Study On The Prevalence And Drug Resistance Of NDM-Producing Bacteria In Some Areas Of China

Since reported by Lancet(the top international magazine in the field of infectious disease)in 2010,New Delhi metal β-lactamase(NDM)-producing bacteria have caused widespread concerns for its astonishing transmission speed and superior drug-resistance.At present,there are more than 20 countries have discovered NDM resistant bacteria worldwide.Except some individual countries in western part of South America and central Africa,other countries and regions have all been reported.The strains carrying the blaNDM gene are resistant to common antibiotics,including carbapenems,and there is no definite treatment.The researchers have found that NDM-1 has 15 mutants from NDM-2 to NDM-16,which are mostly carried by Acinetobacter,Escherichia coli and Klebsiella pneumoniae.Accumulating data indicates that NDM has spread globally causing various types of infections,which constitutes one of the major public health problems.Therefore,the effective control and treatment of NDM resistant bacterial infection has practical significance.Bacterial drug-resistant monitoring is an important component of the prevention and control of NDM-positive bacteria.To identify the prevalence and characteristics of NDM-positive bacteria in real time plays a key role in the control and treatment of NDM producers.Previous studies have reported that the domestic NDM producers are mostly NDM-1 positive Acinetobacter in China.However,little information is available regarding the dissemination of the other types of NDM and other genus in China.In order to better understand the prevalent situation,prevalent trend and transmission mechanism of blaNDM gene in China,1559 strains of non-repetitive carbapenem-resistant bacteria(including 156 hospital sewage sources)from 11 provinces and 25 hospitals in China from 2010 to 2015 were screened for blaNDM gene in this study.The epidemiology and statistical analysis were used to analyze the three distributions of infectious diseases caused by different sources,different genera and different types of NDM producers.The whole genome average nucleotide coincidence(ANI)was used to identify the strains of NDM producers;The genomic multi-site sequence typing(cgMLST)was used to cluster the NDM producers from different strains;VITEK 2 automatic microbial susceptibility analyzer was used to analyze the drug resistance of the blaNDM positive strains;meanwhile,Southern blot hybridization,membrane ligation and bacterial whole genome sequencing were used to investigate the blaNDM gene mapping and transfer characteristics and blaNDM gene surrounding structure in order to identify the molecular characteristics of NDM producers in different regions of China,as well as to explain the possible molecular propagation and prevalence of blaNDM gene.The main experimental results and conclusions of this study are as follows:1.Different types and different strains of NDM positive bacteria were prevalent in some areas of China.A total of 77 blaNDM genes were screened out in 1559 strains of bacteria,the positive rate was 4.9%.1)Temporal distribution characteristics: The positive rate of blaNDM gene in patients with drug-resistant bacteria increased year by year.2)Spatial distribution characteristics: In the northeast,north,east and south of China,NDM positive bacteria distributed in 11 cities.Among them,Beijing,Shanghai,Haikou and Xiamen had more positive strains and higher rate.Besides NDM resistant bacteria are widely found in hospital sewage.3)Population distribution and specimen source distribution characteristics: The number of NDM-positive bacteria caused infection in male was significantly more than that in female,while in children younger than 10 years old and those older than 60 years were significantly more than in those of other age groups.These are indicating that male and elderly patients are susceptible population of NDM-positive bacteria.The fact that blaNDM gene positive bacteria were most frequently detected in respiratory medicine and sputum specimens is suggesting that compared to other modes of transmission,NDM producers are more susceptible to respiratory infections and spread.4)Genotype characteristics: In this study,four genotypes of blaNDM are found,including blaNDM-1,blaNDM-3,blaNDM-5 and blaNDM-13.NDM-5 is the second dominant type,which is found in Beijing,Shenyang,Haikou,Jinan,Shanghai and other cities,suggesting that carrying blaNDM-5 gene strains have also been widely spread in China.5)Genus Category Features: In the case of bacterium,Acinetobacter occupies the top position based on its quantity,followed by Klebsiella pneumoniae and Escherichia coli.In recent years,NDM-positive pneumoniae and NDM-producing Escherichia coli have been prevalent in some parts of China.A NDM-3-positive Escherichia coli and a strain carrying blaNDM-13 gene Salmonella are first identified.And the NDM-1,NDM-3,NDM-5,NDM-13 positive bacteria have spread to intestinal pathogens.2.ANI can successfully identify Acinetobacters species.For the first time,ANI was used to identify the NDM producers.Among the 38 blaNDM positive-strains,5 strains of Acinetobacter baumannii are misidentified as other bacteria by 16 S rRNA,among which,3 are identified as Acinetobacter baumannii,1 strain as Acinetobacter junii and 1 strain as Acinetobacter johnsonii.3.CgMLST is important to study the genetic relationship among NDM-positive strains.CgMLST cluster analysis found that NDM positive pneumoniae Klebsiella pneumoniae and Escherichia coli may have been propagated in populations of different regions.Moreover,NDM-positive Acinetobacter towneri and Acinetobacter calcoaceticus have been spread in different regions and sources.In this study,we established the cgMLST to investigate the genetic relationship of NDM-positive bacteria,which provided a solid foundation for the establishment of NDM producers monitoring platform and molecular typing database.4.NDM-positive bacteria in different regions of China have broad drug resistance and the drug resistance of different types and different strains is different.1)NDM-positive bacteria are multi-drug resistant bacteria,mostly pan-resistant bacteria with wide drug resistance.77 strains of NDM resistant bacteria are containing more than three kinds of antibiotics resistance,as multi-drug resistant bacteria.The resistance rate of all strains to ampicillin,cefazolin and ceftriaxone was as high as 100%,and the resistance rate to cefotaxime and cefepime was 98.7% and to piperacillin was also high as 93.5%,to aztreonam as high as 97.4%;All strains showed high levels of resistance to carbapenems imipenem and meropenem,nitrofurans nitrofurantoin,sulfonamides sulfamethoxazole.The bacteria react more sensitive to aminoglycoside amikacin,with a resistance rate is of 24.7%.2)Drug resistant rates of NDM-5 positive strains were higher than those of NDM-1 positive strains.Comparing different types of NDM-positive bacteria,the drug resistance rate of NDM-5 positive strain was significantly higher than that of NDM-1 strain.3)The drug resistance of different NDM-positive strains is different.Comparing different strains of NDM-positive bacteria,we found that the resistance rate of Acinetobacter bacteria to piperacillin is significantly lower than that of other bacteria.Salmonella bacteria are sensitive to amikacin,ciprofloxacin and levofloxacin.Citrobacter bacteria are only sensitive to tetracycline;Escherichia bacteria are only resistant to amikacin.These results are indicating that the appropriate adjustment of antibiotics and rational application are particularly important for the treatment and prevention of NDM-positive bacteria infection.5.Compared to Acinetobacter,the bla NDM gene carrying plasmids are easily spreading in Enterobacteriaceae,and have higher drug resistant rates.1)The blaNDM gene passes through the plasmid in the bacterial genus and within the bacterial genus: The results showed that all the strains contained 2-5 plasmids,67 strains of blaNDM gene were mapped to the plasmid,and 10 strains of blaNDM gene were located on the chromosome.The largest number of plasmid strains were located in the 30~50kb size,covering multiple strains,and there may be a dominant plasmid between the spread in Enterobacteriaceae and Acinetobacter.11 strains of two plasmids are containing both blaNDM gene,and the drug resistance compared to other NDM resistant bacteria was wider.This is suggesting that multiple drug-resistant gene plasmid may enhance the resistance of the strain.Resistant gene located on the chromosome in NDM-positive bacteria are mostly Acinetobacter.2)The blaNDM gene of Enterobacteriaceae is more prone to horizontal transfer than Acinetobacter: In a transferring experiment of 67 NDM positive strains with blaNDM gene located in the plasmid,50 strains were successively transferred the plasmid with blaNDM gene to the recipient strain J53.Among the strains without conjugating transfer,16 strains were Acinetobacter,and 1 strain was Klebsiella pneumoniae.3)The drug resistance activity of blaNDM-positive combination Enterobacteriaceae is stronger than that of Acinetobacter: Evaluation of the resistance activity of blaNDM-1-positive conjugate bacteria found that all Enterobacteriaceae strains had typical resistance to the original donor bacteria and showed resistance to penicillins,cephalosporins and carbapenems resistant.Most of the Acinetobacter containing blaNDM-1 gene adapter,with the access to the plasmid,did not get the corresponding carbapenem antibiotic resistance phenotype.They only performed a resistance against cephalosporins,and showed none of the original donor bacteria typical resistance.6.The like Tn125 transposable structure plays an important role in the blaNDM gene transmission process,and there is an outbreak risk of NDM positive bacteria in China.1)The bla NDM gene is transmitted through the like Tn125 transposable structure in the bacterial genus and within the bacterial genus: The 17 different bacterial genus and different positions of the blaNDM-1 gene(plasmids and chromosomes)were selected in order to analyze,the whole structure of the blaNDM-1 gene by the whole genome sequencing of the strain.17 strains of NDM positive strains were divided into seven different types of like Tn125 transposable structures,which were different from the complete Tn125 structure of pNDM-BJ01.2)ISAba125 transposase is not a necessary enzyme for blaNDM gene transmission: For the first time,we found there were two strains lost in the upstream and downstream ISAba125 transposonases at the same time,which were Acinetobacter towneri and Acinetobacter lwoffii respectively.Previous reports have said that the ISAba125 transposase was important during the transmission of blaNDM-1 gene,but we found it lost.Consequently,we concluded that ISAba125 transposase was not a necessary enzyme for blaNDM gene transmission.3)The blaNDM-1 gene was first inserted from Acinetobacter spp.in China: In combination of the above studies and related reports,as well as the phenomenon that the bacteria which blaNDM-1 gene ss located in the chromosome are Acinetobacter mostly,the blaNDM-1 gene is the first to be derived from Acinetobacter and it is mostly chimeric in the chromosome of the pathogen of Acinetobacter.When the blaNDM-1 gene was transferred to Acinetobacter through the Tn125 transposon structure.it was transferring to Klebsiella,Escherichia,Salmonella,Enterobacteriaceae at the same time.4)There is a risk of serious public health problem caused by NDM producers in our country.Combining the transfer and binding drug resistance experiments,we found that the blaNDM-1 gene of Acinetobacter,compared to Enterobacteriaceae,was not easily transferred through the genetic level to other strains.This may also be the reason that the serious public health problem cased by NDM positive bacteria has not been emerged in China yet.However,the blaNDM-1 gene of Enterobacteriaceae is mostly located on the plasmid and is resistant to carbapenems after joint transfer.With the production of NDM positive Enterobacteria increased every year,there is an risk of serious public health problem cased by NDM positive bacteria in our country,and blaNDM gene transmission process was requiring key regulatory factors for in-depth research.In summary,this study through the bacterial resistance monitoring research has obtained the incidence of NDM positive bacteria infection and drug resistance,and discovered that different types,strains of NDM positive bacteria was prevalent in some areas of China.The drug resistant of NDM-positive bacteria is broad in some areas China,and the drug resistant of different types,different bacterial genus are different.These results provide important experimental basis for the clinical treatment.And the scientific and effective prevention and control of NDM-positive bacteria caused severe infection.Furthermore,the genetic polymorphism,gene mapping and transfer characteristics and blaNDM gene peripheral structure of NDM-positive bacteria in some areas of China were studied.The blaNDM-1 gene was first derived from Acinetobacter in China.During the Spread within the bacterial genus,it had been transferred to Klebsiella,Escherichia,Salmonella and other Enterobacteriaceae bacteria.blaNDM gene in NDM positive Enterobacteriaceae were more prone to gene transfer and have the typical carbapenem antibiotics drug resistance as the donor bacteria after the transfer.With the production of NDM-positive Enterobacteria increased every year,there is a risk of serious public health problem cased by NDM producers in our country.Hence,improving awareness of the detection monitoring and infection control of NDM positive bacteria,strengthening epidemiological surveillance of the Chinese-made NDM positive bacteria,and vigilanting its epidemic changes,virulence and changes of drug resistant spectrum,are essential to prevent and control the spread of these organisms in China and even in the whole world.