Effect Of Bacteria In Lettuce On Food-borne Pollution Of Noroviruses

Objective Noroviruses are most major causative agents of nonbacterial acute gastroenteritis,which mainly transmitted by fresh fruits and vegetables(lettuce,strawberry,etc.).In this study,high-throughput sequencing was used to analyze core microbiota of lettuce and soil in a field in Hangzhou,and lettuce isolates were screened the expression of histo-blood group antigens.The effects of HBGA-positive bacteria on the persistence of G?.4 No Vs under heat or UV disinfection,and the effects of HBGA-positive bacteria on the residual and infectivity of G?.4 No Vs on the surface of lettuce under UV irradiation and high planting temperature environment were detected to provide theoretical basis for prevention and elimination of No Vs on fresh fruits and vegetables.Methods 1.High-throughput sequencing was used to analyze core microbiota of lettuce sample and soil sample in a field in Hangzhou.Plate isolation was used to isolate bacteria on the surface of lettuce.Lettuce isolates were tested by 16 Sr DNA sequence analysis,and draw a phylogenetic tree.2.Suspension assay was used to detect binding efficiency of G?.4 No Vs on bacteria and the location of binding was determined by transmission electron microscope.ELISA was used to screen the expression of histo-blood group antigens of bacteria.3.RT-q PCR and RNase treatment combined with RT-q PCR were used to detect the effect of histo-blood group antigen-expressing bacteria on G?.4 No Vs persistence under heat and UV disinfection.4.Leaves of lettuce were sterilized by UV and viruses were spot inoculated onto lettuce surface.RT-q PCR and RNase treatment combined with RT-q PCR were used to detect the effect of histo-blood group antigen-expressing bacteria on G?.4 No Vs residual and infectivity on lettuce under UV irradiation and high planting temperature environment.Results 1.Core microbiota of the lettuce sample mainly consists of Pseudomonas,Acinetobacter,Arthrobacter,Shewanella and Sanguibacter,accounting for 31.66%,22.67%,10.23%,6.8%,6.44%,respectively.Core microbiota of the soil sample mainly consists of Arthrobacter,Gp6,Sphingomonas,Renibacterium,Pseudomonas,accounting for 26.77%,16.16%,9.45%,8.88%,3.82%,respectively.A total of 25 strains of bacteria were isolated from the lettuce which mainly include 7 strains of Pseudomonas sp,4 strains of Microbacterium sp,3 strains of Brachybacterium sp,3 strains of Sphingobacterium sp,etc.2.Various binding efficiency was observed for G?.4 No Vs,TEM images showed that No Vs mainly absorb on bacterial cell.ELISA results showed that most of bacteria can express histo-blood group antigens.3.Under UV disinfection,all of bacteria tested have an impact on the persistence of No Vs compared to negative control group(p < 0.05).Under heat disinfection,RT-q PCR results showed that Pseudomonas sp.SC006,Pseudomonas sp.SC023 and Sphingobacterium sp.SC015 have an impact on viral persistence(p < 0.05),while the results of capsid integrity indicated the above bacteria have no effect on viral persistence compared to negative control group(p > 0.05).4.Under UV irradiation,RT-q PCR results showed that Wautersiella sp.SC042 have an impact on viral residual compared to negative control group(p < 0.05),and the results of RNase treatment combined with RT-q PCR showed that Sphingobacterium sp.SC015 and Wautersiella sp.SC042 have impact on viral infectivity on the surface of lettuce compared to negative control group(p < 0.05).Under high temperature planting environment,all of bacteria tested have no effect on viral residual and infectivity within 7 days(p > 0.05).Conclusion The tested lettuce was rich in microbiota,and bacterial dominant microbiota mainly include Pseudomonas,Acinetobacter,Arthrobacter,Shewanella and Sanguibacter,which have a big difference with soil microbiota.Most of the lettuce isolates have an adsorbance with G?.4 No Vs and can express histo-blood group antigens.Bacteria have an impact on G?.4 No Vs' persistence under UV disinfection,while bacteria have no effect on viral persistence under heat disinfection expect for RNA degradation.Under high temperature planting environment,bacteria have no impact on viral residual and infectivity on the surface of lettuce within 7 days.Under UV irradiation,some histo-blood group antigens-expressing bacteria have an effect on viral residual and infectivity,HBGAsexpressing bacteria may facilitate foodborne transmission of No Vs on lettuce.