Determination of incompatibility group plasmids and copy number of the bla NDM-1 gene in carbapenem-resistant Klebsiella pneumoniae strains recovered from different hospitals in Kerman, Iran

Abstract

Introduction. New Delhi metallo-β-lactamase (NDM)-producing Klebsiella pneumoniae has become a serious global health concern. Hypothesis/Gap Statement. Due to the high genetic diversity among NDM-positive K. pneumoniae, we need further surveillance and studies to better understand the relationships between them. In addition, the coexistence of several plasmid replicon types in NDM-positive K. pneumoniae may affect the copy number of bla NDM, the MIC level to antibiotics, as well as increasing the chance of horizontal gene transfer. Aim. The aim of this study was to determine incompatible plasmid groups and copy numbers of bla NDM, and to investigate the genetic relationship of 37 NDM-positive K. pneumoniae in Kerman, Iran. Methodology. The bla NDM-1 gene was detected and confirmed by PCR-sequencing. The plasmid replicon types were determined by PCR-based replicon typing (PBRT) and the copy number of bla NDM-1 was determined by quantitaive real time-PCR (qPCR). Random amplified polymorphic DNA (RAPD)-PCR typing was used to detect genetic relationships between the strains. Results. In this study, 10 different replicon types, including Frep [n=25 (67.5 %)], FIIAs [n=11 (29.7 %)], FIA [n=5 (13.5 %)], FIB [n=3 (8.1 %)], I1-Iγ [n=2 (5.4 %)], L/M [n=7 (18.9 %)], A/C [n=7 (18.9 %)], Y [n=3 (8.1 %)], P [n=1 (2.7 %)] and FIC [n=1 (2.7 %)] were reported. The copy numbers of the bla NDM-1 gene varied from 30.00 to 5.0×106 and no statistically significant correlation was observed between a rise of the MIC to imipenem and the copy numbers of bla NDM-1 (P>0.05). According to RAPD typing results, 35 strains were divided into five clusters, while two strains were non-typeable. Conclusion. The spread of NDM-1-producing K. pneumoniae strains that carry several plasmid replicon types increases the chance of horizontal transfer of antibiotic resistance genes in hospital settings. In this study, 10 different replicon types were identified. We could not find any relationship between the increase of MIC levels to imipenem and the copy numbers of bla NDM-1. Therefore, due to the identification of different replicon types in this study, the type and genetic characteristics of bla NDM-1-carrying plasmids, and other factors such as antibiotic selective pressure, probably affect the copy number of bla NDM-1 and change the MIC level to imipenem.

Carbapenem antibiotic stress increases blaKPC‐2 gene relative copy number and bacterial resistance levels of Klebsiella pneumoniae

“…For the bla KPC‐2 and cpsG standard curves, the mean correlation coefficients ( R 2 ) were 0.997 and 0.996, respectively, suggesting a good fit between the experimental data and the fitted function, and the absolute copy numbers of bla KPC‐2 and cpsG in each KPC‐2‐positive clinical specimen were calculated from the C T values based on the corresponding standard curves. When performing quantitative PCR assays, especially when testing bacteria, 22,23,30,31 since bacterial genomic DNA is single copy, as long as the copy number of a conserved gene in bacterial genomic DNA can be determined, the number of other genes in bacteria can eventually be quantified relative to each other. Because qPCR has the advantages of simplicity, rapidity and accuracy, this experiment strictly follows the quantitative PCR‐related copy number detection method, and the shortcoming is the lack of validation of WGS, digital PCR, classical qPCR and other detection methods, which can be used as a reference for the relative copy number detection of bla KPC‐2 gene.…”

“…Quantitative PCR 19 is an important and widely used analytical tool. Moreover, more papers have reported qPCR methods to detect the relative copy number of bacteria, plants, and even human cells 20–23 …”

“…Moreover, more papers have reported qPCR methods to detect the relative copy number of bacteria, plants, and even human cells. [20][21][22][23] F I G U R E 2 MLST analysis results of 37 carbapenem-resistant Klebsiella pneumoniae strains…”

The emergence of carbapenem-resistance and New Delhi metallo-β-lactamase-1 (bla NDM-1 ) among Salmonella spp. in Kerman, Iran

“…The sequence, annealing temperature, and PCR product sizes of beta-lactamase genes are presented in Table 1 . Klebsiella pneumoniae and Pseudomonas aeruginosa strains harboring some ESBLs, AmpC, and MBLs genes that isolated in our previous studies were used as positive controls in PCR ( 19 – 21 ). PCR reactions in sections 4.2 and 5.2 were done using Taq DNA Polymerase 2× Master Mix RED (Ampliqon, Co, Denmark) according to manufacturer’s protocol.…”

“…The PBRT method is performed with 18 pairs of primers sequence recognizing L/M, N, P, W, T, A/C, K, B/O, X, Y, F, FIA, FIB, FIC, HI1, HI2, I1-Iγ, and FIIA in five multiplex and three simplex-PCR reactions. Klebsiella pneumoniae strains harboring some plasmid replicon types were used as positive controls in PBRT method, these bacteria were identified in our previous studies ( 21 ).…”