| Food safety incidents occur from time to time,caused by pathogenic microorganism are more common,Enterohemorrhagic E.coli O157:H7 infection is a common cause of intestinal disease in human.It can result in diarrhoea,acute intestinal inflammation,haemorrhagic colitis and haemolytic uraemic syndrome.Although antibiotics are conventionally administered against E.coli O157:H7 infections,their use can cause the increasing frequency of drug-resistant pathogenic bacteria.The widespread use of antibiotics can also result in antibiotic-associated diarrhea that is a common problem in hospitalized geriatric patients.Therefore,it becomes necessary to find alternatives to the use of antibiotics as antibacterials.It is widely acknowledged that some lactic acid bacteria can be used in the prevention of development,incidence and recurrence of disease caused pathogen.However,its application may be limited due to lactic acid bacteria bacteraemia were identified in a 10-year survey in Finland,but no L.acidophilus bacteraemia were identified in the survey and previous studies have indicated that L.acidophilus may have antagonistic activity against E.coli O157:H7.Therefore,L.acidophilus strains in our laboratory were used as research subject in this study,to determine whether they have application prospects for the prevention and treatment of E.coli O157:H7-related diseases,by comparison with L.acidophilus NCFM which has showed very good results in the prevention and treatment of pathogen-induced diseases.Firstly,identification of strains was evaluated by morphological characteristics,phe S and 16 Sr RNA gene sequence analyses.Secondly,to study the antibacterial activity and antibacterial mechanism of L.acidophilus against E.coli O157:H7.Thirdly,the bactericidal activity from CFS or viable L.acidophilus against E.coli O157:H7 was also assessed by using co-incubation methods.Furthermore,the inhibition E.coli O157:H7 adhesion and colonization of L.acidophilus strains were also investigated based on competition,exclusion and displacement assays.Finally,the probiotic potential of L.acidophilus was evaluated,based on acid tolerance assay,bile salt tolerance assay,Caco2 cell adhesion assay,antibiotic sensitivity assay and anti-inflammatory effects by IL-8 produced in Caco-2 cells.The results were as follows:All of Lactobacillus strains were determined as L.acidophilus by morphological characteristics,phe S and 16 Sr RNA gene sequence analyses.All of Lactobacillus strains showed a certain degree of bacteriostatic activity against S.aureus ATCC25923,E.coli ATCC25922,Listeria monocytogens CMCC5002,Salmonella ATCC14028 and E.coli O157:H7.Their inhibition zone of KLDS1.0901,KLDS1.0902,KLDS1.1003 or NCFM against E.coli O157:H7 was 11.20±0.10 mm,12.60±0.80 mm,11.23±0.15 mm and 10.20±0.92 mm,respectively.One of the main inhibitory mechanisms that L.acidophilus showed bacteriostatic activity against E.coli O157:H7 may result from the production of organic acids and lactic acid in organic acids plays a major role,R=0.87(Correlation lactic acid with inhibition zone diameters).L.acidophilus had the strongest bacteriostatic activity against E.coli O157:H7 when lactose was the only carbon source in the MRS medium and their inhibition zone was 13.43±1.00,14.33±2.32,14.10±1.99 or 13.43±1.12(mm),respectively.All of L.acidophilus' cell-free fermentation supernatant(CFS)showed strong bactericidal activity against E.coli O157:H7,no viable E.coli O157:H7 was detected when E.coli O157:H7 was co-incubated with the CFS of L.acidophilus for 2 h.However,all the L.acidophilus themselves had no bactericidal activity,there was no significant difference in growth when E.coli O157:H7 was cultured alone in Penassay broth(227.7±14.6 CFU)and when E.coli O157:H7 was co-incubated with L.acidophilus KLDS1.0901(225.3±18.9 CFU)(P=0.82),KLDS1.0902(242.3±6.1 CFU)(P=0.18),KLDS1.1003(249.0±2.6 CFU)(P=0.06)or NCFM(231.0±12.9 CFU)(P=0.70)for 18 h,respectively.L.acidophilus KLDS1.0901,KLDS1.0901,KLDS1.1003 and NCFM strains were effective to displace E.coli O157:H7 from a Caco-2 cell layer in competition and exclusion assays.However,all of the strains showed no significant effect in displacement assay(P>0.05).In general,L.acidophilus KLDS1.0901 and NCFM showed better probiotic potential than KLDS1.0902 and KLDS1.1003.In conclusion,KLDS1.0901 showed the similar ability with L.acidophilus NCFM at antagonistic activity against E.coli O157:H7 and probiotic potential,the results suggest KLDS1.0901 has potential applications for prevention or treatment of diseases associated induced E.coli O157:H7 in vivo and may also have the potential to develop into commercial probiotics. |
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