The Contamination Tracing And Rapid Identification Method Of Listeria Monocytogenes In Mushroom Facilities

Listeria monocytogenes is an important foodborne pathogen worldwide,with a high mortality rate of 20%～30%for immunocompromised persons.We previously showed that21.6%of mushroom samples were positive for L.monocytogenes,indicating that L.monocytogenes may contaminate the production process of mushroom facilities.The aim of this study was to determine the distribution,contamination routes,and persistence of L.monocytogenes in the mushroom facilities and to develop a multiplex PCR to simultaneously detect L.monocytogenes persistant strains(clonal complex 87 and 88)based on comparative genomic analysis.The specific contents are as follows:1.Samples were seasonally collected at different phases according to the production process for Hypsizigus marmoreus within a one-year interval,27.0%(55/204)were positive for L.monocytogenes.A total of 60 representative L.monocytogenes strains were selected for further analysis based on serogrouping and enterobacterial repetitive intergenic consensus polymerase chain reaction(ERIC-PCR)fingerprints.Multiplex PCR showed serogroups I.1(1/2a-3a)and II.2(1/2b-3b-7)accounted for 68.3%(41/60)and 31.7%(19/60),respectively.Clonal complex(CC)8(61.7%,37/60)and 87(23.3%,14/60)were predominant and repeatedly isolated within a one-year seasonal investigation.CC8 strains were isolated from the mycelium-scraping phase to the harvest phase,indicating that ST8and ST87 persist in the key process of this facility.In addition,all strains harbored genes of Listeria pathogenicity island(prf A,hly,mpl,act A,iap,plc A,plc B,)and inl A,inl B gene.Only ST87 strains carried pts A,but none of the strains harbored lls X.All isolates were susceptible to 12 antimicrobials,except for levofloxacin(1.7%,1/60),erythromycin(3.3%,2/60),and clindamycin(88.3%,53/60).The minimum inhibitory concentrations of commonly used quaternary ammonium compounds were≥4μg/m L.2.Samples were seasonally collected at different phases according to the production process for Flammulina velutipes facilities(A and B)within four times,43.9%(79/180)and27.0%(55/204)were positive for L.monocytogenes,respectively.A total of 79/59representative L.monocytogenes strains were selected for further analysis based on serogrouping and ERIC-PCR fingerprints.Multiplex PCR showed serogroups I.1(1/2a-3a)and II.2(1/2b-3b-7)accounted for 3.8%/8.5%and 96.2%/91.5%in A/B facility,respectively.CC87 strains from facility A and CC5 and CC87 strains from facility B were isolated from the mycelium-scraping phase to the harvest phase,indicating that CC5 and CC87 persist in the key production process of these facilities.In addition,all strains harbored LIPI-1 genes and inl A/B gene in facility A and B,ST87 strains carried pts A,no lls X gene was present in L.monocytogenes strains.All isolates from A/B facility were susceptible to 13 antimicrobials,except for gentamicin(2.5%,2/79)and levofloxacin(1.7%,1/59).All strains of L.monocytogenes in facility A/B were susceptible to clindamycin.With 2.5%(2/79)strains of L.monocytogenes in facility A,were susceptible to benzalkonium chloride/benzalkonium bromide.The minimum inhibitory concentrations of commonly used quaternary ammonium compounds were≥4μg/m L in facility B and other strains in facility A.3.A novel multiplex PCR comprised of genes A6K41＿13255(specific for CC87 and88),BCW＿4260＿01987(specific for CC88)and 02-1103＿01073(specific for L.monocytogenes)were designed by comparative genomic analysis.The detection limit of this multiplex PCR for CC87 and 88 were 7.06 fg and 6.10 fg genomic DNA,respectively.This multiplex PCR could accurately detect CC87 and CC88 strains with the interference of different ratios of L.monocytogenes CC8,CC9,CC121,and CC155 strains.Furthermore,this multiplex PCR method could successfully detect X×10~4cfu/m L of L.monocytogenes CC87 and 88 strains in artificially contaminated milk after 9 h enrichment.In addition,this multiplex PCR could accurately detect CC87 isolates in food samples within 48 h as well as conventional MSLT analysis.A multiplex PCR was developed which is rapid,inexpensive,and accurate to simultaneously detect L.monocytogenes CC87 and CC88 strains based on comparative genomic analysis.This study showed that the mycelium-scraping machine was the main source of L.monocytogenes contamination of mushroom products.CC8,CC87 and CC5 strains were the persistant strains that continuously contaminated the production process of mushroom.A multiplex PCR were developed for detecting L.monocytogenes CC87 and CC88 strains based on comparative genomic analysis,which could apply to surveillance the prevalence of CC87 and CC88 strains in both food and food production environments and to evaluate the effect of disinfection measures for controlling the persistent L.monocytogenes contamination.