Dissemination of incompatible plasmid groups among Klebsiella pneumoniae superbug strains harboring New Delhi metallo-lactamase-1 gene, Kerman, Iran

Introduction and objectives: New Delhi metallo-lactamase NDM – producing K. pneumoniae (CRKP) strains defined as superbugs. So far, twenty-one variants of blaNDM gene have been described. The blaNDM-1 gene, is on plasmid incompatibility (Inc) groups including IncX, IncH, IncFII, IncL/M, IncN, IncR, and IncHIB -M/FIB-M. Materials and Methods: this study was done on 37 clinical sample of non-duplicative NDM-producing K. pneumoniae strain. The bacterial isolates analyzed by PCR-sequencing and BioEdit 7.0.0, Lasergene 6 and MEGA 7.0 saftwares. After determination of of blaNDM variants PCR-based replicon typing (PBRT) methods (5 multiplex PCR and 3 simplex PCRs) used for further evaluation of 18 distinct replicons including incompatible plasmid of FIA, FIB, FIC, HI1, HI2, I1-Ig, L/M, N, P, W, T, A/C, K, B/O, X, Y, F and FIIA. Randomly Amplified Polymorphic DNA (RAPD) typing used for Strains clustering and molecular typing Results: all 37 NDM-carrying strains had variant of blaNDM-1 gene which were on the conjugative plasmid. The frequency of plasmids were as ,FrepB (n=25[67.5%]), FIIA(s) (n=11[29.7%]), FIA (n=5[13.5%]) ,FIB(n=3[8.1%]) and for, I1-Ig, L/M, A/C , Y ,P and FIC plasmids reported as (n=2[5.4%]), (n=7[18.9%]), (n=7[18.9%]) ,(n=3[8.1%]),(n=1[2.7%]) and (n=1[2.7%]) respectively. In this study a strains was found that simultaneously carried I1, L/M, Y ,FIC, A/C and FIIS plasmids . Based on typing by RAPD method, our strains were divided into clusters 1 to 5. According to phylogenetic tree construction six strains belonged to cluster 1, five strains to cluster 2, eleven strain to clusters 3, six strains to cluster 4 and finally six strains to cluster 5 Conclusion: The success of the blaNDM-1 gene can not be attributed to a particular plasmid and a specific genus of bacteria. The results obtained from this is and other similar studies show that blaNDM -1 gene located on a wide range of conjugative incompatible plasmid group that leading to quick spread of blaNDM-1 gene among Enterobacteriaceae and other genus such as Pseudomonas and Acinetobacter hence considering the fast rise of superbugs and the danger they pose to human health worldwide, it is required to precisely monitor the molecular epidemiology of the blaNDM-1 gene as a powerful tool in superbagging, to make better counteractions and prevent their spread.

Keywords:

blaNDM , incompatible plasmid , superbugs , PBRT , RAPD-PCR