Study On Antimicrobial And Virulence Characteristics Of Listeria Monocytogenes Isolated In China

The intracelluar pathogen Listeria monocytogenes is the causative agent of listeriosis,a severe invasive illness that has an extremely high mortality rate.Persons with speicific immunocompromising conditions,pregnant women,old people and new borns appear to be particulaly susceptible to invasive listeriosis.It is of great importance to select appropriate antibiotics due to its high morality.This study was in an attempt to clarify (1)antimicrobial resistance characterization of Chinese foodborne Listeria monocytogenes;(2)antimicrobial resistance mechanisms of Listeria monocytogenes and the distribution of resistance genes in this pathogen in China;(3)the correlation between antimicrobial resistance and serology characterization of Listeria monocytogenes as well as the homologisation of the drug resistance isolates;(4)serology and virulence genes distribution of Listeria monocytogenes isolated from ready-to-eat(RTE)food in order to evaluate the injection risk at the first level.1. Antimicrobial resistance characterization of Chinese foodborne Listeria monocytogenesThe susceptibility of 1065 strains isolated from food to Listeria monocytogenes to 15 antibiotics currently used in veterinary and human therapy.Susceptibility tests were performed by using the Broth Microdilution Susceptibility Testing in Clinical and Laboratory Standards Institute. Among the 1065 tested strains,32(3.00%)displayed resistance to one antibiotic,32(3.00%)to two antibiotics and 3(0.28%) to three antibiotic.The foods that were highest rates of antibiotic resistance were frozen rice and flour products.Jilin Fujian and Hebei were the third top among the 22 provinces, the rates of which were 20.00%, 19.05%, 17.07%. This study shows that Listeria monotogenens strains in China are susceptible to the antibiotics commonly used in veterinary and human listeriosis treatment.Considering that Lisiteria monocytogenes is slowly becoming antibiotic resistant,a continued surveillance of emerging antimicrobial resistance of this pathogen is important to reduce the antibiotic resistant strains.2. The study of antimicrobial resistance mechanismsThe antimicrobial resistant determinants was detected by PCR. 59 of 63 tetracycline-resistant strains harbor tetM gene.The int-Tn gene for the integrase of Tn916-Tn1545 was found in 69.49%(41/59)of strains harboring tetM,confirming that tetracycline resistantance in Listeria monocytogenes is due to acquisition of conjugative transposons in a large part.tetS ,ermB,cat and aad6 gene were detected in 2 MDR strains.MefA and cat gene were detected in another erythromycin-resistant and chloromycetin-resistant strains separately.The fluoroquinolones-resistant isolates had no differences in the sequences of susceptible strain in QRDRs and MICs of these strains has a great decrease(a twofold or greater) in the presence of reserpine.It is speculated that the increased MICs of fluoroquinolones is due to the overexpression of efflux pump gene lde.3. The study of antimicrobial resistance strains by serotyping and PFGE analyzingSerotypes and lineage distribution of the drug resistance isolates was analyzed by tradtional method and ASO-PCR.The serotype results indicate 1/2a,1/2c,1/2b,4b and 3 a serotypes(1 strain was not identified by serotype,ND)were distributed in resistance strains.Comparing to the susceptive strains,resistance ones had a significant larger proportion of lineage ?. We could get a deduction that lineage ? strains may have a higher probability to acquire antimicrobial resistance according to its higher recombination levels.67 drug resistance strains and two standard strains were analyzed with PFGE,and PFGE patterns were imported and analyzed with BioNumerics software.All of strains were digested with restriction endonucleases Apa ? and it allowed differentiations of 67 isolates into 32 PFGE profiles and 6 groups;Lineage distribution is closely related with PFGE substypes;It is speculated that the acquisition of antimicrobial resistance is mainly due to horizontal transfer for the reason that strains in the advantage group B isolated from different regions and different kinds of foods; The vast majority of group B were lineage ? and further confirmed that lineage ? strains may have a higher probability to acquire antimicrobial resistance.4. The study of serotyping and lineagne distribution of strains isolated from ready-to-eat(RTE) foodAmong the 226 Listeria monocytogenes isolated from RTE food,serovar 1/2a(41.59%) and 1/2b(40.71%) dominated,followed by sevoar 1/2c(10.62%) and 4b(5.31%).We also got a sevoar 3a and 4c,as well as one ND.The proportion of lieage I was 46.46%(105/226) and lieage ? 53.10%(120/226),and only one lieage ?strain was detected.The rate of human injection related sevoar 1/2a, 1/2b and 4b was relatively high(87.61%),which indicated the potential risk in Chinese RTE food.5. Virulence-associated genes distribution in strains isolated from RTE foodLacking of virulence-associated genes general leads to virulence attenuation.In this study virulence-associated genes were detected by PCR.4(1.80%) isolates lacked prfA gene,3(1.35%) lacked plc A,5(2.26%)lacked actA,2(0.89%) lacked inlB and 1 isolate (0.44%) lacked inlA,inlJ and bsh separately. Only 1 isolate lacked act A and inlA gene which was two virulence-associated genes deletion.Lacking of virulence-associated genes was rare in Listeria monocytogenes strains isolated from Chinese RTE food indicating high risk of it.But it is necessary to make deeper study on the potential pathogenicity by the integration of vivo and vitro analysis as well as epidemiology data to evaluate the exact risk.