| Objective:Carbapenem is the first-line drug to treat multidrug-resistant Enterobacteriaceae. However,widespread of this class of agents has resulted in an increasing incidence of carbapenem-resistant strains.According to CHINET 2009,the average carbapenem-resistance rate of Enterobacteriaceae was more than 1%,higher than CHINET 2008.This study focuses on the resistance mechanisms of Enterobacteriacea resistant to carbapenem,including arbapenemase production and outer membrane protein deficiencies with ESBLs or AmpC production. The significance of this research is not only reduced the transmission or the emergence of bacterial resistance by proper antibacterial agents, but also developed a new drug to overcome the problem.Methods:1,Strains and susceptibility testing:A total of 227strains of Enterobacteriaceae were isolated from a hospital in Tianjin during 2009.03-2010.04,including 41 multidrug-resistant isolates.Antibiotic susceptibility to 15 antibiotics was tested by micro-dilution broth method and disk diffusion on Mueller-Hinton agar.2,Detection of carbapenemase: A modified Hodge test and EDTA cooperation test may assist in confirming the presence of carbapenemase and MBL. The genes coded the carbapenemase were amplified by polymerase chain reaction (PCR), including KPC, IMP-1 group, IMP-2 group, VIM, NDM-1 and OXA-48.3,Research on outer membrane protein:Outer membrane protein (Omp) was analyzed by SDS-PAGE and the coding genes were amplified by PCR. DNA and amino acid sequence were Analysis,and transmembrane segment was predicted.Omp concerned with drug-resistance of Enterobacter cloacae was OmpF(38KDa),of Enterobacter aerogenes was OmpE36(42KDa),and of Klebsiella pneumoniae were OmpK36/35(39KDa/37KDa).4,Detection of otherβ-lactamases:Otherβ-lactamase genes were detected by PCR, including broad-spectrum and exceed-broad-spectrum P-lactamases (TEM, SHV, CTX-M-1 group, CTX-M-2 group, CTX-M-9 group) and plasmid-mediate DHA genes.Results: 1,Antibiotic susceptibility: Six carbapenem-resistant clinical strains were screened from 227 clinical isolates of Enterobacteriaceae, including two strains of Enterobacter cloacae E2414 and E2166, three strains of Enterobacter aerogenes Ea2880, Ea293,and Ea391, one strain of Klebsiella pneumoniae Kp382.The isolation rate was 2.6%, accounting for multi-drug resistant Enterobacteriaceae 14.6%. Six strains were all resistant to ertapenem,cephalosporin antibiotics and enzyme inhibitors with P-lactamases. The resistance to imipenem and meropenem was 50%, to aminoglycosides and fluoroquinolones were 83.34% and 66.67%. Three of the six strains (E2414, Ea391, Kp382) were pan-resistant strains.2,Carbapenemases detection:For modified Hodge test,three strains were positive, including E2414, Ea391 and Kp382, suggesting that they may produce carbapenemase.For EDTA cooperation test,six strains were all negative, suggesting that they did not produce MBL.PCR amplification of common carbapenemase genes, the results were all negative.There strains were positive to modified Hodge test,but did not bring common carbapenemase, suggesting that the three strains may carry other carbapenemase,such as IMI-type carbapenemase,or other resistance mechanisms,such as the absence of outer membrane protein.3,Outer membrane protein research: SDS-PAGE and related outer membrane protein genes analysis indicated that E2414 and E2166 lost a 38KDa omp,and OmpF gene did not amplificate. Ea2880 and Ea293 lost a 42KDa omp,and OmpE36 gene of Ea293 did not amplificate.The OmpE36 gene and the predicted amino acid sequence of Ea2880 were all showed 87% identity with OmpE36 gene of standard strain ATCC13048 published on GenBank. Transmembrane segment prediction showed that Ea2880 was difficient at the seventh segment. Ea391 and Kp382 did not lose a omp,but related outer membrane protein gene did not amplificate.4,Otherβ-lactamases detection:E2414 brought TEM-1 and CTX-M-9 group ESBLs. Ea2880 brought SHV-11 and CTX-M-9 group ESBLs. Ea391 brought TEM-1. Kp382 and E2166 were all negative.Conclusions:1,Six carbapenem-resistant clinical strains were screened from 227 clinical isolates of Enterobacteriaceae, The isolation rate was 2.6%. There strains were pan-resistant strains.2,Six carbapenem-resistant clinical strains did not bring the common carbapenemases,but E2414 brought CTX-M-9 group ESBLs, and Ea2880 brought SHV-11 and CTX-M-9 group ESBLs.3,SDS-PAGE and related omp genes analysis showed that,the six strains lost the omp,or the omp genes,or variation in omp genes,related to resistance.4,In all,the six clinical strains of Enterobacteriaceae resistant to carbapenem might be associated with the loss of omp or the loss of omp genes,or variation in omp genes.ESBLs production had played an important role in the resistance.Keywords:Enterobacteriacea, Carbapenemase, Multi-drug resistanceP-lactamase, Outer membrane protein, Outer membrane protein genes... |
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