Risk Factors And Surrounding Genetic Determinants For The Acquirement Of NDM-1 Plasmid In Carbapenem-resistant Bacteriaceae

Background and Objectives:Regional dissemination mainly contributes to the widespread prevalence of plasmid encoding NDM-1 enzyme?New Delhi metallo-?-lactamase1?;however,the risk factors and surrounding genetic determinants related to the regional prevalence of NDM-1 remain unclear.In this study,we collected and screened NDM-1 enzyme producing strains in Carbapenem-resistant bacteria in the First Affiliated Hospital of Nanchang University.Based upon the epidemic situation of bla_NDM-1 gene,we assessed the clinical risk factors related to the prevalence of NDM-1 producing bacteria strains as well as the prognosis of infected patients.Using the whole genome sequencing and bioinformatics approaches,we systemically analyzed the epidemic status of NDM-1 gene and its potential risk factors as well as the outcomes of infected patients.We also examined the surrounding genetic determinants of NDM-1 plasmid and compared our results with the findings of national and international studies.Our study reveals the prevalence,predisposing factors,and evolutionary history of NDM-1 gene;and also provides experimental evidence for the development of scientific and effective measures for preventing and controlling the prevelance of NDM-1 containing bacteria.Methods:1.We screened Carbapenem-resistant Gram-negative bacteria from specimen recently collected at our hospital?Repetitive strains from the same patient or sample were excluded?.Polymerase Chain Reaction?PCR?was used to identify the bla_NDM-1 positive bacteria.The mixed bla_NDM-1 positive bacteria were cultured and the bla_NDM-1 gene was further screened in bacterial suspension.Due to the complexity of isolated bacteria,we identified the Gram positive and Gram negative bacteria using BIOLOG automatic system,amplified and sequenced the 16 s rRNA to further verify bla_NDM-1 positive bacterial strains.The sensitivity to antibiotics,the MIC values,and the phenotypes of metal enzyme of bla_NDM-1 positive bacteria were detected by E-test method.Based upon these findings in combination with clinical data,we characterized the drug resistant bacterial strains containing bla_NDM-1.2.Using case-control study method,patients were grouped into 3 groups by a 1:1:1 pairing ratio.Group A included the patients who were infected with NDM-1 positive bacteria;group B were cases infected with Carbapenem insensitive bacteria?NDM-1 negative?;and group C were cases infected with Carbapenem sensitive bacteria.We employed pre-designed questionnaires to retrospectively collect medical information from hospital records,including inpatient department,name,gender,age,duration of hospitalization,existing diseases,risk factors,and clinical outcomes.We then performed univariate and multivariate Logistic analyses to identify the potential risk factors for the acquirement of NDM-1 plasmid in Carbapenem resistant bacteria.3.Plasmid conjugation test was used to analyze the hereditary stability of bla_NDM-1 gene.Southern blot was utilized to characterize bla_NDM-1 gene location in the bacterial strains stably inherited bla_NDM-1.Five NDM-1 plasmids were sequenced.The sequences of these plasmids were used to contruct genome sequencing library and to perform splicing,comparison,plasmid gap-filling,verification of differentially expressed sequences as well as annotation of serial gene of the plasmid.We then blasted the plasmid sequences in NCBI and identified differentially expressed locus by Manve software.Results:1.We found 1735 strains of carbapenem-insensitive bacteria,among which 54 strains were bla_NDM-1 positive,including 44 Klebsiella pneumoniae strains,8 Bauman Acinetobacter strains,and 2 strains of Escherichia coli.Using in vitro drug sensitivity assay,antibiotics resistant rate in bla_NDM-1 positive strains was found to be over 50%,especially in Carbapenem.Results from the modified Hodge test showed that 512 strains had positive effects among 1735 strains of Carbapenem-insensitive bacteria.The clinical data demonstrated that 54 strains with bla_NDM-1 were from 43 patients,who were from department of burn,department of respiration,ICU,and department of infectious diseases.The main sources of specimen were sputum,urine,and blood.2.Specimen of group A and B patients showed very high drug resistant-rate to most antibiotics.In contrast,specimen of group C patients showed low drug resistant-rate.Results also showed that the resistant rates of Imipenem,Amikacin,and Levofloxacin resistant carbon left sub were statistically significant between group A and B.When compare group A and B?Carbapenem resistant?with group C?Carbapenem sensitive?,results from univariate Logistic analysis showed that the significant contributors were as follows:?1?the duration of hospitalization,?2?the use of broad-spectrum antibiotics for over 7 days within two months before isolation of the strains,?3?the cases transferred from other hospitals,?4?the use of antibiotics within 14 days before the isolation of the strains,?5?fever peak??,mean?,?6?the types of antibiotics applied,?7?the use of tigecycline during treatment,?8?recovery rate?%?,?9?treatment failure rate?%?,and?10?against-advice discharge rate?%?.Multivariate Logistic analysis identified?2?and?4?as independent risk factors.Univariate Logistic analysis in group A and B found that the use of penicillin and the time from infection to death were significantly different;and multivariate Logistic analysis showed that the history of Carbapenem use was an independent risk factor for acquired NDM-1 plasmid in Carbapenem resistant strains.3.bla_NDM-1 gene was detected by Southern blot in most plasmids isolated from bla_NDM-1 positive bacterial strains and from e.J53 hybrid plasmid.The hydridation rate between bla_NDM-1 positive strains and e.J53 plasmid was 50%,which suggests that bla_NDM-1 positive plasmids can be transferred efficiently among Enterobacteriaceae bacteria and thus serve as carriers for the spread of antibiotics resistant bacteria.We constructed plasmid genome library and performed sequencing quality control as well as genomic conjunction.Two complete plasmid genome sequences?p12 and p11106?were obstained from 5 verified strains.The similarity plot was constructed in Mauve software based upon plasmid sequences from 7 different sepecies.By the comparison of surrounding genetic features,we found that?1?p11106 and p12 were highly similar to pNDM-BTR,?2?p11106,p12,and the other six plasmids differed in 20-30 kb region containing orf00032-orf00043;?3?NDM-1 gene locates in the middle portion?orf00032-orf00043?flanked by two tnpA genes at its upstream and a tnpA gene?orf00052?at 36 kb region of its downstream.Conclusions:1.The antibiotics-resistant gene bla_NDM-1 became epidemic at a certain level in our hospital and other regional hospitals.For example,in our hospital,Klebsiella pneumoniae were found to be the dominate epidemic strains,suggesting that Klebsiella pneumoniae possibly serving as host bacteria play a crucial role in the spread of bla_NDM-1 gene.2.The use of Carbapenem was an independent risk factor for acquirement of NDM-1 plasmid in Carbapenem resistant strains.3.p11106 and p12 plasmids possibly acquired the features of pNDM-BTR plasmid.The NDM-1 containing plasmids were different at the region of orf00032-orf00043 and showed gene polymorphism.NDM-1 located at the middle portion of orf00032-orf00043 region,flanked by two tnpA genes at its upstream and a tnpA gene?orf00052?at a 36 kb region of its downstream,which suggest that NDM-1 was possibly obtained by transposition mechanism and was perminantly kept in the plasmids.