Study On The Resistance Mechanism In Imipenem-resistant Enterobacter Aerogenes

Enterobacter aerogenes(E.aerogenes)which belongs to Enterobacter spp of Enterobacteriaceae is an important pathogen in the hospital-acquired infection.It can cause pneumonia,endocarditis,skin and soft tissue infections,abdominal infection, arthritis,osteomyelitis,urinary tract infection and bacteremia.Hospital infection caused by E.aerogenes has increased in recent years.The resistance toβ-lactams of E. aerogenes generally results from enzymatic degradation antibiotics,which is mainly caused by extended spectrumβ-lactamases(ESBLs)and high-level production of cephalosporinases.These strains are still susceptible to carbapenem.Carbapenem antibiotics have higher stability on the great majority of plasmid- or chromosome-mediatedβ-lactamases,and strong affinity on penicillin binding proteins (PBPs),which results in the extension and dissolution of cell.They also can effectively penetrate into the periplasmic space across bacterial outer membrane and exist after -effect of antibiotics.Carbapenem antibiotics are currently characterized as a kind of antibiotics with the most extensive antimicrobial spectrum,which are characterized by the most antibacterial activity and rapid bactericidal action.However,with the increase of the clinical application,imipenem-resistant E.aerogenes have been isolated in succession,which gave difficulties for clinical treatment and evoked more attention.There are three main resistance mechanisms in carbapenem-resistant Enterobacteriaceae including carbapenemase producing,change of drug action target, and loss or decrease of outer membrane protein(Omp)associated with chromosome- or plasmid-mediated high-levelβ-lactamases production.Carbapenemases were divided into three groups based on functional classification,including the Amber class A(NMC-A,SME-1 to -3,IMI-1 to -3,KPC-1 to -3,SFC-1,GES-2 to -6),class B (IMP-1 to -13,VIM-1 to -8,SPM-1,SIM,GIM),and class D(OXA-23 to -27,-40,-48, -54,and -58).So far,only KPC-2,IMP-1,VIM-2 were discovered in E.aerogenes. Carbapenem antibiotics can combine with gram-negative bacteria PBP1 and PBP2. Disability of antibiotic combination or affinity decline resulting from PBP alternation lead to drug-resistant bacteria production.Imipenem resistance due to PBP2a change has been reported in only an E.aerogenes strain.It is likely that the most important function for the additional membrane layer,the outer membrane,in gram-negative bacteria is to serve as a selective permeation barrier.The hydrophilic channel in outer membrane was named as outer membrane protein.There are two major Omp related to resistance to antibiotics,OmpF with larger molecular weight and OmpC with smaller molecular weight.Resistance to carbapenem can result from the loss or reduction of a major porin or from the expression of a porin altered in the constriction area.The resistance to imipenem was mainly caused by loss or decrease of Omp associated with high-level ESBLs or AmpC production.The alignment of the Omp36 sequence was consistent with the appartenance to the OmpC porin class and allowed the definition of the transmembrane,cell-surface exposed,and pore domains.Previous reports showed that the regulation of Omp36 expression involoved the OmpX regulation cascade.In the channel constriction,Loop 3,which in the known porins structures extends inside the barrel and defines the size of the transmembrane pore,is the most conserved loop. The substitution/deletion of residues in Loop 3 may lead to imipenem not to enter into cell. In this study,the clone relativity,resistance gene and resistance transmission mechanism were analyzed,which can provide the basis for infection control of imipenem-resistant E.aerogenes.It is important to instruct the clinical use of antibiotics and prevent the distribution of resistance strains.1.Study on the resistance of imipenem-resistant E.aerogenesThe four E.aerogenes strains C1-C4 were isolated from a liver transplantation recipient from January to March 2005.C1 and C3 were recovered from abdominal dropsy.C2 and C4 were cultured from blood.C3 and C4 were isolated after 40 days of treatment with imipenem or meropenem in turn.Pulse-field gel electrophesis(PFGE) analysis showed that the four tested isolates were closely related to molecular typing patterns,suggesting that these strains are derived from the single clone.These strains are resistant to multiple antibiotics,with MICs above the break-points for expanded-spectrum cephalosporins,peperacillin-tazobactam,and ticarcillin-clavulanate acid.In addition,the pre-carbapenem therapy isolates C1 and C2 showed imipenem (MICs,0.25μg/ml,respectively)and meropenem(MICs,0.125μg/ml,respectively) susceptibility,while the post-carbapenem therapy isolates C3 and C4 exhibited remarkable resistance to imipenem(MICs,32μg/ml,respectively)and meropenem (MICs,8μg/ml,respectively).MICs to gentamicin,amikacin and minocycline were also resistant in these strains.All of strains remained susceptible to colistin,polymyxin B and tigecycline.β-lactamases analysis of these strains C1-C4 indicates that they contain class A(CTX-M-3,CTX-M-14,TEM-1,SHV-12)and C(DHA-1)β-lactamases,but no carbapenemases.TEM-1,CTX-M-3 and DHA-1 were transferred by broth conjugation. Western blot revealed that C1 and C2 produced OmpE36,but OmpE36 was not expressed for C3 and C4.Sequencing of ompE36 gene PCR products indicated that an additional 1066 bp fragment,IS903-like,inserted after nucleotide position 97 bp of the ompE36 gene of C3 and C4.The MICs of imipenem and meropenem were increased by 32-fold in the presence of the kanamycin gene replacing the ompE36 gene in the strain C1,which suggested that the ompE36 gene seemed to play an important role in carbapenem resistance.2.Clone and expressionon of ompE36 gene in Enterobacter coli(E.coli)M15.According to omp36 gene sequence(GenBank Accession no.AF335467),a pair of primer was designed.Gene amplification in C1 was carried out by polymerase chain reaction(PCR).A 1110 bp fragment was gained.The ompE36 gene in strain C1 had 95% identity to omp36 sequence(GenBank Accession no.AF335467)and 90%identity to ompK36 sequence(GenBank Accession no.AF373860).The deduced amino acid sequence had 90%identity with Omp36(GenBank Accession no.AAK11270).The purified fragment was cloned into pQE30 expression vector and infused with histidine gene sequence.The recombinant plasmid was named as pQE30-△ompE36 and sequenced,then transformed to the host Eneterobacter coli(E.coli)M15.The expression of the fusion gene was induced with isopropylthio-d-galactoside(IPTG)at 30℃.The recombinant△OmpE36 was overproduced in inclusion form.The yield of expression was almost 22.3 percent of the total of tropina.Western blot analysis was carried out to identify the specific reaction of histidine monoclonal antibody to the expression fusion protein.3.△OmpE36 purification and polyclonal antibody preparation.The recombinant△OmpE36 was overproduced in insoluble inclusion form in E. coli M15.△OmpE36 was purified under denatured conditions using Ni-NTA conjugates.The target fraction was collected,ultrafiltered to desalt and concentrate by Millipore Amicon-Ultra 4.The antigen was mixed with equalvolume of Freund's adjuvant.The emulsion was injected into male rabbit.After 10 days of the last injection, the serum containing the polyclonal antibody against OmpE36 was collected.Agrose diffusion test indicated that valence of the antibody was 1:32.The purified OmpE36 and the polyclonal antibody provided the basis for detection of valence of the OmpE36 antibody by indirect enzyme linked immunosorbent assay(ELISA).4.Detection of valence of the polyclonal antibody against OmpE36 by indirect ELISAWell plate(96 well)was coated with the purified OmpE36 and incubated at 4℃for overnight.The plate was incubated with blocking solution.After washing,different dilutions of blocking solution containing anti-OmpE36 antisera were incubation at 37℃. The plates were rinsed three times and 1:2000 diluted alkaline phosphatase-labelled swan anti-rabbit IgG was added to each well.Optical density was read at 492 nm with an ELISA plate reader.The optimum coat concentration,serum dilution,the optimum blocking solution,and the best time for coloration were tested by indirect ELISA.The above results have showed:1.The resistance to imipenem was due to producing multipleβ-lactamases(TEM-1, SHV-12,CTX-M-3,CTX-M-14,and DHA-1)combined with OmpE36 insertional inactivation in the clinical E.aerogenes of our hospital.2.The inditinguish PFGE pattern indicated C3 and C4 derived from C1 and C2,which indicated that the carbapenems exposure to treat the infection has resulted in collateral effect of carbapenem resistance.3.A novel ompE36 gene(GenBank Accession no.EF191076,95%nucleotide acid identity with and 90%amino acid identity with omp36)was detected.4.It is firstly proved that OmpE36 played an important role in carbapenem resistance in vitro by gene knock-out technology.5.Valence of the polyclonal antibody against OmpE36 by indirect ELISA is 1:400 based on the antibody valence 1:32 tested by agrose diffusion test.It is essential and important for detection of OmpC expression in Enterobacteriaceae.