| Objective To screen strains producing metalloβ-lactamases (MBL) in Stenotrophomonas maltophilia isolated from ICU and study the susceptibility to antimicrobial agents. and the molecular epidemiology of these strains. To measure the pI and stability of MBL produced by the strains. To confirm what role of MBL played in the antimicrobial-resisitance of S. maltophilia by sequencing the encoding genes of MBL in strains of 603,710 and 750 and construct the prokaryotic expression vector carrying the MBL gene and expressed in E.coli BL21.Methods Total 422 clinical Gram-negative bacterial isolates from patients in intensive care unit of the First Affiliated Hospital of Chongqing Medical University were collected during January 1998 to December 2001. The susceptibility of 10 antimicrobial agents was measured by E-test. The imipenem-resistant strains probably producing MBL of S. maltophilia were confirmed by double disk synergy and MBL-E test. The genotypes in 13 producing MBL strains of S. maltophilia obtained in this study were examined by ERIC-PCR (enterobacterial repetitive intergenic consensus-PCR). The pI of β-lactamases extracted from strains 603,710 and 750 were determined by isoelectric focusing. And the stability of 13 antimicrobial agents to MBL were studied with spectrophotomertrical method and the inhibitory activity of β-lactamases inhibitors to MBL was observed. The blaMBL gene was amplified by PCR and cloned into pMD18-T plasmid. After sequenced, the recombination was subcloned into pET32a(+) plasmid and expressed in E.coli BL21. The susceptibility between expression vectors and strain 750 to antibiotics were compared. Results The lowest antibiotic-resistant rate (11.6%) in 422 isolates was found to imipenem. The most imipenem-resistant strains were S.maltophilia and Pseudomonas aeruginosa which resistant rate were 83.3%(25/30)and 16.9% (11/65), respectively. all the MBL positive strains detected by double disk synergy was Stenotrophomonas maltophilia ,and the number of them detected by Na2EDTA and 2-mercaptopropionicacid (2-MPA) disk were 4 and 7, respectively. And 13 S. maltophilia and 1 P. aeruginosa strains determined by MBL-E test were resistant to imipenem,ceftriaxone,cefotaxime,amikacin and gentamicin. The results of ERIC-PCR showed that there were ten genotype patterns in 13 MBL positive strains of S. maltophilia and the susceptibility data showed there were eight drug-resistant profiles against six antimicrobial agents. There was no significant relationship among strains sources,antibiotics-resistant profiles and genotype in these 13 strains. Furthermore, Strain 710 and 750 had β-lactamase of pI 6.6 and strain 603 had β-lactamases of pI 6.2 and pI 8.4. Bands with pI 6.6 and pI 6.2 could be inhibited by EDTA were classified as MBL. And the stability of 13 antimicrobial agents to these MBL showed that moxalactam,ceftazidime and aztreonam were hardly hydrolyzed, but the highest relative hydrolysis rate 1400% of penicillin, 680%,420% and 190% of ampicillin,oxacillin and cefazolin were found in comparion of 100% of imipenem. Meanwhile the hydrolytic activity of the MBL could be inhibited by EDTA but β-lactamase inhibitors clavulanate,sulbactam and tazobactam. The 867-bp DNA fragment of MBL encoding gene was amplified from both strains 710 and 750 by PCR and sequenced. Results showed the gene was 99.31% homologous to blaS and blaL1 of MBL L1. After being transformed into the E.coli BL21 and inducted with lmM IPTG, a recombinant protein of about 48 kDa was expressed in the pET32a(+)-blaMBL stsytem. The susceptibility of pET32a(+)- blaMBL system and strain 750 showed MIC 12mg/L and 128mg/L to imipenem and MIC 2mg/L and 2mg/L to ceftazidime, respectively.Conclusion The Gram negative bacteria producing MBL isolated from ICU in our hospital was focused in S. maltophilia, which showed a high hetero-geneotype. The MBL produced by strains 710 an 750 were similar to the that in strain ULA511 reported before. Otherwise MBL in strain 603 may be non- L1 MBL. |
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