The Research About The Resistance Mechnisms Of Stenotrophomonas

Stenotrophomonas maitophilia is opportunistic pathogen, an important organism, is frequently isolated from environmental sources, is opportunistic pathogens. But in the last decade, the gram-negative non-fermenting bacilli has emerged as a relevant nosocomial pathogen, usually associated with infections of immunocompromised patients that has been associated with different human pathologies, that cause nosocomial infection, may be resistant to almost all the clinical antibiotics. The overuse of antibiotics in the clinic and for agricultural uses has resulted in a tremendous selective pressure for antibiotic resistant bacteria. The pathogen is intrinsically resistant to 3 -lactams, aminoglycosides and macrolides, highly resistant to several antibiotics, eg. imipenem. The intrinsic and acquired resistance of S. maitophilia to antibiotics and disinfectants is associated with the outer membrane barrier, efflux pump system, enzymes (beta-lactamases, aminoglycoside modifying, erythromycin-inactivating enzymes) and rapid mutation in targets. The mutidrug-pump of S. maitophilia is the most important factor to intrinsic and acquired resistance. SmeDEF efflux antibiotics or various poisonous materials. Infections caused by S. maitophilia are difficult to treat due to the intrinsic antibiotic resistance phenotype displayed by this bacterium. The study about its resistance mechanism is rare of native or abord.To realize the distribution and find resistance mechanisms in clinical strains of S. maitophilia, the isolations from hospitals in Tianjin, during 2001-2002 were collected. In this study, the multidrug resistance mechanism of S. maitophilia, involved with efflux pump and β -lactams was studied, the findings were innovations of these areas. The regulation mechanisms of the pump SmeDEF -the repressor SmeT and other seqencese involved in antibiotic resistance were investigated in the clinic isolations of S. maitophilia, the new variant sequences possibibly associated with resiatance were subscribed to Genebank.To investigate the clinical feature and resistance profile of S. maltophilia clinical isolates from 8 hospitals in Tianjin, isolated during 2001-2002. The MICs of 24 antimicrobials were determined with S. maltophilia strains by the standard micro dilution method and disc expansion method.In this survey of clinical S. maltophilia isolates, 77.5% patients was with >55y. The patients mostly(95.8%) had serious fundamental diseases, in wich pulmonary diseases were dominant. S. maltophilia may cause infections of immunocompromised patients suffering from cancer, steroids and chemical therapy, diabetes, and in patients after operation or endotracheal intubation, on dialysis. This bacterium may cause infection of the respiratory and urinary tracts, bacteremia, thorax and abdomen fluids, wounds infections. S. maltophilia is an important pathogen in nosocomial that can be associated with several organs. The infection correlated with several pathogens, in which nonformentives was in the majority, in our study.The resistance rate of S. maltophilia to Aztreonam, Imipenem was high, those of cefataxime, cephradine and cefprotone were higher than 90%. Its resistance to quinolone was low, the rate of levofloxacinlt was 15.9%, higher than those of ciprofloxacin and ofloxacin. SMZco was the most efficient one of all antibiotics tested, its resistanc rate was 7.7%. S. maltophilia may be resistant to 0 -lactams, aminoglycosides, macrolides, quinolone and so on, and highly resistant to several antibiotics. Resistance in the hospital acquired strains to several antibiotics was higher than community acquired ones, the antimicrobials were ciprofloxacin, chloramphenicol, ceftazidime and cefoperazone. In the clinical isolates, 90.14% of those were MDR(multidrug resistance).Study the efflux effect in the clinical isolates of S. maltophilia, and weather the expression of SmeDEF efflux pump in S. maltophilia is associated with antibiotic resistance. The MICs of antibiotics were determined with Mueller-Hinton. medium for S. maltophilia strains by the standard agar dilution method. To investigate the presence of an avtive efflux mechanism, the MIC of ciprofloxacin, ofloxacin, norfloxacin, and chloramphenicol were determined in combination with pump inhibitor. The distribution of smeDEF gene was analysized by PCR amplification of the smeD sequence in S. maltophilia.The results show that CCCP and reserpine affects the MIC of all 4 antimicrobials, while the MIC of ofloxacin has the obvious changes. In presence of reserpine and CCCP, the 4 antimicrobials showed a better activity, with MIC50 and MIC90 2-4 times lower. 15 isolates with efflux positively inhibited was determined when the quinolone MIC in the presence of CCCP or reserpine was at least fourfold less than the corresponding MIC without inhibitors. We found that the isolates with efflux positively inhibited have higher resistance rates than the negatively inhibited ones. The efflux pump involved with resistance exists widely in the clinical isolates of S. maltophilia.35 of the randomly selected strains all had the PCR products of the amplified region with the primers Dl and D2. The bands obtained had the expected sizes of 328 bp, which include the 5' end of smeD. No amino acid mutation in the coding region of this sequence was found in the 6 studied strain, the amino acid sequences of the amino terminus of smeD genes included in this study were the same as that of D457/D457R strains reported by others. The mRNA level of smeD was high in the 6 isolates resistant to, and 1 strain sensitive to quinolones with efflux positively inhibited; while the isolates susceptible to quinolones or the ones with efflux negatively inhibited mostly had lower level of that. The transcription level of SmeDEF pump system are correlated with the resistance profile, SmeDEF contributes to resistance to multi-antimicrobials in S. maltophilia.Study the regulation mechanism of the pump SmeDEF expression. Investigate the repressor SmeT and other seqencese involved in antibiotic resistance, of efflux pump SmeDEF in the clinic isolations of S. maltophilia. Amplicate smeD-smeT fragment by PCR, then seguence it. Investigate the smeT 5' fragment by PCR, then seguence it. Adding poly(A) tail to 3' end of total RNA. applying 3'RACE, nested PCR amplicate smeT 3" fragment, then seguence it. Investigate the MIC, EOP of the strains to ciprofloxacin, chloramphenicol with induction by ciprofloxacin, and the mRNA level of smeD by RT-PCR.The amino acid sequence of the forepart of SmeT was conservative in all the 7 strains. The nucleotide sequence of the amplified smeT fragment of S. maltophilia clinical strains, compared with the equivalent region of D457/ D457R, with the highest homology was D397 only with 94 V/I substitution, and the lowest was the resistant strains H474, D479 and D489 with efflux positively inhibited. The latter strains had Asp218Glu substitution, different from the negetively inhibited ones. A26 had several amino acid mutation, compared with other strains. The mutant form of SmeT might not bind to its operator(s) and in these circumstances expression of smeDEF is derepressed. Any studied isolates havn't Leul66Gln mutation within the SmeT protein of D457R previously described, which was reported possibly associated with antibiotic resistance.In antibiotic-resistant strains with positive reaction to pump inhibitor, the intergenic sequences of smeD-smeT were different from those in sensitive ones. The variations in -165- -82nt of smeT and several nucleic acids before the start codon of smeT, possibly were involved with antimicrobial resistance. The sequence downstream to the smeT coding sequence showed obvious difference, of which the isolates susceptible to quinolones with efflux negatively inhibited were completely different with the other strains. The 6 new variant sequences possibibly associated with resiatance were subscribed to Genebank. The possible resistance mechanism of S. maltophilia includes the variations in the intergenic smeD-smeT and/or smeT codon sequences.