Isolation, Identification And The Characteristics To Lysis Enteric Pathogens Of Bdellovibrio

In recent years, food safety incidents are emerging in an endless stream, such as pesticide residues exceeding the standards in fruit and vegetable, illegal addition of chemical raw materials, additives abuse, excessive nourishment and so on. However, foodborne disease remains a major issues of global food safety, including foodborne illness caused by biological contamination and chemical materials residue. Foodborne disease outbreaks, also known as food poisoning, of which 66% are caused by bacterial pathogens. Pathogenic bacteria in the food chain and the resulting pollution are common. Antibiotics played a key role in the protection of human health and life, but a lot of irrational use of antibiotics makes the growing bacterial resistance, and bacterial resistance also boost the popularity of foodborne bacterial diseases.Pathogenic microorganisms which have direct and indirect relationships with food may all contaminate the food, pathogenic microorganisms that can cause significant human diseases and food poisoning are significant for testing, including:Salmonella, Staphylococcus, Streptococcus, Vibrio parahaemolyticus, Proteus, Shigella, avian influenza virus, aflatoxin, viruses, foot and mouth disease virus and so on. Some food in the definition of pathogenic microorganisms may not be detected, while others have limited indicators. Therefore, we must strengthen the studies, prevention and control to the major pathogenic microorganisms. Natural, no residual methods of killing them are urgent in the food production, processing, consumption and other aspects.This paper isolated the new Bdellovibrio strains from Ninghai seawater and mud respectively to compare the lysis properties between Bdellovibrio strains with differene enteropathogenic bacteria. Their 16S rDNAs were identified and their morphologies were examined under electron microscope. We analysised the differences of lysis properties between different Bdellovibrio strains on its host bacteria by comparison the plaque size and their co-culture growth curve, providing the basis related research of prevention and control of foodborne bacteria by Bdellovibrio. The main contents and results are as follows:1 Separation and purification of Bdellovibrio from sea water and mudBdellovibrio are bacterial parasite, host bacteria are the sole source of their nutrition. Most of the current separation of Bdellovibrio bacteria strains use the double plate method, Bdellovibrio lysis host bacteria by the formation of plaque. According to the standard curve equation of host bacteria to converted different OD values to the corresponding colonies number (CFU/mL). In this experiment the medium we used were cultured to late logarithmicon, according to the growth curve found the respective incubation time of late logarithmicon. Among them, Salmonella typhimurium, Fu's Shigella 2a, E. coli and Proteus mirabilis reached the late logarithmicon at 24 h; Vibrio parahaemolyticus reached the late logarithmicon at 36 h; Salmonella paratyphi A, Enteroinvasive E.coli and Vibrio fluvialis reached the late logarithmicon at 48 h. The optimum amount of the host bacteria must form plaque with the shortest time, having the largest number of plaques and the diameter of plaque with no more increase. Besides Vibrio parahaemolyticus with the optimum amount of 0.8 mL, the other host can be cracked with the optimum amount of 1.0 mLWe isolated 6 Bdellovibrio strains in which 3 strains were isolated from the sea water, the strain lysed Fu's Shigella 2a was named Bs2, the strain lysed Enteroinvasive E.coli was named Bs4, the strain lysed Vibrio Parahemolyticus was named Bs7; another 3 strains were isolated from the sea mud, the strain lysed Salmonella typhimurium was named Bm1, the strain lysed Proteus mirabilis was named Bm6 and the strain lysed Vibrio Parahemolyticus was named Bm7. Plaque diameter of Bs2 and Bml was between 1～2 mm, plaque diameter of Bs4 and Bm6 was between 2～4 mm, plaque diameter of Bs7 and Bm7 was between 5～7 mm.2 The identification and preservation of Bdellovibrio from the sea water and mudPrimer 63F and primer Bdg842R can not produce target band at 400 bp, only a lot of primer dimer formation. Primer 63F and primer P3 can not produce target band at 800 bp, there are primer dimer formation. Primer Bac1442R and primer Bac676F can produce a single bright target band at 800 bp, this result not only confirmed the plaque purified isolates were Bdellovibrio, also the 6 strains isolated belong to the genus Bacteriovorax spp..6 Bdellovibrio strains were negative stained by transmission electron microscopy to observe the size and shape of them, their cell were short, in arc-shaped or in rod-shaped, with one wavy flagella.Using the sterile seawater as a protective liquid, in 4℃refrigerator all strains can form plaques at 91 days, that indicate Bdellovibrio still have phage activity, Bdellovibrio in the sterile seawater at 4℃can survive at least for 91 days; and strains stored at room temperature recovery are different, Bs2 can survive at least for 49 days, Bs5, Bs7, Bm1, Bm7 can survive at least for 63 days, Bm6 can survive at least for 56 days. When using saline as a protective liquid,4℃ refrigerator all strains can form plaques at 91 days, that indicate Bdellovibrio in the saline at 4℃can survive at least for 91 days; at room temperature, Bs7 and Bm7 can survive at least for 49 days, Bs2, Bs5, Bml, Bm6 recovery were relatively weak, can survive at least for 28 days. Using the sterile seawater with host as a protective liquid, in 4℃refrigerator all strains can not form plaques at 7 days, that indicate Bdellovibrio lost phage activity; and strains stored at room temperature, besides Bs7 and Bm7 can survive at least for 21 days, others can only survive for 7 days. When using saline with host as a protective liquid, Bs2, Bs5, Bm1, Bm6 in 4℃refrigerator can survive at least for 14 days, Bs7 and Bm7 can not form plaques at 7 days; at room temperature, Bs7 and Bm7 can survive at least for 14 days, others can survive at least for 28 days.3 Properties of Bdellovibrio to lysis enteropathogenic bacteriaBs2,Bs4,Bml and Bm7 reach their max lysis rate at 72h, the rate was 43.56%,71.28%,67.66%,95.81%, respectively; Bs7 and Bm6 reach their max lysis rate at 48 h and 96 h hour, the rate was 98.70% and 74.69% respectively. Also, Bs2,Bs4,Bm1 and Bm7 reach their max PFU number at the 72th hour, the number was 770,507,1064,240, respectively; Bs7 and Bm6 reach their max PFU number at 48 h and 96 h hour, respectively, the number was 279 and 588.Marine Bdellovibrio have different capacities on lysising different host bacteria, Bs7 and Bm7 have the strongest lysis capacities on the Vibrio parahaemolyticus which also were from the marine environment, their lysis rates were both over 95%, followed by the Bm6 to the Proteus mirabilis, lysis rate was 74.69%, while the lysis capacities were relatively weak to other terrestrial environment. Bdellovibrio from different sources have different lysis capacities on the same host strain, Bdellovibrio from sea water can lysis Fu's Shigella 2a and Enteroinvasive E.coli, but Bdellovibrio from mud can not lysis the two host bacteria, but can lysis Salmonella typhimurium and Proteus mirabilis. Bdellovibrio could not completely eliminate the host bacteria in the lysis process, the host number could only be controlled in a certain range, and the lysis ability of Bdellovibrio had a time limit. The number of Bdellovibrio in 168 h also had a significant change.4 Bdellovibrio's prevention and control of foodborne bacteriaBacterial count, coliform and pH values of pork, fish sample groups with Bdellovibrio sprayed rise more slower than the control group. Which bacterial count in 72 h with the lysis rate 51.55% on average of pork, with the lysis rate 62.08% on average of fish. Coliform with the lysis rate 4.03% on average of pork, with the lysis rate 8.88% on average of fish.The volatile basic nitrogen (TVNB) were in almost the same trend between the samples and blank of pork and fish, TVBN value has been in the increase state. At 12 h TVNB in pork and fish had significantly exceeded our requirements for fresh meat of acceptable limits 15mgN/100g.