Preliminary Study Of Two-component Regulatory Gene Of Stenotrophomonas Maltophilia

Part1Preliminary Study of two-componentregulatory gene of StenotrophomonasmaltophiliaObjectiveStenotrophomonas maltophilia genome annotation indicates the existenceof about forty pairs of two-component regulatory genes among its four thousandgenes. The vast majority has not been specifically studied. To research theexpression of two-component regulatory genes of Stenotrophomonasmaltophilia induced by imipenem, and further investigate the function of thetwo-component regulators.MethodsReal-time quantitative PCR was used to determine expression levels of thetwo-component regulatory genes of Stenotrophomonas maltophilia treated with64μg/ml concentration of imipenem. Different expressed two-componentregulatory genes were observed, after imipenem induction of30minutes and2hours. Bioinformatic analysis revealed characteristic of conserved domains ofdifferentially expressed genes.ResultsUp-regulated genes in30minutes or2hours were Smlt1784-1785,Smlt2199-2200, Smlt4208-4209, Smlt4295-4296and Smlt0277-0278,Smlt2233-2234respectively, and Smlt3765-3766genes was both up-regulated in30minutes and2hours. Bioinformatics revealed that these genes containconserved domains of EAL, REC, GGDEF and OmpR, which participate inresistance and other life activities of Stenotrophomonas maltophilia.ConclusionTwo-component regulatory genes probably play a role in the resistance ofStenotrophomonas maltophilia. Part2Comparative and functional analysis of theStenotrophomonas maltophilia WJ66genomeObjectiveStenotrophomonas maltophilia (S. maltophilia) is a non-fermentativeGram-negative bacillus found in natural and hospital environments. In theintensive care unit, infections caused by multi-drug resistant strains, especiallybacteraemia, are associated with increased mortality. Since the whole genomeof S. maltophilia was first sequenced in2008, bioprojects of48other S.maltophilia strains can be found in NCBI. S. maltophilia WJ66is resistant tosulphonamides, fluoroquinolones，but K279a issensitive. In this study, the draftgenome sequence of bacteremia strain WJ66was determined and compared toother S. maltophilia sequences to explore the mechanism of drug resistance.MethodsAfter the DNA was prepared, a whole-genome shotgun (WGS) sequencingstrategy and Illumina HiSeq2000sequencing technology with a paired-end protocol was used to determine the genome sequence of S. maltophilia WJ66.ResultsA total of258contigs, covering a total of4,642,973bp, were generated.The average length of a contig is17,996bp with a G+C content of66.48%. TheN50contig size was33,808bp. The genome includes4,392predicted openreading frames (ORFs) and3ribosomal RNAs (rRNAs) and61transfer RNAs(tRNAs). Genome-wide evolutionary analysis revealed that WJ66was highlyhomologous with the strain K279a. Compared to K279a, automatic annotationanalysis in the Comprehensive Antibiotic Resistance Database showed thatJW66contained one additional antibiotic efflux pump gene, beta-lactamresistance gene, amino glycoside resistance gene, sulphonamide resistancegene, and fluoroquinolone resistance gene. Further analysis revealed thatWJ66contained an amino acid substitution (Q83L) in fluoroquinolone targetGyrA. In addition, WJ66carried a class1integron, with a aadA2gene in theresistance gene cassette. Homology analysis from the pathogen-hostinteraction database showed that WJ66lacks raxST and raxA, which isconsistent with K279a. Ax21and AvrXa21were identified and shown to havehomology to pathogenicity-related genes in Xanthomonas oryzae. In additionthere are eight categories of efflux pumps involved in multidrug resistance.ThisWhole Genome Shotgun project has been deposited at DDBJ/EMBL/GenBankunder the accession AZRF00000000.ConclusionsComparative genomic analyses revealed a considerable diversity among S.maltophilia strains, providing new insights into the differences and similaritiesthat may explain the diverse nature of these strains. Drug-resistant genemutations, drug-resistant enzymes and horizontal drug resistance gene transfer in bacteria contribute to drug resistance, while the regulatory networks ofdifferent virulence genes contribute to differences in virulence. Part3The RNA-seq transcriptomic profiling ofStenotrophomonas maltophilia in response tolevofloxacinObjectiveStenotrophomonas maltophilia has emerged as an important nosocomialpathogen and displays resistance to a variety of antimicrobials. One of theprimary therapy for S.ma infection is fluoroquinolone but its resistance rate isemerging worldwide. To gain insight into the transcriptome of the levofloxacinresistant strain S.ma66after levofloxacin induction, we took a Illumina RNAsequencing approach.MethodsVitro susceptibility test of Stenotrophomonas maltophilia strains WJ66wasdetermined by Etest. Experimental group T with64μg/mL levofloxacin treatmentand control group R with an equal volume of saline, after a half an hourinduction of the second-generation logarithmic growth phaseStenotrophomonas maltophilia bacteria, then total RNA was extracted, digestedgDNA, testing after passing on building a database and sequenced in IlluminaHiSeq2000. ResultsMIC>32μg/mL, according to the latest CLSI the strains was resistance tolevofloxacin, We obtained T22333334bp, R22377778bp Sequencing data, whenmatched to WJ66genome gene coverage were T95.30%and R96.23%. rRNAratio was T0.22%R0.18%, Sequencing data was credible. T compared with Rgroup we found256up regulated genes and703down-regulated genes. GOclustering analysis of different expressed genes showed425genes involved inmolecular functions,384genes involved in biological pathways,227genesinvolved in the cell component. Kegg pathway analysis showed that theflagellar assembly, base excision repair, nucleotide excision repair, mismatchrepair, bacterial chemotaxis, chemotaxis, homologous recombination, DNAreplication, two-component genes, ABC transporters, ten categories maybeparticipate in levofloxacin resistant.ConclusionThe data showed that S. ma is capable of employing diverse responsivemechanisms to adapt to levofloxacin stress. The project should be the firstunknown genome S.ma strain gene expression transcriptome sequencing.DEGs reveals the molecular mechanisms of resistance. Final provide a basicsupport for the clinical development of new drugs.