| [Objective](1) To analyze the risk factors and antimicrobial resistance pattern of S.maltophilia isolated from 6 hospitals of Beijing, and study the difference in resistance rate of isolates from different hospitals. (2) To evaluate the antibiotic effects of SXT combined with TCC, CFS respectively against strenotrophomonas maltophilia. (3) PFGE was used to analyze the homology of 82 stenotrophomonas maltophilia at ICU wards of 6 hospitals in Bejing area. (4) To construct a multi-PCR assay using specific primers of int1 int2 and int3 to screen class 1, 2 and 3integron (intI) among clinical samples and to investigate the distribution of intI of strenotrophomonas maltophilia in China. (5) The presence and genetic content of class I integron were examined with specific primers of intIl and genes cassettes by PCR and subsequently were sequenced. (6) To detect genome DNA and plasmid DNA with multiple PCR and to knowledge the location of genes. (7) 3' sulI resistance gene was examined by PCR and were sequenced. The effect of integron and sulI resistance gene on drug-resistance phenotypic was explored to guide selection of antibiotic in clinic and surveillance of bacteria.[Methods](1) 145 cases of S. maltophilia infection were identified with VITEK-II and analysis. Minimal inhibitory concentratiob(MIC) of 12 antimicrobial agent against 145 isolates were determined by broth microdilution method. (2) The protocol was designed by checkerboard method and the MIC of 2 groups antibiotics against strenotrophomonas maltophilia was determined by broth dilution method, the FIC index was calculated according to MIC results. (3) PFGE was used to analyze the homology of 82 stenotrophomonas maltophilia. (4) Specific primers of int1, int2 and int3 were designed according to the multi-aligment result and the multi-PCR assay was classify the integrons. (5) Gene cassettes were examined by using specific primers of varied regionaccording to intI type by PCR. And then they were sequenced after amplification of bacterium. The result were BLAST on GeneBank(http://www.ncbi.nlm.nih. gov/BLAST). (6) Plasmid DNA of the integrase-positive strains were extracted by their kits. The integrase were located by PCR. (7) sulI gene were amplified using chromosomal DNA as templates and subsequently were cloned and sequenced. The difference carrying class I integrase gene and sulI gene was found between stenotrophomonas maltophilia. ( 8 )To explore the effect of integron and sulI on its resistance phenotypic and to study the resistance mechanism of stenotrophomonas maltophilia.[Results] (1) Most of 145 strains were isolated from sputum(86.2%). 145 strains were mainly from ICU(56.5%), department of respiratory medicine(20.6%). The drug sensitivity tests in vitro showed these strains were resistance to commonly used antibiotics, and drugs whose sensitive rate was over 50% included Doxycycline, Gatifloxacin, Cefoperazone-sulbactam, Levofloxacin, Compound sulfamethoxazol, Ceftazidime-clavulanate and Ticarcillin- clavulanate. The antimicrobial resistance of strains isolated from different wards showed some difference. SXT resistance rate was the highest in tne first hospital of Genaral Hospital of PLA. Of all the infected patients, 56.7% had primary diseases; 92.2% were treated previously with brod-spectum antibiotics; 78.6% underwent invasive examination or treatment. (2) The in vitro antibacterial activity of MICs of combinations were lower than those of 3 drugs tested alone. The range of the FIC index was mainly between 0 - 1.(3) DNA bands of different size from 7-18 were present in the gel, different homology was shown among the 82 strains. Five couples were more than 80% similar, and present in same ICU . Three strain were same clones in Peking university third hospital. 2 couples from the different wards were 80.6%, 79.6% of homology, respictively, others were either of poor homology or no homology. (4) Eighty -two of clinical strains were used in the study; 11 strains presented a 565bp amplicon and it was shown that these strains harbored int1; 3 strains presented a 403bp amplicon and it was shown that thesestrains harbored int 2; 3 strains presents both of a 565bp and a 403bp amplicon and these strain harbored int1 and int2 ; One strain presents was negative for multi-PCR. None of int3 was constructed and confirmed to be practicable by application. (5) The int1 were amplicated by template extracted from Plasmid and 11 integrase-positive strains by PCR. The results were that there are 2 positive strains only in Plasmid. (6) Three sort of different integrons fragment were detected in 82 isolates. An band of 565bp were found in 5 strains and DNA sequencing revealed that the amplicon carried aadB on a class 1 integron, conferring resistant to spectimomycin, streptomycin. GenBank AY 123253. An band of 1091 bp were detected in 3 isolate. The amplicon harbored gene cassette of aadA5 conferring resistant to spectinomycin and streptomycin, and gene cassette dfrA17 conferring resistant to trimethoprim. GenBank DQ838665. One were an band of 2366bp in 1 isolate, which contained aacA4-apbA15-aadA1, conferring resistant to spectimomycin, streptomycin and GenBank Y18050. (7) Among 82 isolates of stenotrophomonas maltophilia, 20% strains were positive, conferring resistant to trimethoprim. Genbank DQ881445. (8) This difference of the MIC positive was statistically significant between producing integron groups among sulfanilamide, kanamycin. Compared with the other antimicrobial agents used in the study, there was no signifant difference between resistance rates for the two grous.[Conclusion] (1)S.maltophilia is resistant to many kinds of antibiotics. The infection caused by S.maltophilia often occur in patients with severe underlying disease and low immunity. (2) The FIC index indicated that synergism and additivity of SXT combined with TCC , CFS respectively against 30 strenotrophomonas maltophilia and primary action against was synergism, there are little autonomy and no antagonism. (3) No clonal outbreak is seed at ICU ward of 6 hospitals in Beijing area, Only there are vertical dissemination of single clone in 6 ICU wards. PFGE was an effective way for drug resistance and epidemic analysis. (4) The multi-PCR assay to screen class 1, 2 and 3 integronsat one time was constructed and confirmed to be practicable by application. It also provides a swift and versatile method for comprehensive and particular research of antibiotic resistance genes carried by integrons in bacteria. (5)Class I integron were detected in 13.4% of clinical isolated, 3 isolated of among harbored class II integrons. There was not been reported in abroad. (6) Only two integrons lacated is positive in Plasmid. These results suggest that plasmids might not play important roles in multidrug resistance of S.maltophilia. (7) Six resistance drug gene cassettes including aadB, aadA5, aphA15, aacA4, aadA1, dfrA17 were found in integron conferring resistance to aminoglycosides and Trimethoprim. Bete-lactams gene cassette does not play important role in integron-mediated drug-resistance. (8) Multiresistance gene cassettes could play an important role in causing the antibiotic resistance on aminoglycosides and trimethoprim. Class I integron and sulI as important resistance-conveying mechanism would at a certain extent limit the use of trimethoprim-sulfamethoxazole.
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