| Listeria monocytogenes remains a significant public health threat,causing invasive listeriosis with severe manifestations of septicemia,meningitis,and abortion with up to 30% of the cases being fatal outcomes.In China,previous research investigations have been concentrated or limited on the prevalence of L.monocytogenes in the retail market and limited to one province or one section(either from the food section or clinical side).There is paucity of information on the genetic diversity,antimicrobial resistance and genomic virulence determinants of L.monocytogenes isolates from different sources in China.In order to fulfill this knowledge gap,we attempted using PCR typing,antimicrobial resistance profiling and genomic sequencing in combination with bioinformatic analysis to decipher the genetic diversity,antimicrobial resistance,virulence factors from Chinese L.monocytogenes isolates from foods,animals and clinical human cases.Efforts were made to compare among different sources of L.monocytogenes strains and between Chinese clinical isolates and those from other countries to see if there are information unique to a particular source or specific in Chinese isolates.1.Genotypic diversity of Listeria monocytogenes from foods,animals and humansA total of 369 isolates of L.monocytogenes(326 from foods,25 from livestock,and 18 from clinical human cases)were used to investigate their genotypic diversity and virulence genes.Multiplex PCR was used to identify the serotypes and lineages.Multilocus sequence typing(MLST)and gene sequencing were applied to probe the genetic relationship among isolates from different sources.The food isolates were dominated by serotype 1/2a-3a(37.4%),followed by 1/2c-3c(26.3%),and 1/2b-3b(16.2%).Serotype 4b-4d-4e and 4a-4c accounted for 10.1% and 9.81% of the food isolates respectively.Majority of the animal isolates belonged to serotype 4a-4c(76%).Of the 18 human isolates,1/2a-3a(44.4%)were dominant followed by 1/2b-3b(27.7%).Phylogenetic lineage identification revealed the dominance of lineage II(63.4%)in foods,followed by lineage I(26.6%).Animal isolates were mostly typed as lineage III(76%).Majority of the human isolates belonged to lineage II(55.5%)followed by lineage I(38.8%).It seems that the food and human isolates had similar patterns of serotypes and lineages,dominated by serotype 1/2a-3a and lineage II.Altogether,369 isolates were grouped into 55 STs via MLST that could be further classified into 26 CCs and 15 singletons.For isolates from foods,ST9(26%)and CC9(27.9%)were the most abundant,followed by ST8/CC8(8.8%)and ST121/CC121(8.8%).ST202/CC69(76%)were dominant in the animal isolates.The human isolates were mostly typed as ST8/CC8(16.6%)followed by ST9/CC9(11.1%).It appears that ST8/CC8 and ST9/CC9 were shared between the food and human isolates.The animal isolates represented a rather different genetic profile of its own.Such clonal relationship suggests the possible transmission of L.monocytogenes from foods to human being,though the number of human isolates was limited.2.Antimicrobial resistance and genetic determinants of Listeria monocytogenesAlthough the antimicrobial resistance prevalence of L.monocytogenes is low compared to other foodborne pathogens such as Salmonella,but this might gradually increase.Therefore,it is important to acquire the profile of antimicrobial susceptibility and understand genetic determinants of resistance.Minimum inhibitory concentrations(MIC)were determined for all isolates(n=369)as above.Forty isolates were chosen for genomic characterization of antimicrobial resistance by whole genome sequencing to elaborate the phenotypic and genotypic correlation.All isolates were found to be susceptible to meropenem,vancomycin,rifampicin,and levofloxacin.Resistance was mostly found in food isolates and attributed to tetracycline,erythromycin,cotrimoxazole,chloramphenicol,and gentamicin,and ampicillin from 2.1% to 5.1% of the isolates tested.The following antibiotic resistance genes(ARGs)were present among 40 phenotypically resistant isolates: bla MOR-2-1 for ?-lactams,aac(6)-aph-2''1 for aminoglycosides;dfr G-1 for sulfamethoxazole trimethoprim;er(D)-1,mef(A)-1,mef(A)-4,msr(D)-2 and msr(D)-3 for erythromycin;tet(D)-1,tet(S)-3,tet(L)-2,tet(M)-6,tet(M)-10,and tet(M)-13 and tet(X)-2 for tetracycline;and cat-5 and cat A2-1 for chloramphenicol.Resistance genes bla MOR-2-1,aac(6)-aph-2''1,cat-5 and cat A2-1 have not been reported previously.3.Genomic determinants of Listeria monocytogenes for invasive infectionsWe performed the genomic virulence determinants of 13 Chinese isolates from patients and made comparative genomic analysis with genome sequences of isolates from other countries(USA,Canada,Italy,and Switzerland).The isolates were from diverse range of sources(blood,cerebral spinal fluid,placenta of abortion cases,and stool).Emphasis was placed on adherence and invasion genes,stress proteins,immune modulator genes,intracellular survival genes,iron uptake,surface protein anchoring,bile resistance,enzymes and toxins.Phylogenomic analysis and genetic correlation among Chinese and worldwide isolates were also presented.The predominant serotype in all 60 isolates was 1/2a-3a(61%),followed by 4b-4d-4e(20%)and 1/2b-3b(18%).All isolates belonged to two lineages with lineage II accounting for 63% of the strains,and lineage I for 37%.There seem to be difference among different sources of isolates with serotype 1/2a-3a and lineage II being more prevalent for isolates from blood and aborted samples,but serotype 4b-4d-4e and lineage I more dominant for isolates from cerebral spinal fluid and stool samples.Overall,all isolates were grouped into 26 different sequence type(STs),25 were assigned the clonal complex(CCs)and one singleton based on querying the MLST database.Clonal complex CC8(covering ST8,ST120 and ST292)was prevailing for isolates from blood,cerebral spinal fluid and abortion samples,while CC1 isolates were also present in cerebral spinal fluid.Those from stool samples were mainly composed of CC1(ST1)and CC5(ST5).The isolates,particularly ST5(CC5),from the stool samples might represent localized infection in the gastrointestinal infections.Phylogenomic analysis showed that CC8,CC7 and CC5 of Chinese isolates were clustered with European isolates(CC8)and Canadian isolates(CC5 and CC7),indicating strong phylogenomic relation between these isolates.The close pattern and resemblance could be due to international trade of various food products or bacteria may frequently transported between places of food production,possibly with reference to imported raw materials.Listeria pathogenicity island-1(LIPI-1),LIPI-2,intracellular survival,surface anchoring,and bile salt resistance genes were detected in all isolates.L.monocytogenes strains from clonal complex CC8,CC7,CC9,and CC415 carried adherence and invasive genes inl F,ami and aut genes,while CC1,CC2,CC4 and CC6 lacked these genes.Importantly,all LIPI-3 genes were present in about one quarter of the isolates(14/60,5 Chinese isolates,and 9 isolates from other countries).These findings might suggest the possible genetic switching or transfer associated with some STs and clonal complexes from one region to other in order to acquire resistance and virulence for persistence under diverse geographical locations,although there was considerable genetic diversity.In conclusion,this study provides evidence of considerable diversity of L.monocytogenes from foods,animals and humans by serotypes,lineages and clonal complexes.Some of the food and human isolates had similar serotypes and lineages,dominated by serotype 1/2a,3a,and lineage II.Antimicrobial resistance was low in the tested L.monocytogenes isolates.However,we did find novel resistance genes bla MOR-2-1,aac(6)-aph-2''1,cat-5 and cat A2-1,requiring continuing surveillance to monitor the spread of these genes in Listeria.CC8,CC7 and CC5 of the isolates from human patients were dominated by 1/2a-3a and lineages II,and there was close genomic similarity of these clonal complexes between Chinese isolates and those from Switzerland,Italy and Canada,suggesting the importance of international trade and traveling in clonal spread of the bacterium.A quarter of the clinical isolates,including some Chinese isolates,also harbored the full sets of LIPI-3 genes,which could be further explored to see if they contribute to increased virulence.The findings in this study provide good scientific basis for surveillance of L.monocytogenes focusing on the food chain,including those imported from other countries,as part of the control strategy to minimize its infections in the susceptible human populations. |
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