|Year : 2016 | Volume
| Issue : 3 | Page : 286-292
Endemic Indian clones of Klebsiella pneumoniae-harbouring New Delhi metallo-beta-lactamase-1 on a hybrid plasmid replicon type: A case of changing New Delhi metallo-beta-lactamase plasmid landscapes in India?
GK Subramanian1, PG Soundari1, V Ramanathan2, P Krishnan1
1 Department of Microbiology, Dr. ALM PGIBMS, University of Madras, Chennai, Tamil Nadu, India
2 Nephrology Unit, Billroth Hospitals, Chennai - 600 028, Tamil Nadu, India
|Date of Submission||06-Aug-2015|
|Date of Acceptance||01-Jul-2016|
|Date of Web Publication||12-Aug-2016|
Department of Microbiology, Dr. ALM PGIBMS, University of Madras, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Purpose: blaNDM genes are MBL genes that confer resistance to carbapenems. Globally, they are associated with diverse clones and plasmids. In this study, we characterised three isolates of Klebsiella pneumoniae-harbouring blaNDM1 from patients undergoing chronic haemodialysis and renal transplantation. Materials and Methods: 3 blaNDM1 -producing K. pneumoniae were isolated from end-stage renal disease patients undergoing haemodialysis and renal transplantation from a nephrology unit. All the three isolates were screened for clinically relevant resistant genes. Plasmid replicon content was analysed by polymerase chain reaction based replicon typing. Conjugation assays were done using azide-resistant Escherichia coli J53 as the recipient strain. Multilocus sequence typing and variable number tandem repeat typing were done to find the clonality. Replicon sequence based typing was attempted to find the diversity of replicon-associated sequences in IncHI3 plasmids. Results: All the 3 blaNDM positive isolates possessed the New Delhi metallo-beta-lactamase-1 (NDM-1) allele with an IncHI3 plasmid which was not transferable in one isolate. The isolates were found to be sequence type 14 (ST14; 2 nos) and ST38 both of which were previously reported to be the NDM-producing K. pneumoniae STs prevalent in India. Replicon sequence analysis revealed limited sequence diversity within the repHI3 and repFIB locus. Conclusion: To the best of our knowledge, this is the first report of IncHI3, a newly assigned enterobacterial plasmid incompatibility group from India. This could either be a case of importation or a widely circulating NDM plasmid type in India.
Keywords: Carbapenemase, IncHI3, MBL, New Delhi metallo-beta-lactamase-1
|How to cite this article:|
Subramanian G K, Soundari P G, Ramanathan V, Krishnan P. Endemic Indian clones of Klebsiella pneumoniae-harbouring New Delhi metallo-beta-lactamase-1 on a hybrid plasmid replicon type: A case of changing New Delhi metallo-beta-lactamase plasmid landscapes in India?. Indian J Med Microbiol 2016;34:286-92
|How to cite this URL:|
Subramanian G K, Soundari P G, Ramanathan V, Krishnan P. Endemic Indian clones of Klebsiella pneumoniae-harbouring New Delhi metallo-beta-lactamase-1 on a hybrid plasmid replicon type: A case of changing New Delhi metallo-beta-lactamase plasmid landscapes in India?. Indian J Med Microbiol [serial online] 2016 [cited 2019 Jun 18];34:286-92. Available from: http://www.ijmm.org/text.asp?2016/34/3/286/188314
| ~ Introduction|| |
New Delhi metallo-beta-lactamase (NDM) is an enzyme that hydrolyses carbapenems. It was first reported from Escherichia coli and Klebsiella pneumoniae isolated from a Swedish patient of Indian origin with a travel history to New Delhi at the Interscience Conference on Antimicrobial Agents and Chemotherapy 2008 which was subsequently published in Yong 2009.  Later in March 2010, Deshpande et al.  published a report on NDM prevalence in a tertiary care hospital in Mumbai highlighting its high prevalence. This was followed by a larger report by Kumarasamy et al., in Lancet Infectious Diseases about the molecular characteristics of NDM from India. 
Since then, NDM has been reported from many countries with some reporting the associated clonal types and the plasmid scaffolds.  Initial reports of NDM plasmids suggested Inc A/C to be the main plasmid scaffold involved with NDM carriage in both E. coli and K. pneumoniae. But subsequently, other plasmid scaffolds were also found to harbour the NDM gene with a few untypeable plasmids.  Whole plasmid sequencing of such untypeable plasmids and subsequent addition of primers to the existing replicon typing scheme lead to the inclusion of plasmid types that were considered to be exclusively associated with blaNDM gene. ,
Among the many unique, large and untypeable plasmids reported to harbour NDM gene, the pNDM-MAR plasmid was reported from a K. pneumoniae isolate from a Moroccan patient without any known contact with the Indian subcontinent. Initially, it was classified as a subgroup of the RepHI1 plasmid family with a replicon that is closely related to the IncHI1B. , Later, this particular plasmid was reclassified as IncHI3. Like other IncH plasmids, it also harboured another novel replicon that is related to the RepFIB. 
Studies highlighting the clonality and plasmid replicon types help to trace the evolution of a particular resistant gene.  In the current study, we report three isolates of K. pneumoniae belonging to sequence type 14 (ST 14) and ST 38 with IncHI3 plasmid associated with the NDM 1 gene from a nephrology ward in Chennai, South India.
Since all the reported IncHI3 plasmids are >250 kb plasmids, technical difficulties often affect the plasmid extraction, purification and subsequent restriction digestion for comparison of similar plasmids.  Since the plasmid multilocus sequence typing (pMLST) scheme of IncHI2 plasmids could not be used for this plasmid type, we evaluated a double locus replicon sequence typing as a preliminary typing method.
| ~ Materials and Methods|| |
The study was cleared by the Institutional Ethical Committee of Dr. ALM PGIBMS, University of Madras (IEC No. PGIBMS/CO/Human Ethical/2011-12/546).
The three isolates of K. pneumoniae included in this study were isolated within a time span of 6 months from three different patients in a nephrology ward from patients undergoing chronic haemodialysis (1) and renal transplantation (2) in a tertiary care hospital in Chennai.
Identification and speciation of the above isolates were carried out by conventional biochemical methods, and the results were confirmed by polymerase chain reaction (PCR) amplification of K. pneumoniae specific 16S-23S ITS region according to published method. 
Antibiotic sensitivity testing, minimum inhibitory concentration determination, and detection of carbapenemase production
Antibiotic sensitivity testing was done by Kirby-Bauer disc diffusion and minimum inhibitory concentration (MIC) determination for meropenem was performed using Ezy MIC strips (Hi-Media). Carbapenemase production was confirmed by the disc synergy/potentiation tests and the modified Hodge test.
Polymerase chain reaction amplification and sequence analysis
PCR amplification of various β-lactamase genes including blaNDM , PMQR genes, aminoglycoside modifying enzymes, 16S rRNA methylase genes and class 1 integrase were performed according to published methods. ,,,,,,, Additional PCR methods were employed for amplification and sequencing of the full ORF of blaNDM  gene and class 1 integron gene cassette array  [Table 1].
|Table 1: Primers used for the amplification of antibiotic resistant genes, class1 integrons and genetic context of blaNDM gene |
Click here to view
Genetic context of blaNDM
Genetic context of blaNDM was investigated by PCR mapping with primers targeting the ISAba125 region upstream to blaNDM . Two forward primers were used targeting both the complete and truncated ISAba125 region in combination with the reverse primer targeting the blaNDM gene  [Table 1].
Polymerase chain reaction-based genotyping of Klebsiella pneumoniae isolates
MLST analysis was performed to find out the potential links between the circulating NDM harbouring K. pneumoniae clones and their plasmid replicon profile.  MLST was performed according to the K. pneumoniae MLST website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html).
Variable number tandem repeat (VNTR) typing which is considered to be a surrogate marker for PFGE in case of K. pneumoniae was done according to published method. 
Serotyping by a PCR-based method targeting the virulent serotypes K1, K2, K5, K54, K57 and K20 was performed according to published method.
To find the approximate number and the relative size of the plasmids, the isolates were subjected to plasmid extraction by modified Dolly's method.  One millilitre of the overnight culture was centrifuged at 15,000 g for 10 min, and the pellet was resuspended in 100 μL of alkaline lysis solution-1 followed by 200 μL of alkaline lysis solution-2. The mixture was inverted 5 times for optimal lysis. Three hundred microliters of phenol: chloroform: isoamyl alcohol was added, and the mixture was inverted 100 times and centrifuged at 15,000 g for 10 min. Eight microliters of the aqueous phase was then directly loaded with loading dye onto a 0.8% agarose gel and electrophoresed at 100 V for 2 h. The gel was then stained in ethidium bromide solution for 10 min followed by a procedure of de-staining in distilled water for 10 min. Plasmid profiles were then visualised in a gel documentation system. The alkaline lysis solution-1 and 2 were prepared according to the method of Kado and Liu, 1981.  K. pneumoniae ATCC BAA-2146 that harbours 4 different plasmids  (141 kb, 118 kb, 85 kb and 2 kb) and a laboratory E. coli strain (J53pUnnamed1 with the gene bank accession number-LQWX01000000) with a plasmid of approximately 260 kb was used for approximate estimation of plasmid sizes.
Conjugation studies were done by performing mating-out assay with E. coli J53  as the recipient and the K. pneumoniae clinical isolates as the donor. It was done at three different incubation temperatures (25°C, 30°C, 37°C) to find the optimum temperature for conjugation which might vary for different plasmids and strains. The selection was done on MacConkey agar plates containing 100 μg/mL of sodium azide and 10 μg/mL of cefoxitin.
Polymerase chain reaction based replicon typing
PCR-based replicon typing (PBRT) was done according to published methods.  Updated replicon primers were then used to detect the presence of recently reported novel replicons. , The IncHI3 and IncFIB M like replicons were then sequenced from both strands to check for potential nucleotide variations within the replicon as a precursor for a possible double locus sequence typing of this type of plasmid.
PBRT and detection of resistant genes by PCR was then repeated for the single plasmid transconjugants from the donor isolates to confirm the plasmid backbone harbouring the respective resistant gene. 
Evaluation of a double locus replicon sequence typing as a preliminary method of typing pNDM-MAR-like plasmid replicon types
Due to the high costs of whole plasmid sequence analysis, which is considered as the most accurate method to characterise and compare plasmids, we explored the usefulness of the polymorphism within the replicon sequences, as an alternative method for tracing the outbreak of related plasmids. Further, replicon sequence typing scheme is already available in the pMLST database for IncF plasmids using the FAB formula for assigning allele type and numbers for each replicon. 
PCR amplicons of repHI3-like and repFIB M-like were sequenced from both strands and were assembled using Sequencher software version 5.3, Gene Codes Corporation, USA. The consensus sequence of all 3 isolates was subjected to Blast analysis. 5 Blast hits corresponding to whole plasmid sequences of 5 K. pneumoniae isolates were retrieved from NCBI for sequence comparison. Out of the 5 plasmids, 3 were NDM harbouring plasmids and 1 was a KPC harbouring plasmid. ,, Phylogenetic analysis was done using Molecular Evolutionary Genetics (MEGA) Software, version 6, USA.
The PCR amplicons were sequenced by Xcelris Labs Pvt. Ltd., Ahmedabad. The sequencing chromatograms were analysed and compared with known alleles. The complete ORF of NDM gene for one of the isolates was submitted in GenBank (KJ150691).
| ~ Results|| |
All the three isolates showed resistance to carbapenems which were later confirmed to be NDM producers by phenotypic and genotypic methods. The resistant gene contents are given in [Table 2] and gel pictures are given in [Figure 1].
|Figure 1: (a) L1, L2, L3 showing the presence 621bp blaNDM specific band. (b) L1, L2, L3 showing the presence 317bp arm A specific band. (c) L3 showing the presence of 472 bp blaOXA 48 specific band. (d) L1, L3, L5 showing the presence of 570bp IncHI3 specific band and L2, L4, L6 showing the presence of 440bp FIBM alt specific band|
Click here to view
|Table 2: Molecular characteristics of the study isolates, transconjugants and the reported IncHI3 plasmids |
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Analysis of the immediate upstream genetic context by PCR revealed a product of approximately 2.5 kb in all the three isolates. Since the predicted full-length ISAba125 amplicon is approximately 1.2 kb, additional insertion was suspected and sequencing by primer walking revealed the insertion of ISEc33 within ISAba125.
MLST analysis revealed that 2/3 isolates belonged to ST14 and 1 isolate belonged to ST38. Both the STs were included within the CC292 clonal group. The 2 ST14 isolates had a meropenem MIC of >32 μg/mL out of which 1 was also positive for blaoxa-48like carbapenemase. VNTR typing assigned the three isolates to distinct VNTR types [Table 3] indicating that they may represent distinct lineages within their assigned ST which is evident from the distinct plasmid profile of the three isolates. All the three isolates were negative for all the tested serotypes.
Plasmid characterization by conjugation assay failed to give any transconjugant in case of a single ST14 K. pneumoniae isolate, whereas transconjugants were obtained with the other ST14 K. pneumoniae isolate and the ST38 K. pneumoniae isolate [Figure 2]. The transconjugants were subjected to MIC analysis which is given in [Table 4]. PBRT with the conventional primers were negative, and all the isolates gave positive PCR amplicons for the 2 hybrid replicons reported in the IncHI3 pNDM-MAR-like plasmid. The transconjugants obtained from the 2 K. pneumoniae isolates harboured both the plasmid replicons and the blaNDM1 gene indicating that the NDM gene was harboured by the IncHI3 plasmid.
|Table 4: Minimum Inhibitory Concentration values for the study isolates and their respective transconjugants |
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The results of plasmid profiling and resistant gene contents are given in [Table 2]. Although each of the three isolates harboured multiple plasmid bands, IncHI3 related replicons were the only known replicons detected in these isolates and the transconjugants. The ST38 K. pneumoniae strain harboured multiple small plasmids which could be ColE1 like plasmids that do not possess a basic replicon for classification. All the three isolates harboured a plasmid of approximately >250 kb which was found to be the IncHI3 plasmid with NDM gene in 2 isolates. The ST14 K. pneumoniae isolate that failed to yield a transconjugant harboured a single plasmid.
Nucleotide similarity search by Blastn gave 5 and 8 hits for RepFIB-like and RepHI3-like, respectively, with the reported similarity ranging from 95% to 100%. The five blast hits that possessed both the hybrid replicons were taken for phylogenetic analysis. The in silico phylogenetic comparative sequence analysis revealed the presence of two different alleles at the RepHI3 loci and three different alleles at the repFIB-like loci [Figure 3]. The different features of the five fully sequenced plasmids from NCBI database is given in [Table 2].
|Figure 3: Phylogenetic analysis of the replicon sequences obtained revealed that there are so far three sequence alleles in the FIB locus and two in the HI3 locus indicating limited diversity within the selected replicon loci. 138ST14, 145ST38 and 199ST14 are the isolates included in the study|
Click here to view
| ~ Discussion|| |
NDM carbapenemases are hypothesised to have been originated in the Indian sub-continent. Unlike KPC and the ST258 K. pneumoniae, NDM has been associated with clonally diverse K. pneumoniae. Initially, they were associated predominantly with the Inc A/C plasmids with a few untypeable plasmids including the first clinical report of NDM in 2009 which also belonged to ST14. Since then, the plasmid backbone harbouring the NDM gene, in many instances were found to be genetically plastic leading to a continuous evolution of both the basic replicon and the accessory genes.
The pNDM-MAR-like plasmid replicon type reported in this study was initially reported from a K. pneumoniae isolate from Morocco with no known contact with the Indian sub-continent. The K. pneumoniae isolate belonged to ST15, which is a single locus variant of ST14, and both are grouped under the diverse CC292.
Sequence analysis by homology search revealed 5 blast hits including the first reported publication. 3/5 blast hits were whole plasmid sequences of NDM-positive plasmids from different geographical regions. Surprisingly, all the 3 plasmids were 250-300 kb plasmids with substantial homology between them and have been reported only from K. pneumoniae. This indicates that the acquisition of NDM gene by this plasmid scaffold might be a recent 'one off' event.
With many plasmids from Indian environmental water samples reported to be untypeable,  the results of our study indicates that this particular plasmid type might either be already circulating in our clinical settings or it could represent a case of importation since it was not previously reported from India.
All the three isolates reported in this study are from patients undergoing haemodialysis and renal transplantation. Since these patients have constant exposure to both the clinical and community settings, they could be a potential source for the dissemination of blaNDM into the community.
| ~ Conclusion|| |
Studies of larger sample size are required to substantiate our preliminary report. We further suggest that since the replicon typing scheme is continuously updated, retrospective analysis with newly reported primers must be done to get the evolutionary picture of NDM harbouring plasmids in India.
Financial support and sponsorship
The study was partly funded by a Junior Research Scholarship awarded by Lady Tata Memorial trust to G. Kaushik Subramanian.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]