrane binding bound proteins, resulting in cell death, in addition to direct DNA damage (Yatvin and Grummer, 1987). The results in Table 7 detected the D10 values for S. typhi. following irradiation and/or heating as follow 0.561, 0.468 and 0.612 these data are agreement with Kim and Thayer (1996) which detected the D10 values for Salmonella typhimurium following irradiation and/or heating as follow 0.596, 0.465 and 0.654.
Figure 5a presented the morphology and ultrastructure of untreated and treated S. typhimrium Fig. 5a revealed short rods of untreated G-ve cells of S. typhimurium, the protoplast could be differentiated into cytoplasmic regions, which was rich in ribosomes of the cells.
| Table 8: | The individual or combined effect of both radiation or radiation
pre heated cells the amino acids contents (mg g-1) of S. typhimirum |
Figure 4b revealed the effect of heat at 65°C for 2 min, appearance of clear central zones which may be small vacuoles or reserved materials to resist the heat effect. Figure 4c showed the effect of gamma irradiation on the bacterial cell of S. typhimurium which leads to more or less effects similar to heat but the size of the cells affected, Enlargement of the cells with dense vacuoles especially at poles of the cells irregular ribosomal aggregation, the cell become lyzed and the cytoplasmic membrane shrank. Figure 4d showed the combined effect of heat and irradiation which induced the appearance of filamentous bacteria due to the failure of cell separation after divisions, cells undergo complete lysis without destruction of the cell wall. These results are agreement with Khaled (2004) who reported that the ultrastructure study for S. typhimurium. irradiated at 2 KGY showed enlargement of the cell size with dense vacuoles and irregular ribosomal with the cytoplasmic membrane shrank.
The results in Table 8 indicated the effect of Gamam irradiation and heat on the amino acids contents (mg/g) of S. typhimirium in this study, the results obtained from the amino acid analysis for the cell of the S. typhimurium. revealed variation not only in the total amino acids contents but also in the amounts of these amino acids. These results are agreement with Casarett (1968), Khaled (2004) showed variation not only in the total amino acids contents but also in the amount of these amino acids.