Study In The Drug Resistance Of Chryseobacterium Indologenes And Chryseobacterium Gleum And The Related Resistant Mechanism Of Chryseobacterium Gleum

OBJECTIVESFirst, to study the incidence of β-lactamses, mainly the ESBLs and metallo-β-la-ctamses of Chryseobacterium indologenes and Chryseobacterium gleum. Second, to explore the difference of drug-resistance between p- lactamse positive and negative Strains. Third, to investigate the multi-drug-resistant mechanism of Chryseobacterium indologenes and Chryseobacterium gleum. Last, to investigate the resistance mechanism of Chryseobacterium gleum to imipenem, focusing on the role of metallo-β-lactam-ses, outer membrane proteins and efflux mechanism, and to instruct clinical application of antibiotics reasonably.METHODSWe collected 25 strains of Chryseobacterium indologenes and 10 strains of Chryseobacterium gleum from 2nd Affiliated Hospital of Zhejiang University. Agar dilution method was applied to detect Minimal Inhibitory Concentrations (MIC) of 15 different antibiotics used frequently. Three-dimensional test was used to detect ESBLs and meta-llo-β-lactamses, and the genes of β-lactamases were amplified with 3 pairs of prim- ers special for Chryseobacterium indologenes and Chryseobacter ium gleum. The drug resistance pattern of those p-lactamase positive and negative strains were analyzed according to NCCLS standards.The clinical isolates 676 and 592 which were sensitive to imipenem (MIC≤4ug / ml) were chosen for drug resistance inducing test. The resistant strains were obtained by increasing the concentration of imipenem in the medium step by step. The resistance-induced strains were named as 676^imp-r and 592^imp-r , respectively. Then, the MICs of 4 isolates were assayed and metallo- β- lactamses were also identified according to the above methods.The outer membrane proteins (OMP) were isolated from the four strains by centri-fugation and were analyzed by SDS-olyacrylamide gel electrophoresis (SDS-PAGE). Then, the differences of outer membrane proteins were compared in theSDS-PAGE profile.To investigate the presence of possible efflux mechanisms, the MICs of imipene -m were also determined in combination with 0.5 ug of CCCP per ml and 10 ug of res-erpine per ml. An efflux mechanism was inferred to be present when the imipenem MIC in the presence of CCCP and/or reserpine was at least four-fold less (2 doubling dilutions) than the corresponding MIC in the absence of these compounds.RESULTS1 .We detected that, among the 25 strains of Chryseobacterium indologenes and 10 strains of Chryseobacterium gleum, 72.0% (18/25) isolates of Chryseobacterium indologenes and 90 % (9/10) isolates of Chryseobacterium gleum were considered as metallo-β-lactamses positive strains, but no isolates were detected for the production of ESBLs in all the stains. The PCR results of IND,CGB and CGA gene showed that IND positive percentage was 20%(5/25), CGB positive percentage was 30.0% (3/ 10) ,CGA positive percentage was 0 %.2. The resistant rate to IMP, AMC, CTX, CRO, CFP,CAZ, ATM, FEP of Chryseobacterium indologenes was above 50%;the rate of resistance for TZP,TIM, SCF, LEV and CIP was 18.5%, 33.3%, 8.3%, 27.8%, 33.3%, respectively. The resistant rate to IMP, AMC, TIM, CTX, SCF, CRO, CAZ, ATM, FEP for Chryseobacterium gleum was above 50%, and the rate of TZP,CFP, LEV and CIP was 46.8%, 30.0%, 44.1%, 44.2%, respectively.3. The resistant rate to IMP, TZP, AMC, TIM, CTX, CRO, CFP, CAZ of the metallo-p -lactamses positive strains of Chryseobacterium indologenes was more than negative strains, and the difference between them is significant (PO.05) . While the resistant rate of FEP, ATM, LEV and CIP has no significant difference.4. Two strains which are imipenem resistant were obtained after 40 cycles of induction, with the MIC to IMP of 676 imp"r and 592 imp"r increased to 64ug/ml. The resis -tant patterns to TZP, AMC, TIM, CTX, SCF, CRO, CFP, CAZ, FOX, AZT, LEV,VA, FEP and CIP have no difference between original isolates and induced isolates.5. CGB gene was identified in all 4 strains of Chryseobacterium gleum, and no di -fference in the SDS-PAGE profile was found between original isolates and induced isolates under the current electrophoresis conditions. The MIC against imipenem in the presence of CCCP or reserpine was not four-fold less (2 doubling dilutions) than the corresponding MICs in the absence of these compounds.CONCLUSIONSl.The incidence of ESBLs of Chryseobacterium indologenes and Chryseobacter -ium gleum was low, but the incidence of metallo-P-lactamses was very high.2. Chryseobacterium indologenes and Chryseobacterium gleum was resistant to carbapenems, penicillins, some cephalosporin. However quinolones (LEV, CIP) and piperacillin containing lactamase inhibitor may be selected in treating Chryseobac -terium indologenes and Chryseobacterium gleum infections.3. High level production of metallo-P-lactamses may be the main mechanism of drug resistance in Chryseobacterium indologenes and Chryseobacterium gleum.4. There was no evidence that efflux mechanism and the outer membrane protein play an obvious role in the Chryseobacterium gleum resistance to imipenem.