Physical and Biochemical Properties of Heat and Cold shocked Bacteria with Special Reference to their Protein Profiles

Abstract

This study about heat and cold shocked bacteria could be a serious problem in foods which are cooled or frozen like meat and dairy products as this could cause serious hazard for consumers. As reported by Enache et al. (2006) that heat treatment is one of the most widely used methods to control the rates of bacterial growth and death and is considered to be one of the most effective food processing technologies for eradicating food borne pathogens. This study aimed to gain the degree of PhD in Microbiology. The work was done in Department of Microbiology Faculty of Veterinary Medicine, University of Khartoum since November 2015. The bacteria under study were: Staphylococcus aureus, ATCC 25923, E. coli, ATCC 25922, K. pneumoniae, ATCC 13883 and Ps. aeruginosa, ATCC 27853. Staphylococcus epidermidis, B. cereus and S. sonnei were provided by National Public Health Laboratory, Khartoum as isolates. Pasteurella multocida was provided by Central Veterinary Research Laboratory, Khartoum. These bacteria were inoculated into nutrient broth tubes and incubated at 37°C for 24 hours. Then cultures for heat shocked were treated in a water bath at 100°C for 5 min, 90°C for 10 min, 80°C for 15 min, 70°C for 20 min and 60°C for 25 min. For cold shocked study, the cultures were treated at 0°C, -15°C and -32°C for 30 min. All biochemical tests were performed according to the methods described by Barrow and Feltham (2003). It was observed that Gram- negative bacteria were more sensitive than Gram-positive bacteria for heat. This appears clearly in S. aureus and B. cereus which were found to be resistant to V 70°C and 80°C. Staphylococcus epidermidis cells were not affected dramatically at 0°C with a slight drop at -32 °C. On the other hand, B. cereus showed a remarkable drop when treated at 37°C and 0°C, while cells treated at -15°C and -32°C behaved normally. Cells treated at -32°C showed an increase in degree of positiveness towards various biochemical tests. As far as E. coli, there was discrepancy in the results. Cells treated at -32°C showed strong reaction towards all tests applied apart from those obtained by dulcitol, glycerol and mannitol, while cells treated at 0°C, showed a drop in all reactions. Shigella sonnei showed a severe drop in reaction when subjected to glycerol and mannitol tests. Cells treated at -15°C showed strong reactions towards mannitol, rhamnonse, trehalose and MR tests. Klebsiella pneumoniae showed strong reactions in all tests applied apart from those treated at 0°C. Like all organisms tested, Ps. aeruginosa showed a remarkable drop at 0°C followed by -15°C. Pasteurella multocida cells did not react properly when cells were treated at all temperatures. The oxidase test scored a doubtful result throughout all tests applied indicating the severe damage of the oxidase enzyme. For heat and cold shock tests, the protein profiles and their concentrations were estimated. From the chromatogram of S. aureus, apparent changes were noticeable upon raising of temperatures from -32°C to 100°C. The protein bands were focused between 75 kDa to 11 kDa. In case of S. epidermidis, unlike S. aureus, there was no apparent electrophoretic differernces in the protein. On the other hand, there was an increase VI in protein concentrations by more than 140% as the tempreture rises from -32°C to 100°C. Protein bands were focused between 75 kDa to 11 kDa. Although there was an apparent variation in the protein composition of B. cereus with gradual increase of temperature, there were no clear electrophoretic bands discrepancies from -32°C to 100°C. Escherichia coli showed no changes till 80°C, after which sudden increase was noticed till 100°C. Shigella sonnei, Ps. aeruginosa and P. multocida showed more than 100% decrease in mg/ml of isolated protein as temperature was raised from 37°C to 100°C. Klebsiella pneumoniae incubated at 37°C showed a sharp increase in the protein content after which a sharp decline was noticed. After that, protein started to increase, which peaked at 100°C. For study of pathogenicity of P. multocida for rabbits, the culture was shocked at -15°C for 30 min and ten serial dilutions were done in duplicates. The dilutions, which did not show visible colonies on the recovery media were centrifuged and inoculated i/p in four rabbits. Rabbits inoculated with VBNC bacteria died within 24 hours. Low and high temperatures have no effect on DNA.