| Objective:Nowdays,due to the abuse of antibiotics,multidrug resistant bacteria are increasing,threatening human health.Both multidrug resistant Salmonella enterica serovar Typhi(S.Typhi)and carbapenem-resistant Enterobacteriaceae(CRE)are two types of bacteria that can cause serious infections and even death.Therefore,it is of great significance to understand their resistance mechanisms for the prevention and monitoring of their clinical infections.This paper first focused on the function of the traditional bacterial resistance regulators Ram R and Ram A in S.Typhi,to investigate their roles on ampicillin resistance in S.Typhi.In addition,due to the wide use of carbapenem antibiotics clinically in recent years,CRE strains have spread globally.The study intended to analyze the molecular epidemiological characteristics of CRE in Suzhou,eastern China,and to provide a strong theoretical basis for treating and monitoring CRE infections in this region.Recently,the discovery of the plasmid-mediated polymyxin resistance gene mcr-1 and its spreading to CRE strains have indicated the emergence of pan-resistant bacteria,which has attracted worldwide attention.This paper aimed to analyze the molecular characteristics of mcr-1 and its expression characteristics in CRE strains from six different regions in China,and lay the foundation for the in-depth study on the resistant mechanism of mcr-1.Methods:1.Study on the regulating roles of ramR and ramA on ampicillin resistance in S.Typhi(1)Preparation of ramR deficient mutant(ΔramR): upstream and downstream gene fragments F1 and F2 of the ramR gene were amplified by PCR,then F1 and F2 were ligated into F3 with deficiency of ramR,and a recombinant vector of the suicide plasmid p GMB151 and F3 was constructed and transferred to S.Typhi wild strains.Finally,we screened for ΔramR using the signature of sac B gene on the suicide plasmid.(2)Construction of ramA over-expression strain and empty plasmid control strain: the ramA gene fragment was amplified and constructed on the high expression plasmid p BAD33.The recombinant plasmid p BAD33-ramA and the empty plasmid p BAD33 were electrically transferred to the S.Typhi wild strain,respectively,which were the ramA over-expression strain(GIFU10007(p BAD33 ramA))and the empty plasmid control strain(GIFU10007(p BAD33)),respectively.(3)The expression of ramA in ΔramR and GIFU10007(p BAD33 ramA)under ampicillin Stress: total RNA of S.Typhi were extracted from GIFU10007,ΔramR,GIFU10007(p BAD33 ramA)and GIFU10007(p BAD33)after ampicillin stress for 30 min.The expression level of ramA in each strain was determined with q RT-PCR method,and the difference of their expression was compared.(4)The bacterial growth curves under ampicillin stress: growth curves of S.Typhi GIFU10007,ΔramR,GIFU10007(p BAD33 ramA)and GIFU10007(p BAD33)under ampicillin stress conditions were measured.Then we compared the differences of their growth curves,which means the difference of their tolerance for ampicillin.2.Resistance molecular epidemiological analysis of CRE clinical strains in Suzhou(1)Identification and drug sensitivity analysis of strains: we used Phoenix-100(BD)automatic microbial analyzer and gramnegative bacteria drug sensitivity identification compound card to identify CRE strains and antimicrobial susceptibility of CRE strains,then we amplified 16 S r RNA by PCR and sequence blasting to verify strains.(2)Screening of drug resistance genes: carbopenemase genes(bla KPC,bla NDM,bla VIM,bla IMP,bla OXA-48),extended-spectrum β-lactamase genes(blaCTX-M,bla SHV,bla TEM),Amp C(bla CMY,bla ACT,bla DHA)and porin genes(omp K35/omp F,omp K36/omp C)were selected by PCR,and were identified by gene sequencing.(3)Multilocus sequence typing(MLST): we used PCR method to amplified housekeeping genes of E.coli,Klebsiella pneumoniae and Enterobacter spp.,and carried out paired-end sequencing,then the sequences of the genes were uploaded to the MLST database site to obtain the Sequence Type(ST)results.(4)Pulsed-field gel electrophoresis(PFGE)analysis of clinical strains: PFGE experiments were performed on strains that were untyped by MLST to investigate the epidemiological relevance of these CRE clinical isolates.(5)Plasmid conjugation transfer experiment: recipient bacteria and carbapenemase producing bacteria were co-cultured to perform conjugation transfer experiment,and screen conjugators by PCR.(6)S1-PFGE: bla KPC-haboring conjugative plasmids were performed S1-PFGE experiments to analyse the molecular characteristics of bla KPC-harboring plasmids.(7)Sequence analysis of plasmids: the conjugators of plasmids carried the representative carbapenemase genes(bla KPC-2,bla KPC-3,bla NDM-1,bla VIM,bla IMP-4)were sequencing.3.Study on CRE strains carried polymyxin resistance gene mcr-1 from multi regions(1)Analysis of rsistance genes: we first used PCR method to screen the colistin resistance gene mcr-1 in CRE clinical isolates from multi regions,then mcr-1 positive strains were analysed for other resistance genes such as carbapenemase genes.(2)MLST analysis: As the above method 2.3,MLST analysis was performed on the CRE strains carried mcr-1.(3)Plasmids conjugation transfer experiment: the mcr-1 positive CRE strains were used as the donor strain,the experiment was carried out as the above method 2.5.(4)Sensitivity testing: Phoenix-100(BD)was used to test the drug sensitivity of mcr-1 positive CRE strains and conjugators,then the characteristics of their resistance spectrum were analyzed.(5)PCR-based Replicon Typing(PBRT): according to the different types of plasmid replicons,plasmids can be divided into 18 incompatible plasmids and Inc X,Inc I2,so we typed mcr-1-harboring plasmids by PCR.(6)Plasmid sequencing and whole-genome sequencing: different types of plasmids in the conjugators were extracted for plasmid sequencing,and mcr-1 positive clinical bacteria which could not obtain conjugator were performed whole genome sequence analysis.(7)Analysis of the expression level of mcr-1 in clinical strains: the total RNA of CRE clinical isolates was extracted under no stress conditions.q RT-PCR was performed using absolute quantitative method to analyze the basal expression level of mcr-1 in these CRE clinical strains.(8)Analysis of mcr-1 expression levels in clinical strains under polymyxin stress: after stimulation with polymyxin for 20 min and 120 min,and the expression of mcr-1 was determined by absolute quantitative RT-PCR.Then differences in the expression levels of mcr-1 between different strains after different times of stress were analyzed.(9)Analysis of the expression levels of mcr-1 in conjugators under polymyxin stress: with polymyxin stimulating for 20 min and 120 min,absolute quantitative RT-PCR were performed to analyze the expression of mcr-1 in the same genetic background.(10)Determination of polymyxin MICs of clinical strains containing mcr-1 and their conjugators: polymyxin MICs were determined by constant broth dilution method to found the relation between colistin MICs and the expression level of mcr-1.Results:1.Study on the regulating roles of ramR and ramA on ampicillin resistance in S.Typhi(1)By PCR and sequence analysis,we successfully constructed ramR gene-defective mutant(ΔramR),ramA high-expression strain GIFU10007(p BAD33 ramA)and empty plasmid control strain GIFU10007(p BAD33).(2)Under ampicillin stress conditions,the ramA expression level of GIFU10007(p BAD33 ramA)was 43-fold higher than that of GIFU10007(p BAD33),but the expression levels of ramA in ΔramR and GIFU10007 had no statistical differences.(3)According to the results of growth curves under ampicillin stress,the growth of GIFU10007(p BAD33 ramA)was significantly better than GIFU10007(p BAD33),and there was no differences in the growth rates of ΔramR and GIFU10007.2.Resistance molecular epidemiological analysis of CRE clinical strains in Suzhou(1)We collected 70 CREs,among which 40 K.pneumoniae belonged to at least 10 STs,with ST11 being the most common;2 Klebsiella oxytoca belonged to two different STs;7 Enterobacter spp.shared 3 STs;4 E.coli had 4 different STs;15 Serratia marcescens were divided into two major PFGE groups,while the 2 Klebsiella ornithinolytica contained two different PFGE patterns.And 52 carbapenemase-producing Enterobacteriaceae(CPE)were identified,which contained bla KPC-2(n=42),bla KPC-3(n=1),bla NDM-1(n=6),bla NDM-5(n=1),bla IMP-4(n=3)and bla VIM(n=2);54 CRE were detected with ESBLs and/or Amp Cs,including bla TEM(n=42),bla SHV(n=26),bla CTX-M(n=50),bla DHA(n=19),bla CMY(n=2)and bla ACT(n=2).We also detected mutations in the porin gene among 20 strains.(2)We successfully obtained 15 conjugators of bla KPC-harboring plasmids,including bla KPC-2 and bla KPC-3.Addtionally,we also obtained conjugators p NDM_E600,p IMP_E600 and p VIM_E600.There were at least three kinds of plasmids carrying bla KPC,containing ~70 kb,~95 kb,~130 kb,and ~150 kb,among which the ~95 kb length plasmid was Inc F and was the most;the plasmids carrying bla NDM-1,bla NDM-5,bla IMP-4 and bla VIM were Inc A/C,Inc X3,Inc H and Inc I1,respectively.bla KPC-2 is harbored by a NTEKPC-1a p Kp048-like element,while bla KPC-3 is carried by a novel NTEKPC-II element with a bla TEM inserted 94 bp upstream of bla KPC-3.bla NDM-1 was embedded in a class 1 integron,forming a novel mobile element.3.Study on CRE strains carried polymyxin resistance gene mcr-1 from multi regions(1)From CREs in multi regions,we isolated 5 mcr-1 positive E.coli strains,SZ01(ST2448)and SZ02(ST2085)from Su Zhou,CDA6(ST167)from Cheng Du,BJ10(ST156)and BJ13(ST457)from Bei Jing,and 2 K.pneumoniae containing mcr-1,SZ03(ST25)and SZ04(ST25)from Su Zhou.The conjugators SZ01-mcr-1-E600,SZ02-mcr-1-E600,CDA6-mcr-1-J53,BJ13-mcr-1-J53,SZ03-mcr-1-J53,SZ04-mcr-1-J53 were obtained,except the conjugator of BJ10.And the plasmids carring mcr-1 in SZ01,CDA6,SZ03 and SZ04 all belonged to Inc X4(p MCR1_Inc X4),while the plasmids carrying mcr-1 in SZ02 and BJ13 were Inc I2(p MCR1_Inc I2).We found that all plasmids carrying mcr-1 shared an identical 2.6 kb mcr-1-pap2 element and mcr-1 in E.coli BJ10 was located on a Tn9-like composite transposon(Tn Apl),which was integrated in the chromosome.(2)Without colstin stress,the mcr-1 expression levels in p MCR1_Inc X4 varied from 1.81×10-5 to 1.05×10-4(pmol/μg total RNA),and the expression levels of p MCR1_Inc I2 were 5.27×10-5(SZ02)and 2.58×10-5(BJ13),respectively.In addition,the expression of chromosomal mcr-1 in E.coli BJ10 was 5.49×10-5.(3)After colistin treatment,mcr-1 in p MCR1_Inc X4 increased 2-and 4-fold at 20 min and 120 mins,respectively,in all p MCR1_Inc X4-harboring strains,except for SZ01,which had a lower expression after 20 min and restored to baseline levels after 120 mins.In contrast,mcr-1 expression of p MCR1_Inc I2 in SZ02,BJ13 and the chromosomal mcr-1 in BJ10 remained at baseline levels after 20 and 120 mins.(4)In E.coli E600,mcr-1 expression of p MCR1_Inc X4 and p MCR1_Inc I2 were similar and decreased after colistin treatment for 20 min.However,mcr-1 in p MCR1_Inc X4 was up-regulated after 120 min,while mcr-1 in p MCR1_Inc I2 was down-regulated.Conclusion:1.The over expression of ramA in S.typhi may increase the bacteria resistance to ampicillin;ramR neither inhibited the transcriptional expression of ramA,nor involved in the regulation of ampicillin resistance of ramA,which is different from other bacteria.2.The bacterial species and STs of CRE strains in Suzhou were abundant,and the resistance mechanisms such as carbapenemases,ESBLs,Amp C,mutated porin,and plasmids had significant diversity.3.The CRE strain carrying mcr-1 has spread to hospitals in different regions of China,and this study first introduced the chromosomal mcr-1 in CRE.And we found that almost all plasmids carrying mcr-1 had an identical 2.6 kb mcr-1-pap2 element.In addition,under the stress of colistin,mcr-1 has different expression characteristics in different plasmids and bacterial hosts with different genetic backgrounds,suggesting that its expression regulation is complex and needs to be further study. |
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