Subtyping,Distribution Of Virulence Genes And Biofilm Formation Of Food-borne Listeria Monocytogenes Isolates In Shanghai

Listeria monocytogenes(Lm)is an important food-borne pathogen which is widely distributed in the environment.It can cause listeriosis in human with a high fatality rate of at most 30% by contaminated food.Lm can form biofilms when adhering to a variety of surfaces in food processing environments.It has been observed that the resistance of biofilms to sanitizers is increased compared with what is normally seen with planktonic cells.Thus,the bacterial cells in biofilm can be regarded as a potential contamination source in food processing systems with increased risk to food safety.In this study,the population structure of eighty-six Lm isolates from Shanghai by Multilocus sequence typing(MLST)and the distribution of virulence-associated genes and markers was screened by PCR,as well as the biofilm formation and relative gene expression in Lm exposed to a hypochlorite sanitizer,XY-12 were also studied.The results are as follows:1.MLST was used to characterize the subtype of eighty-six Lm food-borne isolates from Shanghai.The result of MLST showed that these Lm belonged to fifteen Sequence Types(STs)and it contained two clone complexes–CC7 and CC155,STs correlated well with serotypes.The most common STs in Shanghai were ST8,ST87,ST121 and ST155.The Lm STs were correlated to different food categories,the most widely distributed STs were ST87 and ST121,and they could found in four kinds of food,including raw meat,cooked meat,aquatic products and frozen food.2.Eighty-six food-borne Lm strains were investigated for distribution of virulence-associated genes and markers including six genes belonged to LIPI-1(prfA,plcA,hly,mpl,actA and plcB)and four genes from LIPI-2(inlA,inlB,inlC and inlJ)and another two virulent genes iap and clpE and Listeriolysin S(LLS)-positive strains and epidemic clone III(ECIII)by PCR.The results revealed that eighty-six isolates carried these twelve genes.Four isolates were LLS-positive strains by detection of llsX using PCR.Sixteen isolates harbored ECIII-specific markers.These findings suggest that food-borne Lm isolates from Shanghai could be pathogenic,which is threating to public health.3.A microtiter plate assay was used to assess the biofilm formation of eighty-six Lm isolates.It revealed the mean biofilm production ability of lineage I strains was greater than that observed in lineage II strains but not significant(P>0.05).XY-12,a hypochlorite sanitizer was used to threat biofilm formed and planktonic Lm cells,and it revealed that biofilms were more resist than planktonic cells.The transcriptional change of five selected genes(sigB,fri,kat,perR and recA)in biofilms and planktonic cells exposed to XY-12 was characterized by RT-qPCR.It demonstrated that,the expression of fri and sigB was significantly up-regulated in planktonic cells,which may contributed to the survival of planktonic cells,however,but this change did not happen in biofilm formed cells when exposing to XY-12.In conclusion,the most common STs of Lm in Shanghai were characterized as well as the distribution of virulence genes among these isolates.The biofilm formed cells were more resistant to the sanitizer than the planktonic ones with different transcriptional changes on five response genes when exposing to XY-12.