|Year : 2015 | Volume
| Issue : 1 | Page : 30-38
Complete sequencing of an IncX3 plasmid carrying blaNDM-5 allele reveals an early stage in the dissemination of the blaNDM gene
M Krishnaraju1, C Kamatchi1, AK Jha1, N Devasena2, R Vennila1, G Sumathi3, R Vaidyanathan4
1 Department of Biotechnology , Dr. M.G. Ramachandran Educational and Research Institute, Chennai, India
2 Department of Microbiology, Institute of Child Health and Hospital for Children, Chennai, India
3 Department of Microbiology, Sri Muthukumaran Medical College, Hospital and Research Institute, Chennai, Tamil Nadu, India
4 Department of Microbiology, Institute of Surgical Gastroenterology and Liver Transplantation, Government Stanley Medical College and Hospital, Chennai, India
|Date of Submission||16-Jul-2013|
|Date of Acceptance||14-May-2014|
|Date of Web Publication||5-Jan-2015|
Department of Microbiology, Institute of Surgical Gastroenterology and Liver Transplantation, Government Stanley Medical College and Hospital, Chennai
Source of Support: None, Conflict of Interest: None
Purpose: The aim of the present study was to perform molecular characterisation of the blaNDM plasmids and to understand the mechanism of its spread among pathogenic bacteria. Materials and Methods: Seventy-six non-repetitive carbapenem-resistant isolates which were collected during Nov 2011 to April 2013 from four hospitals in Chennai were analyzed for the presence of the blaNDM gene by PCR. Further, the genetic context of the blaNDM gene was analyzed by PCR specific to ISAba125 and bleMBL gene. One of the blaNDM plasmid was completely sequenced in the Illumina HiSeq platform. Results: Twenty-three isolates consisting of 8 Escherichia coli, 8 Klebsiella pneumoniae, 3 Klebsiella oxytoca, 3 Acinetobacter baumanii and 1 Pseudomonas aeruginosa were found to carry the blaNDM gene. In 18 isolates the blaNDM gene was associated with a bleMBL gene and the ISAba125 element. The complete sequencing of pNDM-MGR194 revealed an IncX3 replication type plasmid, with a length of 46,253 bp, an average GC content of 47% and 59 putative ORFs. The iteron region contained the blaNDM5 gene and the bleMBL , trpF and dsbC genes downstream and an IS5 inserted within the ISAba125 element upstream. Conclusion: This is the first report where the blaNDM gene insertion in a plasmid is not accompanied by other resistance gene determinants. These observations suggest that the IncX3 plasmid pNDM-MGR194 is an early stage in the dissemination of the blaNDM .
Keywords: bla NDM , ISAba125, IncX3, metallo carbapenemase
|How to cite this article:|
Krishnaraju M, Kamatchi C, Jha A K, Devasena N, Vennila R, Sumathi G, Vaidyanathan R. Complete sequencing of an IncX3 plasmid carrying blaNDM-5 allele reveals an early stage in the dissemination of the blaNDM gene. Indian J Med Microbiol 2015;33:30-8
|How to cite this URL:|
Krishnaraju M, Kamatchi C, Jha A K, Devasena N, Vennila R, Sumathi G, Vaidyanathan R. Complete sequencing of an IncX3 plasmid carrying blaNDM-5 allele reveals an early stage in the dissemination of the blaNDM gene. Indian J Med Microbiol [serial online] 2015 [cited 2020 May 31];33:30-8. Available from: http://www.ijmm.org/text.asp?2015/33/1/30/148373
| ~ Introduction|| |
The emergence and dissemination of Enterobacteriaceae resistant to carbapenems is a clinical and epidemiological problem in patient care and public health. Carbapenem resistance due to the presence of blaNDM metallo-β-lactamase has been of concern since numerous studies have suggested that the Indian subcontinent is an important reservoir for the blaNDM gene. ,,, Currently 12 alleles of the blaNDM have been identified suggesting a rapid evolution of the gene (blaNDM1-12 ). The information on the alleles is available at: http://www.lahey.org/Studies/other.asp accessed 10 th June 2014. The analysis of the genetic context of the blaNDM gene has led to the hypothesis that the Transposon Tn125 which has two copies of ISAba125 could be responsible for dissemination of the bla NDM gene  most likely originating from the chromosome of Acinetobacter baumanii. Several studies have shown the presence of an IncX3 plasmid harbouring the blaNDM gene in China  and UAE. 
In the present study, blaNDM -carrying isolates were identified from carbapenem-resistant isolates from Chennai, India and the genetic structure surrounding the blaNDM was analyzed by PCR. One of the plasmids carrying the blaNDM was completely sequenced and analyzed.
| ~ Materials and Methods|| |
Bacterial isolates and antimicrobial sensitivity tests
A total of 76 carbapenem-resistant Gram-negative isolates from four different hospitals in Chennai were collected during the period Nov 2011 to April 2013. Antimicrobial susceptibility testing was performed by disk diffusion assay on Muller-Hinton agar plates with the following antibiotics: β-lactam antibiotics-cefotaxime (30 μg), cefepime (30 μg), cefoxitin (30 μg), amoxycillin-clavulanic acid (30 μg), and non-β-lactam antibiotics-nalidixic acid (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg) discs and the results were interpreted as recommended by CLSI guidelines.  Imipenem resistance was determined using 10 μg discs and interpreted as per the EUCAST guidelines. 
The blaNDM gene was identified by a PCR using the following primers NDM-Forward: 5' GGT TTG GCG ATC TGG TTT TC-3' and NDM Reverse - 5' CGG AAT GGC TCA TCA CGA TC-3' which amplifies a product of 621 bp. A Klebsiella pneumonaie isolate harbouring the blaNDM sequenced (Acc. No. JX401535) in our laboratory was used as a positive control. The blaVIM , blaIMP and blaOXA-48 genes were identified by gene specific PCR primers.  For ESBL gene detection, PCR was carried out on the plasmid DNA from each of the clinical isolates, using blaTEM /blaSHV /blaOXA-1-like multiplex PCR and a blaCTX-M multiplex PCR including phylogenetic groups 1, 2 and 9.  The genetic region around the blaNDM gene was analyzed by PCR using ISAba125A and bleo-Rev primers.  For PCR reactions, Taq DNA polymerase from M/S Bangalore Genie, Merck Millipore, Bangalore, India and dNTP stock from M/S Cinnagen, Tehran, Iran were used for all PCR reactions.
Phenotypic assays for NDM carbapenemases
The blaNDM isolates were identified by biochemical methods.  The presence of a secreted carbapenemase was confirmed by the modified Hodge test with the following control strains [Figure 1]. E. coli ATCC 25922 was swabbed on the Mueller-Hinton agar plate, K. pneumoniae ATCC BAA 1705 was the positive control for secreted carbapenemases and K. pneumoniae ATCC BAA 1706 for the negative control. The synergy test was used to identify the EDTA-sensitive metallo-carbapenemases using the CLSI 2012 guidelines. 
|Figure 1: The scheme for the AST, genotypic and phenotypic tests done to identify the blaNDM producing clinical isolates|
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Plasmid extraction and transformation
From each of the blaNDM -positive isolates, total plasmids were extracted using the alkaline lysis method  and transformed into E. coli DH5α selecting for growth in ampicillin (100 μg/ml). K. pneumoniae isolate MGR194 was found to give the highest number of transformants. These transformants were analyzed by AST to antibiotics listed in the AST section, followed by a blaNDM -specific PCR. One of the blaNDM -carrying transformants was selected for further analysis by sequencing.
Plasmid sequencing and Bioinformatic analysis
Total plasmid was extracted using Qiagen Plasmid Mega Kit using the manufacturer's instructions. The whole plasmid sequence was determined using the Illumina HiSeq 1000 platform. A DNA library was prepared and sequenced in a paired end (2 × 100 bases run) and sequencing was performed by Centre for Cellular and Molecular Platforms, Bangalore, India. The raw sequencing data was pre-processed using FASTX-toolkit. The reads mapping to K. pneumoniae genome were filtered before assembly using Bowtie2.  The de novo plasmid assembly was performed using Velvet assembler  for a range of k-mers and the assembly with highest N50 value was selected for downstream analysis. The plasmid sequences were annotated by the RAST Server  and each predicted protein was further compared against the NCBI non-redundant protein database using BLASTP.  Insertion elements and Transposons were identified by homology search against the IS finder database.  The Geneious Pro (Version 6, Biomatters Limited, Auckland, New Zealand) was used for construction of a schematic plasmid map. The distance between the dsbC and the umuD gene was confirmed by a PCR reaction to find out whether it corresponds to the assembled sequence. The following primers were used: umuD Forward 5'- CTG ATG ACG ATG AGG ACG GG-3' and dsbC- Reverse 5'- CTC GGG TGA AGT CGG GAA AA to amplify a region of 2629 bp. Comparisons with other sequences were made using Easyfig Version 2.1 software. 
The GenBank accession number for pNDM-MGR194 plasmid is KF220657.1. The GenBank accession number for the region around the blaNDM in pNDM-MGRK8 is KF220658.
This study focused on bacterial strains that were isolated by the hospitals for treatment of patients. This study was completely anonymous and no identifiable information was obtained from the patients. This study was approved by the Institute Ethics Committee.
| ~ Results|| |
Identification of blaNDM-positive isolates
Out of the 76 non-repetitive carbapenem-resistant bacterial isolates from Chennai collected during the period Nov 2011 to April 2013, analyzed by a PCR for blaNDM found carbapenemases, 23 isolates were found to harbour the blaNDM metallo-carbapenemase [Figure 1] and [Figure 2]. These isolates consisted of 3 Acinetobacter int baumanii, 8 Escherichia More Details coli, 8 Klebsiella pneumoniae, 1 Pseudomonas aeuruginosa and 3 Klebsiella oxytoca [Table 1]. All the blaNDM isolates were positive for the Hodge test and the synergy test for inhibition by EDTA confirming the presence of a secreted metallo-carbapenemase. These isolates showed resistance to a broad spectrum of cephalosporins including the beta-lactamase inhibitor clauvulanic acid. All the isolates also showed resistance to fluoroquinolone antibiotics norfloxacin or nalidixic acid. In addition to the blaNDM , blaVIM gene was also found in one each of A. baumanii, E. coli, P. aeruginosa and K. oxytoca isolate. Class D blaOXA carbapenemase was found in another E. coli and a K. pneumoniae isolate. The isolates were also analyzed with ESBL-specific primers since they are routinely present in the clinical isolates and contribute to higher MIC against the cephalosporin antibiotics. [Table 1] shows the distribution of the ESBL genes. Multiple ESBL genes were found in each of these isolates. The blaOXA-1 like gene was found in 16 isolates, blaTEM gene in 15, blaCTX-M1 in 13, blaSHV in 7, blaCTX-M2 in 4 and blaCTX-M1 in 6 and blaCTX-M8/25 in 3 isolates. The ESBL genes are likely to contribute to a higher MIC for the β-lactam antibiotics and may be present on the same plasmid or on different plasmids in the same isolate.
The analysis of the genetic region around the blaNDM and the complete sequencing of plasmids carrying the blaNDM can give a clue to its origin, spread and acquisition of other resistance genes. In order to study NDM plasmids, K. pneumoniae isolate MGR-194 from which carbapenem resistance and the blaNDM could be transferred by a plasmid transformation into a sensitive E. coli DH5α was chosen for further analysis. The blaNDM transformants were resistant to all β-lactam antibiotics and carbapenem but had lost the resistance to ciprofloxacin and norfloxacin. The blaNDM -carrying plasmid from the isolate K. pneumoniae MGR-194 was named pNDM-MGR194.
pNDM-MGR194 is an IncX3 plasmid carrying blaNDM-5 within the iterons
The complete sequencing of the plasmid pNDM-MGR194 revealed that the plasmid had a length of 46,253bp, an average GC content of 47% and 59 putative ORFs. BLAST homology analysis showed that this plasmid had 100% query coverage with pNDM-HN380,  a 54,035 bp IncX3 plasmid isolated from K. pneumoniae isolates from multiple regions in China (Acc. No. JX104760).  pNDM-MGR194 was found to have the typical backbone of IncX3 plasmids such as the replication, partitioning, plasmid maintenance, transcriptional activator and putative DNA transfer proteins' in addition to a conjugation/type IV secretion system. Analysis of the blaNDM gene in this plasmid showed that it differed from blaNDM-1 by two point mutations at positions 262 (G to T) and 460 ((A to C), leading to two substitutions at positions 88 (Val to Leu) and position 154 (Met to Leu) corresponding to blaNDM5 allele (http://www.lahey.org/Studies/other.asp accessed 24 th December 2013). [Figure 3] shows an 8248 bp fragment showing the genetic context of the blaNDM5 in this plasmid. Upstream the blaNDM-5 , there is an ISAba125 element interrupted by an IS5 element. Downstream the blaNDM-5 are the genes for bleMBL which encodes a bleomycin-resistant protein, trpF gene and dsbC gene. Further downstream is an IS26 element. This region has a markedly high GC content suggestive of horizontal transfer.
|Figure 3: The Iteron region (8248bp) carrying the blaNDM region from pNDM-MGR194 (Acc. No. KF220657) is shown here. The location of the ISAba125A and bleoRev primer which amplifies a 2942 bp fragment is shown by arrow. ΔIS5 in refers to the deletion observed in the 1743 bp PCR product from the ISAba125 and bleo Rev amplification in pNDM-MGRK8 (Acc. No. KF220658)|
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The IncX3 plasmids are broad host range plasmids containing iterons.  The genetic load region containing antibiotic-resistant genes are present downstream the resolvase gene. There are atleast 5 IncX3 plasmids in the database of which 4 have antibiotic-resistant gene within the iteron [Table 2]. Plasmid pEC14_35  (Acc. No. JN935899) without any antibiotic-resistant gene was isolated from a human patient in 1989 in the U.S.A.  Plasmid pIncX-SHV  (JN247852) which carries blaSHV-11 , adjacent to IS26, is from a K. pneumoniae ST258 isolate from Italy.  Plasmid pNDM-HN380 carries the blaNDM-1 gene and blaSHV12 within the iterons. Plasmid pNDM-HN380 differs from pNDM-MGR194 in the following [Figure 4]. (1) an insertion of 7874 bp spanning blaSHV12 till the truncated cutA1 gene. This region includes two mobile element regions- an indirect repeat of an IS26 element after the blaSHV12 and an IS91 family transposase. Secondly, an IS5 transposase which is in between the blaNDM and the ISAba125 sequence is inverted with respect to pNDM-MGR194. Thirdly the NDM allele in the Indian isolate is the blaNDM-5 allele. There has been a report of an blaNDM-7 allele from a Yemeni patient hospitalized in Germany where the genetic region around the blaNDM is identical to pNDM5-MGR194. 
|Figure 4: A comparison of the iteron region between pNDM-MGR194 and the pNDM-HN380 (Acc. No. JX104760) is shown here. Blue color region denotes high similarity in Blast. The yellow color regions represent the inversion|
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|Table 2: Completely sequenced IncX3 plasmids and the antibiotic - resistant genes present are listed here. The transposon elements in the iteron region were identified by performing a blast against the IS Finder database|
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The other two IncX3 plasmids are pKpS90  and pKPC-NY79.  They both carry the blaKPC gene in the iteron region and have been isolated from France and China, respectively.  In pKpS90 KPC-2-encoding Tn4401 transposon and a bla SHV-12 -encoding region have been inserted. These comparisons suggest that antibiotic-resistant cassettes are acquired in pIncX3 plasmids with the help of specific transposon elements. For example, blaSHV gene is associated with IS26, blaKPC with ISKpn6 and ISKpn7, blaNDM with ISAba125.
In order to find out if the genetic region surrounding the blaNDM is different in each of the isolates obtained in our study, we analyzed the region surrounding the blaNDM by PCR using ISAbaA and bleo-Rev primers.
The blaNDM gene is associated with an upstream ISAba125 element and downstream bleMBL gene in 18 isolates
The genetic region around the blaNDM has always been associated with a downstream bleMBL gene and an upstream ISAba125 element which provides a strong promoter. In order to find out if this region is similar, we performed PCR reactions using primers specific to ISAba125 (ISAba125A primer) and downstream gene trpF (bleo-Rev primer). We found two sizes of PCR products in the isolates [Table 1]. The 2942 bp amplicon found in pNDM-MGR194 was observed in four more isolates. In 13 isolates, a 1743-bp PCR fragment was observed. In five other isolates, no amplification was observed with these two primers suggesting a variation in the genetic region. An amplicon size of 1743 bp is possible if the 1195 bp IS5 element and a 4-bp CTAA repeat at the position of IS5 insertion seen in pNDM-MGR194 is not present in these isolates [Figure 3].One of the amplified products from K. pneumoniae isolate MGR K8 was sequenced. Sequencing revealed that this was indeed the case. The blaNDM allele corresponded to NDM-1. This genetic structure has been observed in blaNDM carrying plasmids such as pNDM-AB from A. baumanii from food of animal origin,  pNDM-BJ01 and pNDM-BJ02 from A. lwoffii clinical isolates. 
| ~ Discussion|| |
Carbapenem resistance can be conferred by carbapenemase enzymes or alteration in the membrane properties such as an increased efflux of carbapenems or decrease in the porins. Of these, plasmid borne and transposon associated carbapenemase genes such as the blaNDM are notorious for their rapid spread and dissemination. In this study, we found that 23 (30%) out of the 76 carbapenem-resistant isolates harboured the blaNDM gene. Of these 18 isolates were associated with an upstream ISAba125 element and downstream bleMBL gene. Six of the isolates possessed additional carbapenemase-resistance genes-class B blaVIM or class D blaOXA carbapenemase genes. All the isolates were resistant to quinolone antibiotics and possessed multiple ESBL genes. The ESBL genes may be present in the same blaNDM -carrying plasmids or could be in other plasmids in the same isolate.
Complete sequencing of one of the isolates revealed that the blaNDM gene was present within the iteron region in an IncX3 type plasmid of 46,253 bp. IncX plasmids are self-transmissible plasmids which were earlier believed to be of low prevalence and hence not included in the routine replicon typing.  However, more recently there have been reports of IncX3 plasmids carrying blaNDM genes. ,, The analysis of the plasmid pNDM-MGR194 shows the mobile region which has the blaNDM gene in association with an upstream ISAba125 and downstream bleMBL , trpF and dsbC gene often observed together in plasmids of various replicon types. No other antibiotic-resistance gene was found in this iteron region of pNDM-MGR194. In comparison to pEC14_35, which represents the prototype IncX3 plasmid, pNDM-MGR194 has an insertion of 10,106 bp between the umuD gene and the Zinc mpr gene. When compared to the iteron region in pIncX-SHV and pNDM-HN380, pNDM-MGR194 has the shortest iteron region and does not harbour any other ESBL gene or other resistance determinants. The efficiency of transmission of such a small iteron is not known. However, the carbapenem-resistant clinical isolate K. pneumoniae MGR194 harbors blaOXA-1 like, blaCTX-M1 and blaCTXM-2 in addition to blaNDM5 . It is likely that these resistance determinants are present in other plasmids in the same isolate.
In pNDM-MGR194, an IS5 element was inserted in the ISAba125. An IS5 insertion in the opposite orientation has been observed in pNDM-HN380, IncX3 plasmid from China  and plasmids from isolates reported in United Arab Emirates.  Analysis of carbapenem-resistant isolates from UAE has reported a 50 kb IncX3 plasmid carrying blaNDM-1, an IS5 element and blaSHV-12 in three different genera. It is likely that these plasmids may be similar or related to pNDM-MGR194.
It has been suggested that blaNDM gene has been acquired into Acinetobacter spp from the environment and then has spread to the enterobacteraceae with the help of the ISAba125 element.  The association of ISAba125 element with blaNDM gene in 18 isolates supports this hypothesis.
The first NDM completely sequenced was pNDM-HK, which is an IncL/M plasmid and it carries the blaNDM-1 along with blaTEM-1 and blaDHA-1 .  The blaNDM-1 was first reported in an untypable plasmid which in addition to blaNDM-1 also carried the blaCMY-4 .  All the blaNDM -carrying isolates that have been reported carry other resistance gene determinants.  This is the first report where the blaNDM gene insertion in a plasmid is not accompanied by other resistance gene determinants. These observations suggest that the plasmid pNDM-MGR194 is one of the early steps in the evolution and spread of blaNDM gene. The short iteron may not get transmitted efficiently. However, after the acquisition of other resistance determinants from other plasmids, this region might transmit more efficiently. The analysis of the region around the blaNDM and the plasmid replicon type of more isolates will throw light into its mode of spread.
Numerous variants of blaNDM have been identified. Of them blaNDM-1 , blaNDM-4 , blaNDM-5 and blaNDM-6 have been identified from patients who had a history of hospitalisation in India. ,, Our study shows the presence of the blaNDM-5 and blaNDM-1 allele in isolates from India. Further analysis of the blaNDM -carrying plasmids will be useful in understanding the role of IncX3 plasmids in the spread of the resistance determinants.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]
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