Study On Antimicrobial Resistance And Genetic Polymorphism Of Acinetobacter Spp. And Molecular Detection Of NDM Producers In China

Acinetobacter spp. is non-fermentation bacteria and one of the opportunistic causing nosocomial infection pathogens. It often causes infection when the body immunity is reduced. Acinetobacter baumannii have strong vitality and widely spread in the hospital environment. In recent years, with the abuse of broad-spectrum antibiotics, the multidrug resistant(MDR) Acinetobacter baumannii isolates emerged, which often cause outbreaks of nosocomial infections and have brought great difficulties to the clinical treatment. Especially the emergence and prevalence of MDR strains carrying NDM-1 have caused great concern in the global fight against antibiotics abuse and the problem of bacterial resistance.This study bases on military infectious disease pathogen monitoring platform,from 2010 to 2014, 300 strains of suspected Acinetobacter spp. has been collected from clinical patients and hospital environment in different regions of our country such as Beijing, Shenyang, Suzhou, Gansu, Xiamen, Nanjing, etc., and detected by16 S r RNA sequencing technology, and 292 strains are identified as Acinetobacter spp., among which 251 are Acinetobacter baumannii, 19 are A. calcoaceticus, 19 are A. junii, and 3 are A. lwoffi; 117 are separated from Suzhou, 117 from Shenyang, 33 from Gansu, 12 from Beijing, 2 from Jinan, 3 from Xiamen, 4 from Guangzhou and 4from Nanjing.In order to understand the drug-resistance characteristics of Acinetobacter spp. in our country, antibiotic susceptibility testing was performed to the 226 strains of bacteria. The drug sensitivity result shows that Acinetobacter spp.from Beijing, Shenyang and Suzhou generate resistance in different degree to 8 kinds of clinically preferred treatment for A. baumannii. Resistant rate from high to low in turns are ceftazidime 96%, followed by norfloxacin and cefepime of 92%, imipenem of 88%, cefoperazone of 87%, piperacillin of 84%, ticarcillin/clavulanic acid of 83%and amikacin of 77%. In addition, there are 211 strains of multidrug-resistant(MDR)Acinetobacter spp., which accounts for 97% of the total tested amount, 7 strains are from Beijing accounting for 3% of the total amount, 94 strains are from Shenyang accounting for 45% of the total amount, and 110 strains are from Suzhou accounting for 52% of the total amount. We identified 150 strains of MDR Acinetobacter spp.,which are resistant to more than 5 kinds of antibiotics, among which 57 are from Shenyang and 93 are from Suzhou. The results above show that, the prevalent Acinetobacter spp. in hospital of our country at present experience serious drug resistance. Also notice that the drug-resistant Acinetobacter spp. has a high sensitivity on lactamase inhibitors and aminoglycosides so far. These two kinds of drugs could be clinically considered as the treatment for the infections caused by Acinetobacter spp., and drugs which have had resistance should be considered to suspend in clinical use, so as not to cause more serious resistant consequences.In order to further explore the genetic polymorphism and genetic relationship of Acinetobacter spp. in different regions, the study does molecular typing analysis to the 292 strains of Acinetobacter spp. by using the method of pulsed field gel electrophoresis(PFGE), which shows good discrimination, resolution and repeatability, and could accurately reflects the correlation of pathogens’ epidemiology.Clustering analysis to the 267 strains of electrophoresis-succeed Acinetobacter spp. is made by using the Bio Numeries software; they can be divided into 52 cluster groups according to a cutoff value of 63%, with a total number of 170 kinds of PFGE patterns; the similarity is between 25% and 100%, which indicates the high genetic polymorphism of Acinetobacter spp. in our country, and there are many different patterns of Acinetobacter spp. prevalent in China. Same PEGE banding pattern is found in one strain of Shenyang and one strain of Gansu. Another set of data shows that 7 strains of bacteria, including 2 strains from Shenyang, 4 strains from Gansu and1 strain from Suzhou, have exactly the same PFGE banding pattern. Thus it can be seen that the same PFGE banding pattern in different regions may probably be caused by the spreading of a same clone Acinetobacter spp. in different regions.Experimental results show that the Acinetobacter spp. epidemic in the same department of hospital has the same banding pattern. The PFGE banding patterns of 9strains were found the same in the ICU of hospitals in Gansu, and the Acinetobacter spp. with the same PFGE banding pattern was found in different wards of those hospitals. The PFGE banding patterns of 9 strains from ICU in Gansu were found the same to the PFGE banding patterns of the separated two stains of A. baumannii of respiratory medicine. It indicates that the Acinetobacter spp. can be epidemic and transmitted in different wards by way of cross infection in hospitals.At present, the drug-resistance bacteria producing NDM have been disseminated all over the world, and become the “superbug bacteria”, which seriously threaten the public health. In order to know well the epidemic status of the NDM bacterial strains produced by Acinetobacter spp. in China, this research carried out bla NDMgene screening by way of PCR amplification on 572 strains of Acinetobacter spp. collected from Xiamen, Beijing, Guangzhou, Nanjing, Jinan, Shenyang, Suzhou, Gansu during2010 to 2014. The result found that 45 strains are bla NDM positive, which occupies 8%of the total number. Among the 45 strains, 42 were NDM-1 producing bacterial strains.The positive rate of the produced NDM-1 in Beijing is the highest, among the foresaid Acinetobacter spp. producing NDM-1, 7 strains are separated from hospital wastewater. In addition, 1 strain of bacteria in Suzhou was tested as NDM-5 positive.In the former reports, NDM-5 is mainly found in Escherichia coli, while seldom NDM-5 is found in A. baumannii. It is especially noteworthy that this experiment also found 2 strains of A. baumannii that may carry new-type bla NDM sequence, and they are respectively from Xiamen and Beijing. Through sequence alignment, it is found that a point mutation occurred, resulting in the amino acid changes from Gln97(CAG)into Pro97(CCG). From the PFGE clustering figure, we can see that these two strains have differences, so their sequences still need further verification and determination.Selecting 23 strains of bacteria to position the bla NDM gene through S1nuclease-PFGE-Southern blot hybridization, it is found that the bla NDM-1 genes of 12 strains of bacteria exist in the plasmid with the same size, about 30-50 kb. In addition,this research also carried out the complete genome sequence analysis for the first acinetobacter producing NDM-1, XM1570 isolated from a patient from Xiamen.Phylogenetic analysis showed that the isolate XM1570 was closely related to A.calcoaceticus strain PHEA-2. The bla NDM-1 gene is located on plasmid p XM1, and lies in the complex transposon Tn125 which contains two insertion sequences ISAba125, respectively, and lies in the upstream and downstream of bla NDM-1 gene.These two insertion sequences have the difference of two base pairs. Tn125 often coexists on the plasmid with aph A6, and locates in the downstream of aph A6 gene.The plasmid sequence has a high level of similarity with the plasmid sequence carrying bla NDM-1 gene reported at home and abroad currently.From the study and analysis we can get the following significant results:1. The detection of drug sensitivity is very important to guide the clinical medication, making reasonable and effective treatment scheme;2. The rational usage of antibiotics can reduce the infection of MDR Acinetobacter evidently;3. The application of PFGE may be helpful to find the genetic relationship between strains, to find the source of infection in time for its control, and to prevent nosocomial infections caused by A. baumannii;4. High detection rate of A. baumannii with NDM-1 strains in the hospital sewage in Beijing remind government paying attention to the reasonable disposal of sewage;5. A novel bla NDM gene was identified, provisionally designated as bla NDM-15.Emergence of several types of bla NDM hints variations happening in our country, and more attention should be paid.6. The bla NDM-1 gene often locates in bacteria’ plasmids which is prevelent in China; For example, the bla NDM-1 gene from the first reported NDM-1 producing Acinetobacter strain was located in the plasmid with high probability to transfer. The gene bla NDM-1 is likely to spread to other pathogenic bacteria by conjugation, and it is important to strengthen the monitoring of NDM positive bacteria in clinical.