Study On The Molecular Mechanism Of Multi-drug Resistance In Shigella Spp.

Bacillary dysentery caused by Shigella spp. is the third gravest contagiousdisease among the contagious diseases in our country. Recent investigation indicatedthat infectious diarrhea caused by Shigella spp. still made a gigatic health burden fordeveloping countries and is still the emphasis of the prevention and cure of contagiousdiseases. The extensive use of antimicrobials resulted in the phenomenon ofantimicrobial resistance emerging, even multi-drug resistance and Shigella spp.obtaining antimicrobial resistance more and more rapidly, which not only makes thenew challenge to the prevention and cure of Shigella spp., but also contributes to thedissemination of antimicrobial resistance. Shigella spp. was considered a kind ofantimicrobial resistance strains which threatened people's health by WHO in 1996.Thus making a profound and systematical research on the mechanism of antimicrobialresistance and the strategy of solving the problem of antimicrobial resistance is a hottopic for bacillary dysentery.The bacterium can have the antibiotic resistance against the different structure orthe different function mechanism antibiotics, namely multi-drug resistant. Researchon antibiotic resistance of Shigella spp. indicated that the mechanism of antibioticresistance for aminoglycoside antibiotics mainly results from the produce ofAAC(3)Ⅱenzyme; and forβ-lactam antibiotics, mainly results fromβ-lactamase. Quinolone antibiotics target on the DNA gyrase (gyrA, gyrB) gene and topoismeraseⅣ(parC, parE) gene and hamper DNA replication, thus preform antibacterial activity.If its target genes mutate, antibiotic resistance against quinolone antibiotics can beinduced. The decrease of the expression of outer membrane protein Mr43,000 inShigella spp. and the point mutation of marA gene in mutidrug resistance persisterinduce membrane related antibiotic resistance. Researchs also revealed that acrA geneof the multi-drug resistant pump systems is highly expressed in clinical mutidrugresistance strains, which demonstrated the active -effiux mechanism induced by acrAgene contributing to the clinical multi-drug resistance. Based on the study ofintegron-related antibiotic resistance, classⅡintegron roled mainly. These variousmechanisms seldom acted respectively, but interacted and co-operated to determinethe level of Shigella spp. antibiotic resistance against one or more antibacterials. It isthe co-operation that causes the high mutidrug resistant level ofShigella spp..The ways of bacterium obtaining the antibiosis resistance have following severalways: by the chromosome change Shigella spp. can obtain the antibiosis resistancemainly through the medicine own inducted vertical way, but by the transmission ofR-ptasmid and transfer of antibiotic resistance transposons mainly through thehorizontal genetic transferred way. The bacterial multi-drug resistance regardless ofwhich way is a complicated process in which multiple genes and proteins participates,but the present researches have limited in dividual genes and the proteins of onemechanism, not only hard to explain the complete bacterium multi-drug resistantphenomenon, but also cannot reveal the molecular foundation and the mechanism ofbacterium multi-drug resistant comprehensively. At present, systematic, allroundstudies and appraisal data from sensitive Shigella spp. strains to persisters still lacked,this topic designed on the whole researched the mechanism of Shigella spp. antibiosisresistance from the entire genome, proteomics's angle.The Shigella spp. sensitive to antimicrobials was selected to be the object.Subinhibilory concern ration induced test and conjugation experiment were adopted toconstruct multi-drug resistant Shigella spp. strains, nucleinic acid foundation ofShigella spp. multi-drug resistance was illuminated from aspect of chromosomal mutation, plasmid transmission, transposon by Suppression subtractive hybridization,dot blotting hybridization and gene sequencing; two-dimensional electrophoresis wasapplied to illuminate the categories and function of related proteins of multi-drugresistant Shigella spp. strain. Synthetic evaluation on the main ways of antibiolticresistance obtained and coexistent mechanism of antibioltic resistance providedscientific data for molecular epidemiological surveillance of Shigella spp., researchand exploitation of novel antimicrobials and guidance on clinical applyingantimicrobials.Methods1. Detection of Minimal inhibitory concentration.Improved Kirby-Bauer method was adopted to select Shigella spp. F. sensitiveclinical isolates and Escherichia coli multi-drug resistant clinical isolates of fourkinds of antibiotics Cefalotin, Norfloxacin, Gentamycin, Cotrimoxazole. Minimalinhibitory concentration of shihella sensitive strain was detected by agar dilutionmethod.2. Subinhibilory concern ration induced experimentShigella spp. sensitive strain was cultivated 12 generation in LB medium of 1/2MIC, multi-drug resistant strain of more than 4 times MIC was induced multi-drugresistant strain.3. Conjugation experimentThe selected Shigella spp. sensitive strain as the recipient, while multi-drugresistant Escherichia coli as the donator, genetic transferred multi-drug resistantShigella was constructed by conjugation experiment.4. Construction of DNA Subtractive Library of induced and genetic transferredmulti-drug resistant strain of Shigella spp.(1) Bacterial genome extract kit was adopted to extract whole genome DNA ofShigella spp. sensitive strain, induced and genetic transferred multi-drug resistantstrains, then Shigella spp. sensitive strain genomic DNA as Driver. induced andgenetic transferred multi-drug resistant strains genomic DNA as Testers. Testers andDriver respectively digested with Rsa I, a four-base cutting restriction enzyme that yields blunt ends.(2) The Testers DNA were then subdivided into two portions, and each wasligated with different DNA adaptors (1 or 2R). Adaptor' outer side and the first PCRprimer had the same sequences, left with the second PCR(nested-PCR) primer,which are the same series to screen PCR amplified products subsequently.Moreover adaptor containing T7 promotor and a series of important restrictionenzyme cutting site to facilitate subsequent clone and sequencing. 2% gelselectrophoresis to identify efficiency of ligation, more than 25% was efficient.(3) Twice subtractive hybridization: excessive Driver DNA samples haverespectively mixed with two Tester DNA samples. Then mixed the two hybridsamples, while adding a new degenerative Driver DNA for second hybridization,which further enriched by differences DNA.(4) Twice PCR reaction: for the first PCR products, which have two differentadaptor, can be amplified exponentially. A secondary PCR amplification isperformed using nested primers to further reduce any background PCR products andto enrich for differential sequences.(5) Subtractive efficient detection: second PCR products of subtractive andunsubtractive as templates, 23S rRNA 5' and 3' primers as primers carried out PCRamplification to evaluate subtractive efficiency. 2% gels electrophoresis to identifytwice PCR products and subtractive efficiency.(6) Subtractive hybridization products of induced and genetic transferredmulti-drug resistant genome ligated with pMD18-T carrier and transformed highperformance competent cell. cloning proliferation, Blue and White test screened thepositive clone. All of the positive clones were preserved in 30% glycerine, thensubtractive libraries were constructed.5. Screening of multi-drug resistant related genes.Some of positive clones were indentified by PCR and screened by dot blottinghybridization multi-drug resistant related gene were sequenced with M13 two-wayprimers, and the sequence were analysised with homology compare. 6. Proteomic analysis of multi-drug resistant Shigella spp.(1) Two-dimensional electrophoresis(2-DE)were employed to compare thedifferential expression proteins between multi-drug resistant strain and sensitivestrain of Shigella spp.. Transonic and Urea-CHAPS-DTT schizolysis method wasadopted to extract the whole mycoprotein of sensitive strain, induced and genetictransferred multi-drug resistant strain. The protein content of supernatant wasquantified by Bradford protein assay system.(2) First-dimension isoelectric focusing was carried out with separate wholeproteins samples containing about 100μg proteins. After IEF finished, each IPG stripwas incubated for 15min with equilibration solution(containing 1% DTT) andequilibration solution 2 (same as equilibration 1 and 2.5% iodoacetamide insteadingof 1% DTT), then the IPG strip was placed in contact with the top surface ofSDS-PAGE. Separation in the second dimension was carried out at a power setting of18W/gel. The protein spots were visualized in gel by silver staining (analytical type)and Coomassie brilliant blue staining (preperative type) when electrophoresis ended.(3) 2-DE gels were captured by transmission scanning (LabScan). Target gelswere analyzed with ImageMaster software including spot detection, backgroundsubtraction, matching, etc. And after rechecking it by hand to confirm its change, thedifferent expressing spots were found.(4) Spots were excised from the gels and stored at -80℃. The Some ofdifferential proteins were digested in gel by enzyme and the mass of generatedpeptides were measured by matrix assisted laser desorption ioniza-tion time of flightmass spectrometry(MALDI-TOF-MS).The data obtained from peptide massfingerprinting (PMF) were searched using the Internet available database.(Mascothttp://prospector.ucsf.edu/) and Profound (http://129.85.19.192/profound-bin/WebProFound.exe) software were amptied to search the protein which peptides canmatch to the known protein in NCBInr protein database.Results1. Construction of induced and genetic transferred multi-drug resistant strains MIC of Shigella spp. sensitive strain of CF, NOR, GM, SMZ were 32m g/L, 0.5mg/L, 2mg/L, 512mg/L by agar dilution respectively.(1) Results of multi-drug resistance induced by 1/2 MIC: MIC of multi-drugresistant strain of CF and NOR were 6-fold of sensitive strain, that of GM and SMZwere 8 -fold.(2) Conjugal transfer multi-drug resistant: MIC of Shigella spp. genetictransferred multi-drug resistant strain of CF, NOR, GM, SMZ were 256mg/L, 4.0mg/L, 20mg/L, 5120mg/L by agar dilution respectively, drug susceptibility testindicated that there were no bacteriostasis cingula of 4 drugs.2. Construction of Shigella spp. induced and genetic transferred multi-drugresistant genomic subtractive library.Suppression Subtractive Hybridization was adopted.(1) Testers genomic DNA degested completely by Rsa I ligated with adaptors 1and 2R, the PCR product generated using one gene-specific primer (23S rRNAForward Primer) and PCR primer 1 was about the same intensity as the PCR productamplified using two gene-specific primers (23S rRNA Forward and Reverse Primer),the band intensity for these PCR products differs by 2-fold, it indicated that theligation was more than 50%.(2). Different sequences enriched generously through twice subtractivehybridization and twice suppressed PCR, amplified DNA band of induced and genetictransferred multiantibiotic resistance subtractive hybridized products appearssmear(100～2000bp), to agree with anticipated results.(3) Through contrasting known gene 23S rRNA's abundance before and aftersubtractive hybridization explained the subtractive efficiency. The subtractiveefficiency of this experiment indicated that. 23S rRNA amplificated products could beobserved in subtractive hybridized induced and genetic transferred multi-drugresistant products after 28 circulation, but in unstracted products, 23S rRNAamplificated products could be observed at the 18th circulation, this results illustratedsubtractive efficiency of two samples were high. (4) Shigella induced multi-drug resistant subtractive hybridized geneticsequences were connected with PMD 18-T carrier and transformed clone, altogetherobtained 1512 positive clones, randomly picking 216 positive clones had madebacterial PCR verification, 202 clones includes the insertion fragment, thetransformation efficiency was 93.52%; Shigella genetic transferred multi-drugresistant obtained 1080 positive clones. The random picking 192 positive clones hadmade bacterial PCR verification, 181 clones included the insertion fragment, thetransformation efficiency was 94.27%.3. The results of screening multi-drug resistant related genes.(1) Screening induced multi-drug resistant related genes by dot blottinghybridization of nested PCR products of 80 positive clone from induced multi-drugresistant genomic subtractive library, 12 difference genetic sequence were detected.Through sequencing and retrieving, 11 genetic sequence were obtrained except one, 5unknow sequences weren't retrieved homologization sequence on BLAST, they mightbe some novel gene related induced multi-drug resistant; the rests were 16S ribosomalRNA gene(the first 62 and the last 41 base pair were 16S ribosomal RNA gene, butthe middle sequence were unknow), hypothetical Rep protein, typeⅠsite-specificdeoxyribonuclease (Hsds), predicted membrane fusion protein of efflux pump,SEcpl-like transposase (tnpA), ISabal transposase. Among them transposase genewere two copies.(2) Screening genetic transferred multi-drug resistant related genes by dotblotting hybridization of nested PCR products of 80 positive clones from genetictransferred multi-drug resistant genomic subtractive library, 10 difference geneticsequence were detected. Through sequencing and retrieving, 5 unknow sequencesweren't retrieved homologization sequence on BLAST, they might be some novelgenes related genetic transferred multi-drug resistance: the rests were Klebsiellapneumoniae Tn3926 Some of transposase gene. hypothetical protein and Bacilluslicheniformis's Gnt protein, Some of gyrb gene for DNA gyrase B subunit, 23SrRNA ribosomal ribo nucleic acid. Among them DNA gyrb gene appeared twice. 4. Proteomics analysis of Shigella spp. multi-drug resistant.(1) Results of whole mycoprotein two-dimensional electrophoresis of Shigella spp.sensitive strain, induced and genetic transferred multi-drug resistant strain: wholemycoprotein two-dimensional electrophoresis atlases of three kinds of Shigella spp.strains were obtained successfully. After three repeated experiment, 946±37 proteinspots in whole mycoprotein 2-DE gels of sensitive strain, 1013±157 protein spots inthat of genetic transferred multi-drug resistant strain and 1096±189 protein spots inthat of induced multi-drug resistant strain were detected.(2) Analysis of differential expression protein between Shigella spp. sensitive strainand induced multi-drug resistant strain: 2-DE gel atlas of induced multi-drug resistantstrain as a reference gel matched with sensitive strain, 48 differential expressionprotein spots were detected, among them 8 spots only exited in induced multi-drugresistant strain, 8 spots only exited in sensitive strain, express amounts of 26 spots ofinduced multi-drug resistant strain were higher than that of sensitive strain, butinversely in another 6 spots. 5 proteins related to induced multiantibiotic resistancewere identified by peptide mass fingerprinting: ATP binding cassette transporterprotein,γ-glutamyltranspeptidase, aspartate carbamoylransferase expressed higherin induced multi-drug resistant strain; But chain elongation factor EF-Tu and andsingle strand DNA binding protein expressed higher in sensitive strain.(3) Analysis of differential expression protein between Shigella spp. sensitive strainand genetic transferred multi-drug resistant strain: 2-DE gel atlas of genetictransferred multi-drug resistant strain as a reference gel matched with sensitive strain,43 differential expression protein spots were detected, among them 8 spots onlyexited in genetic transferred multi-drug resistant strain, 6 spots only exited in sensitivestrain, express amounts of 22 spots of genetic transferred multi-drug resistant strainwere higher than that of sensitive strain, but inversely in another 7 spots. 7proteins related to genetic transferred multiantibiotic resistance were identified bypeptide mass fingerprinting, two new proteins, CRISPR-associated protein andGroel-Groes-Adp7; ATP binding cassette transporter protein, Cysteine synthase and Predicted periplasmic or secreted lipoprotein protein expressed higher in genetictransferred multi-drug resistant strain; chain elongation factor EF-Tu and single strandDNA binding protein expressed higher in sensitive strain.Conclusions1 Induced and genetic transferred multi-drug resistant strains of Shigella spp. fromthe same sensitive strain were constructed successfully, the homology andcomparability were high.2 Induced and genetic transferred multi-drug resistant genomic subtractive librariesof Shigella spp. were constructed successfully by suppression subtractivehybridization.3 Screening results of Some of difference genes revealed 16S rRNA mediatedaminoglycoside antibiotics resistance, plasmid drug-resistance regulated by Repprotein, active efflux mechanism, R-M system and transposon drug-resistancewere related to induced multi-drug resistance; Meanwhile 23S rRNA mediatedmacrolides(Mis) antibiotics resistance, transposon drug-resistance, gnt operon,DNA gyrase b subunit mediated Quinolone antibiotics resistance were related togenetic transferred multi-drug resistance.4 Transposon drug-resistance existing in both vertical induced and horizontalgenetic transferred multi-drug resistance was discovered firstly.5 5 novel sequences related induced multi-drug resistance and 5 novel sequencesrelated genetic transferred multi-drug resistance were found.6 Proteinaceous 2-DE atlas of clinical isolates of Shigella was constructed firstly,946±37 protein spots were obtained.7 Proteomics analysis of some of difference proteins revealed that someimportant cellular metabolism enzymes expressed highly. ATP binding cassettetransporter protein affected seriously in mechanism of vertical induced andhorizontal enetic transferred multi-drug resistance of Shigella spp..8 It was found that CRISPR-associated protein and chaperonin of Hsp60(Groel-Groes-Adp7) existed only in genetic transferred multi-drug resistant strain.maybe they represent for some new mechanisms of horizontal genetic transferred multi-drug resistance.9 Analysis of genomics and proteomics confirmed multi-drug resistance of Shigellaspp. was a complicated process which was participated by many mechanisms andadjusted by various kinds of metabolic enzymes.