Study On Resistance Mechanisms And Epidemiology Of Carbapenemase-producing Gram-negative Bacilli

Background Due to the uncontrollable abuse of various antibiotics,multiple numbers of drug resistance and pan-resistant bacteria are increasing.It has become a world-wide problem and a huge challenge to the clinical diagnosis and treatment.Carbapenem antibiotics are the last defense for the serious infections of multiple resistant Gram-negative bacilli.Threrefore,the rapid spread of carbapenem-resistant gram-negative bacilli gains more and more concern.The production of carbapenem is the main cause of carbapenem antibiotics resistence.The carbapenemins include A,B and D enzymes in the Ambler molecule classification,which refer to all enzymes that are capable of hydrolyzing Ampicillin or meropenem and other carbapenem antibiotics such as a class of ?-lactamase.Most carbapenemase genes are located on transferable gene elements such as plasmids,integrins,etc.Both the same and different species can obtain the resistence gene via the progress of bonding,conduction,transformation,transportation,etc.resulting in more productions of carbapenemase from bacteria.The carbapenem-resisted gram-negative bacilli demonstrated resistance not only resistant to ?-lactam antibiotics but also most of the quinolones,aminoglycosides and other antibiotics resistant,which is the characteristic of multi-drug resistance and even pan-resistant characteristics.Therefore,it is significant to detect whether the bacteria are capable to produce carbapenemase with a rapid and effective method in order to control the infection and spread of these bacteria.In the contemparory clinical practice,the generally used method for the detection of carbapenem is modified Hodge test(the modified Hodge test).This is a phenotypic experiment,which is the American Clinical Laboratory Standardization Association(CLSI)recommended phenotypic validation test.In addition,imipenem-EDTA double-paper co-test and Etest MBL commercial confirmation test strips are also specifically for the detection of group B carbapenem enzyme [1].However,the specificity and sensitivity of these methods are being challenged as the combination and variety of drug-resistant genes contained in resistant strains.A better detection method of carbapenem-producing strains is urgently needed.The United States Clinical and Laboratory Standards Institute(CLSI)introduced the Carba NP trial [2] as the Enterobacteriaceae,Pseudomonas aeruginosa and non-actinobacteria produced carbapenemase phenotypic test as confirmatory test in 2015.The test of phenotypic was first reported by Professor Patrice Nordmann,France's Southern School of Forensic Medicine,in 2012.[3] Since then,the method had been successfully optimized and developed for the detection of Pseudomonas aeruginosa and the indicator phenotypic by Carba NP test ? [4].Such test can directly determine the group of carbapenase-producing strains,and it is simpler and faster compared with the improved Hodge test.With the rapid development of molecular biology and bioinformatics analysis,the whole genome sequencing technology has been used widely.Whole genome sequencing is the sequencing of the whole genome sequence of unknown species.At present,the whole genome sequencing technology mainly includes second generation sequencing technology(NGS)and third generation sequencing technology.The second generation sequencing technology enables rapid and low-cost genome sequencing,with equipment suppliers primarily 454(Roche),SOLi D(ABI)and Solexa.The third-generation sequencing technology was introduced in 2011,and its single-molecule real-time sequencing technology(SMRT)was completely different from the second generation sequencing,developed by Pacific Biosciences,which was capable to read up to 3kb.The emergence of high-throughput sequencing technology has led to significant changes in the genome-derived approach,especially in the field of microorganisms with small genome information,which has become an irreplaceable tool for the identification of pathogenic microorganisms and their pathogenesis mechanism.In this study,we used the Carba NP test recommended by CLSI to screen the carbapenemase-producing Gram-negative bacilli in our hospital.The multiplexed PCR method was used to screen the positive gene to investigate the prevalence of carbapenemase production in gram-negative bacilli in our hospital,and to explore the advantages and disadvantages of Carba NP test.Four isolates of carbapenem-producing Klebsiella pneumoniae which were obtained from the same patient,were used to test the drug sensitivity between the bacteria and their parents.The homology of four strains was compared.Then,one of the multiple resistant strains carrying bla KPC-2 was sequenced by high-throughput sequencing.Through the bioinformatics analysis,the resistance mechanism of Klebsiella pneumoniae,the resistance gene environment was studied well.Objective 1.To understand the molecular mechanism of carbapenemase-producing multidrug-resistant gram-negative bacilli in the First Affiliated Hospital of Guangzhou Medical University.2.To understand the effect of Carba NP test on carbapenem production by Gram-negative bacilli.3.Drug resistance and homology analysis of four strains of Klebsiella pneumoniae in the same patient.4.The gene environment of a KPC-positive Klebsiella pneumoniae strain was analyzed by high-throughput sequencing.Methods 1.From 2010 to 2013,59 strains of multidrug-resistant Gram-negative bacilli was collected from the First Affiliated Hospital of Guangzhou Medical University.All strains were identified and tested by VITEK2 microbiological automatic identification.The one that is resistence to at least one carbapenem antibiotic is considered as the suspected carbapenemase-producing strain after the screening for three or more antimicrobial resistance of Gram-negative bacilli strains.Its epidemiological characteristics and other data would be analyzed.2.The carbapenem-producing strains were screened by Carba NP test,and the related genotypes of the positive strains were tested by multiplex PCR.The main genotypes of carbapenemase were studied and the advantages of Carba NP test for the detection of carbapenemase were explored.3.Four strains of Klebsiella pneumoniae conjugates or transformants which were carrying the drug resistance bla NDM-1 or bla KPC-2,were obtained via conjugation or electroporation experiments The extraction of plasmid DNA was performed and it was analyzded via digestion technology.4.The total DNA of multiple resistant strains LJ1,LJ2,LJ3 and LJ4 were extracted and the homology of plasmids were analyzed by ERIC-PCR.Seven housekeeper genes of Klebsiella pneumoniae were detected by PCR,and the MLST online tools ST typing was performed to analyze the homology of these strains.5.Extract the total DNA of LJ4 and perform high-throughput sequencing with Illumina Miseq and Pac Bio RS II platforms.Assembled by Celera Assembler software.In its PBc R pipeline,error correction is performed using the data of Illumina Mi Seq Reads.RAST online tools for gene annotation,Res Finder and NCBI BLAST online tools for drug resistance gene analysis,NCBI BLAST online tools for resistance gene genetic analysis,Plasmid Finder online tools for plasmid sequence analysis.Results 1.The epidemiological and antimicrobial susceptibility of 59 strains of multidrug-resistant Gram-negative bacilli were analyzed.The resistant rates of 59 strains of multidrug-resistant Gram-negative bacilli to imipenem,meloveram and urazine were 62.71%,61.02% and 64.41% respectively.The strains were mainly derived from sputum 35.6%(21/59)bile 5.1%(3/59),secretions 8.5%(5/59),ascites 5.1%(3/59),urine 13.6%(8/59),pleural effusion 3.4%(2/59),blood 23.7%(14/59),drainage fluid 5.1%(3/59).The main gene types of multiple resistant Gram-negative bacilli were NDM-1 gene and KPC-2,and the bacteria that produce carbapenemase were more common with Citrobacter freundii,Pseudomonas aeruginosa and Klebsiella pneumoniae.2.A total of 33 carbapenemase strains were identified by Carba NP test,12 strains of class A enzyme and 21 strains of class B were detected via Carba NP test and multiplex PCR test for comparative analysis.Various carbapenemase gene were detected by PCR,including KPC(12 strains),IMP(7 Strain),NDM(12 strains),VIM(strain 3).Compared with PCR,the sensitivity and specificity of Carba NP test were 97.06% and 100%,respectively.3.Three Klebsiella pneumoniae strains carrying bla NDM-1 obtained conjugants successfully by conjugation experiment,though a Klebsiella pneumoniae strain carrying bla KPC-2 was unsuccessful via this method.It was obtained the transformants by electric conversion finally.Four strains were analyzed by S1 endonuclease.The results showed there were three plasmid types.4.LJ1,LJ2,LJ3 and LJ4 were homology analysed by ERIC-PCR.LJ1 and LJ2 were highly homologous but showed different bands with LJ3 and LJ4.LJ3 and LJ4 were not found in any of the 18 incompatibity groups,while LJ1,LJ2 were found to belong to Inc FII plasmid incompatibity group.The results of MLST showed that LJ1 and LJ2 were new type of ST1416,LJ3 was ST20 and LJ4 was ST11.5.A carbapenem-producing strain LJ4 was selected by PCR.The strain was confirmed to be carrying the bla KPC-2 gene and subjected to high throughput sequencing.The plasmid p CT-KPC from LJ4 was a 151466 bp cyclic molecules,including the 120783 bp plasmid backbone,which had coding,replication initiation,transfer,maintenance and stabilization functions as well as inclusion of two resistant gene coding regions(MRR).Plasmid p CT-KPC had a GC content of 53.81%,a total of 233 identified ORFs,and 138 coding genes were highly similar to known functional proteins.Plasmid p CT-KPC was a chimera consisting of p HN7A8,p KPC-LK30 partial plasmids and an additional tra region,the junctional transition gene cluster tracer was associated with bacterial transfer and conjugation function.6.The study of plasmid structure showed that plasmid p CT-KPC carried bla CTX-M-65,blafos A3,blarmt B,bla SHV,bla TEM1 b and bla KPC-2 resistant genes.The Fos A gene was widely spread between poultry and pets,the results of this study showed that the pathogen plasmid isolated from the patient is highly homologous to the plasmid structure of the pet-derived strain.Moreover,the possibility of homologous recombination between pets and poultry and human-borne bacteria was not excluded.Conclusions 1.In our hospital,carbapenemase-producing bacteria mainly are Citrobacter freundii,Pseudomonas aeruginosa and Klebsiella pneumoniae,suggesting that if the detection of such multiple drug-resistant strains is positive,we should pay attention to the screening of the carbapenem enzyme they produced for the rational use of antibiotics.2.Carba NP test for carbapenemase-producing multi-resistant Gram-negative is simple,easy,fast and accurate,that it can be used in clinical laboratories.3.The plasmid that carry ESBLs,fos A,rmt B and KPC genes are of great clinical importance.The bla NDM-1 and bla KPC-2 genes carried by the plasmid can be propagated among the same bacteria by means of conjugation or transformation.These multidrug resistance genes may spread horizontally between bacteria.4.High-throughput sequencing is an effective,accurate and comprehensive method for gene environment analysis and bacterial classification to facilitate the study of bacterial resistance mechanism.