| Enteroaggregative Escherichia coli(EAEC)can form "stacked bricks" on the surface of intestinal epithelial cells.It is one of the leading causes of diarrhoea in children in third world countries and the second leading cause of traveller's diarrhoea.It is mainly transmitted through feces.An outbreak of gastroenteritis and hemolytic uremic syndrome in Germany in 2011 caused nearly 4,000 people infected,which were thought to be caused by Shiga-Toxigenic Escherichia coli(STEC),actually this outbreak was caused by an EAEC mutant containing a virulence gene stx2a.Antibiotics are commonly used to treat bacterial diseases,but the number of bacteria with antibiotic resistance is growing,which becomes a challenge to public health.Bacteriophage(Phage)is a bacterial virus that can kill bacteria.It is widely distributed in the world,from soil to sea and even in antarctic glaciers.Phage are mainly composed of a protein coat and the DNA inside the coat.They can be divided into lysogenic and lytic ones.Lytic phages can infect bacteria with the processes of adsorption,DNA injection,assembly,and release,leading to host bacteria lysis and large number of offspring production in a short time.It is reported that phages could destroy the biofilm of bacteria.Nowadays,with the frequent emergence of drug-resistant bacteria,phages are promising to be used as a therapeutic agent or a a environmental disinfectants against bacterial infection.The aim of this study was to isolate and identify phages with high lytic effect on EAEC strains,study their biological characteristics,and evaluate the efficacy of these phages against bacterial infection as a disinfectants.1.Identification and characterization of EAEC phagesIn 2018,305 samples of animal feces were collected either from Nanjing,Yunnan provinces,or the animal room of our school.Using EAEC strain CVCC232 as a host bacteria,totally 8 phages were isolated.The lytic effect of these phages was measured,and two phages ?11 and ?13 with high lytic effect were pick out for further study.The host range of phage ?11 and ?13 were tested against 167 E.coli strains were tested.The results showed that ?11 can lyse 155 strains,while ?13 can lyse 46 strains.?11 and ?13 presented clear and neat plaques with diameter of 3 mm and 1 mm on LB plates.The plaque of ?11 showed a halo ring,while ?13 did not.The electron microscopy results showed that the two phages belonged to Myoviridae.The highest titer of ?11 and ?13 was 5.4×109 pfu/mL and 3.6×109 pfu/mL,respectively.The optimal multiplicity of infection(MOI)of ?11 and ?13 was both 10.The results of pH temperature stability showed that ?11 could survive at 70? for 30 min,while ?13 could survive at 60? for 1 h.Both ?1l and ?13 could survive in pH 3-11 range.One-step growth curve showed that the incubation period of ?11 and ?13 was both 15 min;burst period of ?11 and ?13 was 65 min and 35 min;and the burst size of ?11 and ?13 was 52 and 21,respectively.2.Evaluation the effect of phages against host bacteria as disinfectants.A closed plastic box was used to simulate a closed space to compare the efficacy of spraying and atomizing phages in killing host bacteria in the environment.The results showed that both spraying and atomizing phages killed more than 99%of the bacteria in 30 min.Next,phage were spray to the floor and faeces,the results showed that ?11 and ?13 could kill 99.3%and 91.2%host bacteria on the floors respectively.And both of ?11 and ?13 can kill above 99%of bacteria in feces.And Cocktail showed more effective in killing host bacteria in faeces.The lytic effect of the two phages against biofilm of host bacteria were also examined,the results showed that?11,?13 and cocktail could lyse 73%,30%and 83%of the biofilm.The emergence rate of resistant bacteria was about 10-3 after co-cultured with phage for 5 h.The tolerance to farm-like environment of phages were tested,like temperatures of 26?and 37?,faecal with pH 6.0 and 8.5,and direct sunlight.The results showed that the titer of ?1l was decrease from 1.6×109 pfu/mL to 2.4×108 pfu/mL and 4.5×109 pfu/mL to 1.5×109 pfu/mL when exposure to 37? and pH 8.5 for 7 d.While the titer of ?13 was decreased from 8.8×108 pfu/mL to 1.7×107 pfu/mL and 1.1×109 pfu/mL to 2.9× 108 pfu/mL when exposure to 37? and pH 6.0 for 7 d.The titer of?11 and ?13 were decreased from 1.6×109 pfu/mL to 2.8×108 pfu/mL and from 4.8×108 pfu/mL to 4.5×107 pfu/mL after exposure to direct sunlight 6 h.In conclusion,phage ?11 and ?13 stronger resistance to the environment,and both of which had a good lytic effect on the host bacteria in the environment,indicating that phage ?11 and ?13 has the potential to be used as disinfectants.But we have to notice that bacteria resistance to phages can be emergent very quickly,which means more effective phages need to be isolated and phage cocktails should be developed to combat this problem. |
PDF Full Text Request