Correlation Analysis Of PBP2X M341I Substitution In Clinical Isolates Of Streptococcus Suis For Resistance To Penicillin Antibiotics

Streptococcus suis is an important zoonotic pathogen and antimicrobial drugs are the first choice for the prevention and control of streptococcal disease in pigs.During our monitoring of the resistance of Streptococcus suis,we found that Streptococcus suis was resistant to macrolides-lincosamids-streptogramin type B（MLS_B）and tetracycline antibiotics.The drug resistance is becoming more and more serious,and it is found that the drug resistance rate of strains toβ-lactam antibiotics is also rising slowly,which has attracted our attention.It has been reported that Streptococcus pneumoniae,Streptococcus agalactiae and other Streptococcus bacteria are resistant toβ-lactam antibiotics.The main mechanism of resistance toβ-lactam antibiotics is the mutation of penicillin-binding proteins（PBPs）.We have also found amino acid substitutions in the PBP2X of Streptococcus suis that are similar to the reported resistance-related mutation sites of Streptococcus,but whether these amino acid substitutions are related to the resistance of Streptococcus suis toβ-lactams remains to be confirmed.Therefore,this thesis investigates the molecular typing ofβ-lactam antibiotic-resistant Streptococcus suis to understand its epidemiological characteristics and transmission features based on the resistance data base of 107β-lactam-resistant Streptococcus suis strains screened by the laboratory among isolates between 2016 and 2018.In addition,we focus on the role of amino acid substitutions at PBP2X resistance-associated sites in the resistance of Streptococcus suis toβ-lactam antibiotics.The main research contents are as follows.1.Molecular typing of penicillin-resistant Streptococcus suis and analysis of differences in PBPs lociThe susceptibility of 107 clinically isolated strains ofβ-lactam antibiotic-resistant Streptococcus suis to 18 other drugs was studied using the CLSI-recommended drug sensitivity test method.The results showed thatβ-lactam antibiotic-resistant Streptococcus suis also showed varying degrees of resistance to other antimicrobials.Among them,the strains had the highest resistance rates of more than 95%to macrolide antibiotics（erythromycin,tilmicosin and telbivudine）,lincosamides（clindamycin and lincomycin）and tetracyclines（tetracycline and doxycycline）.Resistance rates to aminoglycosides（chloramphenicol and florfenicol）,aminoglycoside antibiotics（gentamicin and daikonomycin）,fluoroquinolone antibiotics（meprobamate and enrofloxacin）,truncated siderophore antibiotics（tamoxifen and vonimycin）and linezolid ranged from 45%to 79.4%.The strains still maintained a low resistance rate to rifampicin（13.1%）and vancomycin（5.6%）.It indicates that Streptococcus suis developed more serious resistance to commonly used antimicrobials.According to the capsule sequence cluster（CPS）sequence information in the WGS sequence,94 of the strains belonged to 33 serotypes of Streptococcus suis,with NCL1（14 strains）being the most abundant,followed by 30（10 strains）.7 housekeeping genes were compared for multi-locus sequence typing（MLST）,87 strains can be successfully divided into 59 ST types,of which 24 are new ST types,and the larger number was ST-1317（7 strains）and ST-1319（6 strains）.By goeburst analysis,ST-1068,ST-1077 and ST-1320,ST-1143 and ST-1324,ST-1086 and ST-1325,ST-1063 and ST-1085 had higher affinity.Most of them were distributed in different farms,indicating that Streptococcus suis had mutated during the transmission process and had a small spread in Jiangsu.By comparing the sequences of PBPs of drug-resistant strains and P1/7 strains,it was found that PBP1a,PBP1b,PBP2a and PBP2b of drug-resistant strains were not known to mediate penicillin resistance,although there were a few differences,while PBP2X had reported mutation sites associated with resistance toβ-lactam antibiotics in Streptococcus mainly including V399I,T340M,M341I,I373V,M401T,T551S,A371V and others.The PBP2X evolutionary tree showed that strains with differences in the M341I,I373V,and M401T loci were located in the same branch,and these strains showed high levels of resistance to penicillin,suggesting that these three site substitutions play an important role in mediating high levels of penicillin resistance.Since the substitutions in the conserved motifs SXXK,KS/TG and SXN of PBPs largely determine their affinity toβ-lactam antibiotics,and our results show that the branch of M341I substitution in Streptococcus suis PBP2X S₃₃₉TMK is related to high-level penicillin resistance,the role of PBP2X M341I substitution in mediating high-levelβ-lactam resistance will be further studied.2.The expression of PBP2X of Streptococcus suis and their affinity withβ-lactam antibioticsTo investigate whether the M341I substitution of PBP2X can decrease the affinity of PBP2X andβ-lactam antibiotics,site-directed mutagenesis was used to replace methionine（Met,M）in the PBP2X conservative motif S₃₃₉TMK of P1/7 with isoleucine（Ile,I）,and Ile in the PBP2X conservative motif S₃₃₉TIK of clinical isolates of resistant strains GMJ14 and FJSM4 with Met,and then the PBP2X of the above strains and sensitive strains P1/7,resistant strains GMJ14 and FJSM4 were expressed in Escherichia coli.The expression was induced by Isopropyl-β-D-thiogalactoside（IPTG）and verified by SDS-PAGE,indicating that GST-PBP2X was abundantly expressed in the supernatant of each strain.The GST fusion protein was purified using a GST affinity chromatography column,the GST tag was removed by TEV enzyme,and the PBP2X protein with activity and high purity was desalted by a desalting column.The fluorescence intensity changes of the above expressed various PBP2X were measured after co-incubation with penicillin sodium,amoxicillin,ceftiofur and cefotaxime sodium,respectively,at excitation light 280 nm and emission light 320 nm,and the acylation efficiency was calculated to evaluate the affinity of PBP2X with the above drugs.The results showed that the amino acid replacement of the M341 site of PBP2X of sensitive strain P1/7from methionine to isoleucine decreased its acylation efficiency with penicillin and amoxicillin by 2 fold and 1.8 fold,respectively,with significant differences in the changes before and after the replacement（P<0.05）,but the changes in the acylation efficiency with ceftiofur and cefotaxime were not significant（P>0.05）.The drug-resistant strains GMJ14and FJSM4 revert mutation of PBP2X,where isoleucine（I）at site341 is revert mutated to methionine（M）,increased their acylation efficiency with penicillin and amoxicillin by 1.3-2.2 fold with significant differences（P<0.05）,but the change in acylation efficiency with ceftiofur and cefotaxime was not significant（P>0.05）.The above results confirm in both positive and negative aspects that M341I substitution of PBP2X in Streptococcus suis leads to a decrease in its affinity for penicillin antibiotics,but has no significant effect on the affinity for cephalosporin antibiotics.In summary,this thesis analyzed the epidemiological characteristics and transmission features of penicillin-resistant Streptococcus suis and the association between locus differences in PBPs and penicillin resistance,and further confirmed that the M-I amino acid substitution at locus 341 of Streptococcus suis PBP2X leads to reduced affinity forβ-lactam antibiotics,especially penicillins,explaining the reduced susceptibility of these strains to penicillin antibiotics.The results of this study can lay the foundation for the final elucidation of the resistance mechanism of Streptococcus suis to penicillin antibiotics.