Study On The Expression Levels Of MexAB-OprM And OprD2 Among Carbapenems-resistant Pseudomonas Aeruginosa Isolates

1.BackgroundPseudomonas aeruginosa belongs to G^- bacterium pseudomonas and distributes widely in nature.It is a common conditional pathogen in clinic.PA infection is especially serious in the patient with various kinds of malignant tumor or immunodeficiency.Close attention is paid to PA because of its high isolating rate,high drug resistant rate,high fatality rate and blow up prevalence in clinic in recent years.PA's drug resistant mechanism contains the natural one and the acquired one.Its drug resistance is also becoming serious gradually.But the antibacterials that can be used to control PA infection are too limited.Carbapenems antibiotic is a kind ofβ-lactam antibiotics with broad antibacterial spectrum.It has fairly high antibacterial activity to G⁺,G^-,aerobic and anaerobic bacterium.Its MIC and MBC are contiguous.It has some extent PAE to G^-bacteria and is steady to the majority ofβ-lactamase including ESBLs.There is no across drug resistance between carbapenems and the third-generation cephalosporins antibiotics. So it is an important antibiotic to treat multi-drug resistant PA infection in clinic.But the drug resistant PA increases vigorously with the wide application of carbapenems antibiotic recently.The drug resistant rates of separated PA in China in 2005 to meropenem and imipenem have reached 32.1%and 31.3%respectively.This brings galactic difficulty to clinical anti-infection therapy.The study on PA drug resistant mechanism to carbapenems antibiotic is helpful to trituration of new drugs.It also can help clinician to make reasonable administration program and prevent the appearance and dissemination of drug resistant bacteria.It has been known that the drug efflux pump exists in many kinds of bacteria.There are ATP-Hydrolysing drived type efflux pump and transmembrane protons gradient energy drived type efflux pump.The latter includes the MFS(major facilitator super) family,the SMR(small multidrug resistance) family,the RND(resistance nodulation cell division) family,the MATE(multidrug and toxic compounds extrusion) family and the ABC(ATP binding cassette) family.The RND efflux pump plays key important role in PA's drug resistance in these five families.It has been demonstrated that there are MexAB-OprM,MexCD-OprJ,MexEF-OprN,MexXY-OprM,MexJK-OprM and MexGHI-OpmD.They are all tripartite structure with a membrane embedded efflux protein(MexB,D,F,Y,K,I),a membrane fusion protein(MexA,C,E,X,J,H) and an outer-membrane channel protein(OprM,J,N,OpmD).MexAB-OprM and MexXY-OprM play very important role in PA's natural drug resistance.MexXY-OprM mainly mediates the natural drug resistance to aminoglycoside antibiotics.So far,it is not thought that meropenem diffuses by special channels.The main reason why PA is resistant to meropenem is the excess expression of MexAB-OprM efflux pump,which can bring the entered meropenem out of thallus.So that the decreased accumulation of drug in thallus will induce PA to be drug resistant to meropenem.There are many kinds of outer membrane protein in PA.They can form small pathways.Antibacterials enter the bacteria by these pathways.Antibacterials can't arrive at action target sites in bacteria when they conform or lose.OprD₂ is the specific pathway by which the small molecule imipenem enter the bacteria quikly with selectivity.It has been demonstrated that there is no relation between the PA's drug resistance to imipenem and the conformation of penicillin-binding protein(PBP).OprD₂ is the only helpful channel protein to the permeation of antibacterials.It can form specific bind site of imipenem.PA will become drug resistant to imipenem when OprD₂ decreases or loses.So it is considered that the reduction or loss of OprD₂ is one main mechanism that causes PA's drug resistance to imipenem now.2.Materials and methodsA total of 655 clinical isolated PA were collected from 28 hospitals among 16 Chinese cities.Agar dilution was used to determine the MICs of imipenem and meropenem to PA before and after the addition of efflux pump inhibitor CCCP. Real-time-RT-qPCR was used to determine the expression levels of MexAB-OprM and OprD₂,SDS-PAGE and Western-blot were used to detect OprD₂.3.ResultsThe non-sensitive percentage of the collected PA to imipenem and meropenem is 47.48%and 40.76%.After adding CCCP,this percentage did not change obviously.52.04 %(51/98) meropenem-resistant PA isolates express MexAB-OprM excessively.43.70% (59/135) and 14.81%(20/135) imipenem-resistant PA isolates' OprD₂ expression levels are decreased and absent respectively.SDS-PAGE and Western-blot approve this result. 4.ConclusionPA's drug resistant rate to carbapenems antibiotic represented by meropenem and imipenem is extremely serious in Chinese part areas.This may be related to the use of antibiotics and the management in the hospital.This study shows that the important reasons why PA is resistant to meropenem and imipenem are over-expression of MexAB-OprM and expression reduction or absence of OprD₂ respectively.