Study On The Resistance Mechanisms To Carbapenems And Molecular Epidemiology Of Acinetobacter Baumannii

In the last decades,an increasing incidence of nonfermentative gram negative bacillus emerged,especially Acinetobacter spps.and Pseudomas aeruginosa. Acinetobacter spps is an opportunistis pathogen,it is found in many health care environments,soil and water.A.baumannii currently became one of the most important clinical pathogen,such as E.coli,P.aeruginosa and staphylococcus aerues.A.baumannii is emerging as a cause of numerous global outbreaks,displaying ever-increasing rates of resistance.There are reported of MDR and PDR A.baumannii from Europe,North America,China,Japen and Korea.Carbapenems are often the only active agents against many multi-drug resistant strains.The emergence of carbapenems resistance of carbapenem resistance in A. baumannii has becames global concern.INSPEAR(the international Network for the Study and Prevention of Emerging Antimicrobial Resistance) called the occurrence of carbapenem resistance in A.baumannii a sentry event of global resistance and urged essential intervention both in epidemiology and miocrobiolgy.The genus Acinetobacter comprises at least 33 described genomic species,of which Acinetobacter calcoaceticus,A.baumannii,Acinetobacter genomic species 3 and Acinetobacter genomic species 13 sensu Tjernberg and Ursing means that they are indistinguishable in most routine clinical diagnostic laboratories,where they are grouped together as the A.calcoaceticus-A.baumannii(Ac-Ab) complex. The mechanisms underlying resistance to carbapenems in A.baumannii are still poorly understood,but they would be expected to be similar to those described in other Gram-negative bacteria:production of carbapenem-hydrolysingβ-lactamases, alterations in penicillin-binding proteins,decreased outer membrane permeability caused by the loss or reduced expression of porins,overexpression of multidrug efflux pumps。Carbapenemases are a class of b-lactamases that is able hydrolyse one or more carbapenems,including class A,D and B enzymes in ambler molecular classification. In Acinetobacter spp.class D carbapenemases are most popular.Among A.baumannii, OXA-23-like;OXA-24-like;OXA-51-like;and OXA-58 genes are predominant. Recently it has been suggested that enzymes belonging to the OXA-51-like subgroup are intrinsic to Acinetobacter baumannii.Carbapenemases play a role in the resistance mechanism together with absence of OMPS and activation of efflux pumps.Altered penicillin-binding proteins may also contribute toβ-lactam resistance in A.baumannii.Modification of penicillin-binding-proteins(PBPs) as a source of imipenem resistance in A.baumannii has been investigated only rarely.The outer membrane proteins(OMP porin) responsible for the influx of carbapenem b-lactamases antibiotics in the nonfermentative gram-negative pathogen Acinetobacter baumannii.Their changes can reduct of transport into the periplasmic space.Resistance to both imipenem and meropenem in multidrug-resistance clinical strains of Acinetobacter baumannii is associated with the loss containing the carO, which is a heat-modifiable proteins with 29-kDa molecular.An AdeABC efflux system has been identified in A.baumannii.This effluxmechanism belongs to the RND family of efflux systems.Its role in resistance to aminoglycosides,chloramphenicol,fluoroquinolones,trimethoprim and cefotaxime has been demonstrated clearly.However,its effect on carbapenem susceptibility was not initially evaluated.Various genotypic methods have been developed for the typing of acinetobacters, including ribotyping,macrorestriction analysis by pulsed-field gel electrophoresis (PFGE),randomly amplified polymorphic DNA(RAPD) analysis,and total genomic fingerprinting by AFLP(amplified fragment length polymorphism analysis).Among these,PFGE is regarded as the"gold standard"of epidemiological typing.The increasing use of PFGE not only as a research tool but also as an aid in routine epidemiological analysis in clinical diagnostic laboratories has resulted in the development of protocols for the typing of even the same species of bacteria.This study was designed to investigate the epidemiology and mechanisms of carbapenem resistance in Acinetobacter baumannii of clinical strains of Nanfang hospital.170 strains of A.calcoaceticus-A.baumannii(Ac-Ab) complex were collected from clinical strains of Nanfang hospital from 2005 to 2008.This study were also designed to investigate a rapid identification of Acinetobacter baumannii by multiplex PCR.All statistical analysis was carried out by SPSS 13.0.The first section:Rapid identification of Acinetobacter baumannii by multiplex PCROBJECT:Acinetobacter baumannii has become a serious cause of nosocomial infections. Rapid identification of this pathogen is required so that appropriate therapy be given and outbreaks controlled.At the same time,it can make preparation fou the after exeriperments.METHOD:In this study,170 strains of A.calcoaceticus-A.baumannii(Ac-Ab) complex were recovered from Nanfang hospital.PCRs were performed using the specific Acinetobacter baumannii primers and the internal control primers specific for the recA gene of all Acinetobacter spp.Through experiment,170 strains of A. baumannii were indentified.RESULTS:The one-tube multiplex PCR method was used to examine 170 clinical isolates. Each reference strain and clinical isolate yielded a 425-bp internal control amplicon corresponding to the recA gene,but only 138 A.baumannii isolates yielded the 208-bp fragment of the ITS region.No amplicons were obtained with bacteria belonging to other genera,including Escherichia coli,Pseudomonas aeruginosa and Staphylococcus aureus,et al.DISCUSSION:The one-tube multiplex PCR strategy is a rapid and convenient means for identification of A.baumannii.It cost only 2 hours.Rapid identification of this pathogen is required so that appropriate therapy can be given and outbreaks controlled.The one-tube multiplex PCR technique provides an option to make a fast identification of A.baumannii。The second section:Study of the AdeABC efflux pump among isolates of Acinetobacter baumannii which were resistant to carbapenemOBJECT:Efflux pump play important roles in the resistant mechanism of gram negative bacterium,but there still has difference opinions on that whether efflux pump play role in the resistant mechanism of A.baumannii to carbapenems.Our study proved if that efflux pump play roles in the resistant mechanism of A.baumannii to carbapenems from two aspects of phenotype and genetype.METHOD:Our study firstly detected efflux pump phenotype of the 138 strains of A. baumannii by with and without adding the efflux pump inhibitor carbonyl cyanide 3-chlorophenylhydrazone(CCCP),The positive criteria for phenotypic detection of efflux pump is that MIC of meropenem decreases at least 4-fold when CCCP was added;then we chosed detected pump-encoding gene adeB by using quantitative real-time polymerase chain reaction.RESULTS:1.The bacteria grew in the Mueller-Hinton Broth with CCCP displayed that the meropenem MICs decreased 4- to-8 fold in 28 strains out of 138 strains,indicating efflux may be involved in meropenem resistance.Among which,there were 15 strains out of the 39 strains resistant to meropenem which the meropenem MICs decreased 4-to -8 fold,and there were 13 strains out of the 99 strains sensitive to meropenem which the meropenem MICs decreased 4- to-8 fold.The difference was significant between the two groups by Chi-square test.(x~2=12.477~b,P=0.01).2.Efflux pump-encoding gene adeB were detected through real-time PCR to determining the expression level of adeB which were was calibrated by 16S rRNA of A.baumannii in the same specimen.According to the analysis of the detected copy numbers,we calculated the difference of expression rate.The expression rate of adeB of the sensitive strains was 0.8991±1.17239,which the expression rate of adeB of the sensitive strains was 21.1010±21.44315.The difference was significant between the two groups by t- test.(t=4.403,P=0.000)CONCLUSION:Our study indicated efflux may be involved in meropenem resistance among A. baumannii of NanFang hospital.Increased expression of AdeABC efflux pump may play an important role in reduced meropenem susceptibility among A.baumannii in NanFang hospital.The third section:Study of the outer membrane protein CarO of Acinetobacter baumannii resistant to carbapenemOBJECT:The objective of the present study was to investigate the outer membrane protein CarO of in isolates resistant or sensitive to carbapenem through detecting it on the level of gene expression and protein expression.METHOD:We detected the expression rate of the gene carO on the level of gene expression through real-time PCR.On the level of gene expression,we prepared and purified rabbit anti- CarO polyclonal antibody firstly,then analyzed the outer membrane protein CarO of A.baumannii isolates by Western blotting.Expression rate were calculated by grey scale values.Statistical analysis was carried out by t- test.RESULTS:1.The expression rate of gene carO were detected through real-time PCR to determining the expression level of gene adeB which were was calibrated by 16S rRNA of A.baumannii in the same specimen.According to the analysis of the detected copy numbers,we calculated the difference of expression rate.The expression rate of adeB of the sensitive strains was 0.8991±1.17239,which the expression rates of adeB of the sensitive strains was 21.1010±21.44315.The difference was significant between the two groups by t- test.2.We constructed the anti- CarO polyclonal antibody successfully,then we compared expression rate using grey values of the outer membrane protein CarO in isolates resistant with in isolates sensitive to carbapenem.The expression rate of CarO of the sensitive strains was 717.515±395.84,which the expression rates of the outer membrane protein CarO of the sensitive strains was 255.103±23.26767. The difference was significant between the two groups by t- test.(t=2.857,P=0.001)CONCLUSION:The expression rates of gene carO of the sensitive strains were lower than that of the gene carO of the resistant strains,while the expression rates of CarO of the sensitive strains were higher than that of the gene carO of the resistant strains.It shots that some factors such as outside environment or antimicrobial,result RNA of the resistant strains in translational level accumulate which can't translate in normal,while the RNA of the sensitive strains translate in normal.The forth section:The study of distribution of blaoxa genes among Acinetobacter baumanniiOBJECT:Our study was designed to detect blaoxa genes among 96 strains of Acinetobacter baumannii isolated from patients of NanFang hospital through multiplex PCR,so we could identified the genes encoding the four subgroups of OXA-carbapenemases among the isolates and detected the distribution of blaoxa genes Acinetobacter baumannii.METHOD:DNA was extracted from the strains by boiling method.A multiplex-PCR assay was run using the primers of OXA-51,OXA-58,OXA-23 and OXA-24.RESULTS:Among 96 isolates,9 carbapenem-resistant isolates and 33 carbapenem-sensitive isolates were positive for the OXA-51;38 carbapenem-resistant isolates and 6 carbapenem-sensitive isolates were OXA-51 plus OXA-58;1 carbapenem-resistant isolates and 1 carbapenem-sensitive isolates were OXA-51 plus OXA-23;8 isolates lacked blaoxa genes.There were no isolate positive for OXA-23 or OXA-58.CONCLUSION:Our findings indicated that three years recently,38 out of the 96 Acinetobacter were OXA-51 plus OXA-58,which were different with other related study results.that speculated that area and time were different.The fifth section:The study of molecular of Acinetobacter baumannii by Pulsed Field Gel ElectrophoresisOBJECT:To analyze the relationship of A cinetobacter baumannil by pulsed field gel elect -rophoresis(PFGE).Molecular typing plays an important role in the study of the epidemiology of A.baumannii and in coping with its epidemic spread.METHOD:A standard procedure for pulsed-field gel electrophoresis(PFGE) of macrorestrict-ion fragments of Acinetobacter baumannii was set up and validated for monitoring of epidemic strains.Method of reporting:The DNA restriction pattern that is designated the outbreak pattern is usually reported as type A;the isolates whose restriction patterns are indistinguishable from that pattern are reported as representing the outbreak strain.Patterns that are closely or possibly related to the outbreak pattern are considered subtypes of A and are designated type A1,type A2,etc.Isolates with closely or possibly related restriction patterns are reported as probably or possibly epidemiologically related,respectively.Patterns that differ substantially from the outbreak pattern and that are classified as unrelated are designated type B,type C,etc. Isolates with unrelated patterns are considered unrelated epidemiologically. RESULTS:Pulse field gel electrophoresis(PFGE) was performed to analyze the relateness of 138 isolates of A.baumannii recoverd from NanFang hospital.According to the PFGE DNA patterns,27 distinct clones of A.baumannii were identified.Total of 104 isolates belonged to the popular three clones.87 isolates belonged to clone A;18 isolates belonged to clone B and 9 isolates to clone C.24 strains with unrelated patterns are considered unrelated epidemiologically.CONCLUSION:Some isolates with the same genetic basis spread in different department of NanFan hospital,suggesting that the spread of isolates plays an important role in the increase of CRABS.So,it is important to monitor and control the spread of A. baumannii isolates,especially CRABS.In conclusion,the standard procedure enabled us to generate PFGE fingerprints with excellent inter-laboratory reproducibility and can be used to study the geographic spread of epidemic A.baumannii isolates.