Skip to main content
  • Research note
  • Open access
  • Published:

Lipid A-Ara4N as an alternate pathway for (colistin) resistance in Klebsiella pneumonia isolates in Pakistan



This study aimed to explore mechanism of colistin resistance amongst Klebsiella pneumoniae isolates through plasmid mediated mcr-1 gene in Pakistan. Carbapenem and Colistin resistant K. pneumoniae isolates (n  = 34) stored at − 80 °C as part of the Aga Khan University Clinical Laboratory strain bank were randomly selected and subjected to mcr-1 gene PCR. To investigate mechanisms of resistance, other than plasmid mediated mcr-1 gene, whole genome sequencing was performed on 8 clinical isolates, including 6 with colistin resistance (MIC  >  4 μg/ml) and 2 with intermediate resistance to colistin (MIC  >  2 μg/ml).


RT-PCR conducted revealed absence of mcr-1 gene in all isolates tested. Whole genome sequencing results revealed modifications in Lipid A-Ara4N pathway. Modifications in Lipid A-Ara4N pathway were detected in ArnA_ DH/FT, UgdH, ArnC and ArnT genes. Mutation in ArnA_ DH/FT gene were detected in S3, S5, S6 and S7 isolates. UgdH gene modifications were found in all isolates except S3, mutations in ArnC were present in all except S1, S2 and S8 and ArnT were detected in all except S4 and S7. In the absence of known mutations linked with colistin resistance, lipid pathway modifications may possibly explain the phenotype resistance to colistin, but this needs further exploration.


Beta-lactams have always been the backbone of antibiotic regimens targeting infections with Enterobacteriaceae including K. pneumoniae [1]. With the increase in resistance to these agents [1, 2] there is interest in using colistin (polymyxin E) for treating such infections. Colistin targets lipopolysaccharides (LPS) and phospholipids present in the outer cell membrane of Gram-negative bacteria, and competitively displaces divalent cations from the phosphate groups of membrane lipids leading to pore formation and disruption of the outer cell membrane, leakage of cytoplasmic contents, and bacterial death.

A worldwide increase in resistance to colistin has been reported globally [3, 4]. Such resistance can either be acquired, or occur as a result of intrinsic factors [5]. Mobile colistin resistance (mcr) genes acquired through plasmids were first reported in China in 2015 [6]. The first colistin resistant E. coli with mcr-1 was reported from Pakistan in 2017 [7].

The intrinsic factors contributing to colistin resistance mainly involves the modification of the LPS moiety by the addition of positively charged molecules L-Ara4-N and PEtN [8]. The addition of positively-charged molecules result in the decrease of negative charge in the outer membrane thus reducing its affinity to interact with cationic antibiotics including colistin [9]. Alterations in LPS most commonly occur due to mutations in bacterial genes including mgrB, phoP/phoQ, pmrA, pmrB, pmrC, and crrABC [10,11,12].Increasing colistin resistance amongst K. pneumoniae emphasizes the need to understand mechanisms contributing to such resistance. This study is therefore aimed to explore mechanism of colistin resistance amongst K. pneumoniae isolates from Pakistan, and to study the role of plasmid-mediated (mcr-1 gene) and chromosomal factors contributing to such resistance.

Main text


Study subjects

The clinical isolates (n  = 34) were randomly selected from the Aga Khan University Clinical Laboratory strain bank stored at − 80 °C and used anonymously. The source of the selected strains included sputum (n:1), tracheal aspirates (n:9), blood (n:9), urine (n:8), tissue (n:4), pus (n:2) and cerebrospinal fluid (n:1). All the study isolates were pure and were not passaged for more than 3 times.

Identification of bacterial isolates and susceptibility testing

The isolates were identified by conventional biochemical methods followed by API-E® (BioMérieux, France). Susceptibilities performed by disc diffusion (Kirby-Bauer) method on Mueller Hinton agar [13] and VITEK2® system (BioMérieux, France) showed resistance against beta-lactams and carbapenems (imipenem/meropenem).

Colistin susceptibility was performed using broth micro-dilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines, M07-A10, 2019 [14]. Minimum Inhibitory Concentrations (MIC) were performed in 96-well polystyrene microtitre plates using Cation-Adjusted Mueller Hinton Broth and colistin sulfate powders (Sigma-Aldrich, Inc.) Colistin concentration of 0.03–16.0 µg/ml was used [15]. ATCC E. coli 25922, ATCC P. aeruginosa 27853 and NCTC E. coli 13486 were used as controls. Results were read and interpreted after 16–20 h at 35 °C using colistin cut-offs (≤ 2 µg/ml  =  intermediate;  ≥ 4 µg/ml  =  resistant) for Enterobacteriales [14].

DNA extraction

The selected isolates (n  = 34) were inoculated into Brain heart infusion for 24 h. 1 ml of the broth was used for DNA extraction as per QIA®amp DNA extraction kit (QIAGEN, USA) manufacturer’s protocol. The quality of the extracted DNA was examined using Qubit v.2.0 fluorometer (Life Technologies, USA). The extracted DNA was stored at − 80 °C till further processing.

mcr-1 gene real-time PCR

Real‐time PCR was performed on the extracted DNA in duplicate. 20‐μl reactions were set up containing Platinum® SYBR® Green qPCR Supermix‐UDG (Invitrogen), 150 nm forward and reverse primers and 2 μl of DNA on a BioRad CFX 7500 thermal cycler. Sequence specific primers were used for mcr-1 [16] gene and the house keeping gene rho, [17]. Primer sequences are provided in Additional file 1: Table S1. NCTC 13846 E. coli was used as positive control.

Whole genome sequencing

Eight K. pneumoniae (CRKP1-CRKP6) strains were selected for whole genome sequencing (WGS) with six resistant and two intermediate to colistin. We had clinical data on only 4 isolates: one colistin intermediate isolate failed microbiological clearance for 3 years despite treatment, while three colistin resistant isolates achieved microbiological clearance between 7 and 11 days.

DNA was shipped to Eurofins scientific SE, Luxembourg for sequencing. Sequencing was carried out on the Illumina NovaSeq 6000 platform using 2 × 150 Sequence mode. WGS was performed with the genome coverage of 99%. Genetic analysis of reads was done using the KmerGeni tool which generated an approximate of 5.1 Mbp contigs. The de novo assemblies were evaluated by QUAST and Icarus tools. The evaluation was based on the alignment of the de novo assemblies on the reference genome (HS11286). The contigs were annotated with RAST to look for resistance genes.

Data submission

The raw sequences generated after sequencing were submitted to NCBI-SRA website under the accession numbers SAMN16684225–SAMN16684230. This Whole Genome Shotgun project genome assemblies were deposited at DDBJ/ENA/GenBank under the accession JAEMV(N/O/P/Q/R/S)000000000. The version described is JAEMVX010000000. The complete data details are available in the BioProject PRJNA674952.

Data analysis

The raw Illumina reads were passed through quality check using FASTQC and then assembled with SPAdes-3.13.0 software using spade and plasmid spade scripts to generate raw chromosomal and plasmid contigs. The generated assembly quality assessment was conducted using QUAST ( Continuous chromosome sequence was generated by overlapping raw contigs assembly against the reference genome by abacas.1.3.1 perl script that closed gaps on shotgun assembled contigs against the reference genome based on alignment between assembly and reference to identify syntenies of contigs with the reference.

Multi-Locus Sequence Typing (MLST) was determined using K. pneumoniae Sequence Typing web-based tool (PasteurMLST) ( The MLST was performed using the seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB and tonB) according to the protocol described by Diancourt et al. [18]. RAST server ( and Center for Genomic Epidemiology server ( were used for Chromosomal and Plasmid sequence annotations and downstream analysis.

Phylogenetic analysis

The phylogenetic inference was done by aligning the eight isolates genome with the reference using MegaX software ( The aligned sequences were then converted into a Phylodendogram using the software package Clonal Frame version 1.1. The dendogram was then estimated under the maximum likelihood (ML) principle in PhyloXML ( The tree was colored and edited using FigTree ( The tree was further magnified to show closely related genome using SNP (single nucleotide polymorphism) cluster generated by Pathogen detection Browser (


mcr-1 gene PCR

A total of 34 colistin resistant K. pneumoniae isolates were included. These strains were investigated for the presence of mcr-1 gene. However, PCR conducted revealed absence of mcr-1 gene in all isolates tested.

Alternate mechanisms involved in colistin resistance

WGS was performed to further understand the mechanism of colistin resistance. Mutations in genes involved in lipid-A and Ara-4 N pathways were revealed through a variant analysis of non-synonymous single nucleotide variants (ns-SNVs Table 1).

Table 1 Lipid A pathways related variants identified in colistin-resistant Klebsiella pneumoniae isolates

The mutations detected in PagP gene (I189F) were present in all isolates. In Pho R gene, mutations identified included: A424_V425insSerAla present in all isolates except S3, S4 and S8; L65F present in all isolates except S2 and S7. Additionally, M45I mutation was also identified in isolate S7.

Modifications in Lipid A-Ara4N pathway were detected in ArnA_ DH/FT, UgdH, ArnC and ArnT genes. ArnA_ DH/FT included mutations T185A, S18A, L260I and D205N. Mutations detected in UgdH were N354D, V17I, N354D. Mutation detected in ArnC was S10fs and in ArnT was A55G.

Mutations in ArnA_ DH/FT gene were detected in S3, S5, S6 and S7 isolates. UgdH gene modification was found in all isolates except S3, mutations in ArnC were present in all except S1, S2 and S8 and ArnT were detected in all except S4 and S7.

Multi locus sequence typing of K. pneumoniae

MLST revealed that the isolates belonged to ST37 (n=1), ST147 (n=3), ST14 (n=1), ST 11 (n=1), ST39 (n=1) and ST 17 (n=1) sequence types (Table 2).

Table 2 The general genetic characteristics of the Klebsiella pneumoniae strains

Phylogenetic analysis

The dendrogram was plotted (n = 41,172 as per 6th March 2021) using NCBI Pathogen Detection database (Fig. 1). S3, S5 and S6 (ST147) did not cluster with the reference strain, however, S4 (ST37) was the closest, followed by S2 (ST11) and then S1 (ST14) and S8 (ST17).

Fig. 1
figure 1

Dendrogram colistin-resistant—Klebsiella pneumoniae isolates, along with magnified sub-clades for S1 and S4 based on SNP cluster was plotted using Pathogen Detection Browser

In addition to the above, clustering with other sequences deposited in the database was also observed. S4 clustered with clinical strain CFSAN059641 isolated in 2020 in Pakistan and also with the other clinical strains deposited (PDT0009285111). S1 clustered with clinical strains isolated in USA (2020), and also with others from Australia, Canada and India (2019).


Although novel treatment approaches are being explored [20,21,22,23], increasing prevalence of colistin resistant K. pneumoniae, continue to pose a serious global threat.

In Pakistan mcr-1 gene has been detected in 23.3% E. coli (n  = 120) and 40% of K. pneumoniae (n  = 60) [24]. Our study however revealed absence of mcr-1 gene in all isolates tested. These findings are consistent with earlier studies also reporting absence of mcr-1 gene in colistin resistant clinical study isolates [25] and suggest presence of factors other than mcr-1 gene contributing to colistin resistance in these isolates.

Alternative mechanisms suggested for colistin resistance include: decreased drug permeability by porin loss, resistance to antibiotic penetration through bio-film formation [26] and mutations leading to alteration in antibiotic binding sites and efflux pump [27]. In our isolates WGS showed the modifications in PagP and PhoR genes of lipid A pathway. Mutations in PagP has been shown to be associated with colistin resistance [28, 29]. Also, mutations in the phoB-phoR operon have shown to contribute to the anti-microbial resistance by downregulation of PhoE [30]. Mutations found in genes of Lipid A-Ara4N pathway including Arn_DH/F, ArnC, ArnT have been reported in K. pneumonia [28]. We additionally detected mutations in UgdH gene of Lipid A-Ara4N pathway which has been shown to be implicated in colistin resistance in E. coli [31]. Gram negative bacteria develop resistance against cationic antimicrobial peptides by masking negative charges of the lipid A phosphate substituent through the addition of L-Ara4N positively charged-moieties. Briefly, L-Ara4N is transferred to lipid A by a lipid carrier in a reaction catalysed by ArnT. The synthesis of lipid carrier linked to L-Ara4N is catalyzed by UDP-Glc 6-dehydrogenase (Ugd), ArnA, ArnB, ArnC, and ArnD [32]. This pathway is well-explained in studies in E. coli and S. enterica sv. typhimurium [33]. It is important to note that the mutations found in Lipid A and L-Ara4N pathway were present in all isolates comprising of 6 colistin resistant and 2 colistin intermediate strains. One of our colistin intermediate isolate with mutations in both Lipid A and A-Ara4N pathway, failed to achieve microbiological clearance over 3 years, despite treatment. There is inherent variability in testing methods for colistin susceptibility, hence the susceptible category has been removed by CLSI [14]. Colistin monotherapy is also discouraged to avoid excessive reliance on even the currently recommended broth dilution susceptibility testing method. Hence, learning more about molecular mechanisms of colistin resistance might be helpful for taking clinical decisions in future.

The MLST done showed that eight K. pneumoniae isolates belonged to 6 different sequence types (ST11, ST14, ST17, ST37, ST39 and ST147) hence pointing towards the presence of considerable genetic diversity among them. Previous studies have shown that ST11 and ST14 have been detected amongst clinical isolates from Pakistan [25]. Globally, ST11 has been reported in Korea [34], Tunisia [35], and Egypt and linked with the presence of CTX-M gene [3]. ST 14 has been shown to be circulating in Dubai [36], India [37], Pakistan [25] and described to carry NDM-1 and CTX-M [38]. ST 17 are also reported to be mostly extended spectrum beta lactamases (ESBL) carrying clones [39]. ST 147 detected has been shown globally to be linked with resistance [15, 40, 41]. ST37 has been reported with carbapenem resistance and ST39 [42] with ESBLs.

This study highlights the significant challenges posed by multi-drug resistant K. pneumoniae strains to global health and emphasizes the need to identify factors contributing to resistance towards their better treatment and control.


Although the study highlights mechanism of colistin resistance alternate to mcr-1 gene in clinical isolates in Pakistan, it can be further strengthened by increasing sample size and by performing functional studies to validate the role of mutations found in Lipid A and L-Ara4N pathway in contributing colistin resistance.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.



Deoxyribonucleic acid


Extended spectrum beta lactamases




Multi drug resistant


Mobile colistin resistance 1


Multi-locus sequence typing


National collection of type culture collection strain


Whole genome sequencing


  1. Behzadi P, Garcia-Perdomo HA, Karpinski TM, Issakhanian L. Metallo-ss-lactamases: a review. Mol Biol Rep. 2020;47(8):6281–94.

    Article  CAS  Google Scholar 

  2. Reyes J, Aguilar AC, Caicedo A. Carbapenem-resistant Klebsiella pneumoniae: microbiology key points for clinical practice. Int J Gen Med. 2019;12:437–46.

    Article  Google Scholar 

  3. Woodford N, Zhang J, Warner M, Kaufmann ME, Matos J, Macdonald A, et al. Arrival of Klebsiella pneumoniae producing KPC carbapenemase in the United Kingdom. J Antimicrob Chemother. 2008;62(6):1261–4.

    Article  CAS  Google Scholar 

  4. Wang R, van Dorp L, Shaw LP, Bradley P, Wang Q, Wang X, et al. The global distribution and spread of the mobilized colistin resistance gene mcr-1. Nat Commun. 2018;9(1):1179.

    Article  Google Scholar 

  5. Zhou K, Cattoir V, Xiao Y. Intrinsic colistin resistance. Lancet Infect Dis. 2016;16(11):1227–8.

    Article  Google Scholar 

  6. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmid-mediated colistin resistance mechanism mcr-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis. 2016;16(2):161–8.

    Article  Google Scholar 

  7. Mohsin M, Raza S, Roschanski N, Guenther S, Ali A, Schierack P. Description of the first Escherichia coli clinical isolate harboring the colistin resistance gene mcr-1 from the Indian subcontinent. Antimicrob Agents Chemother. 2017.

    Article  PubMed  Google Scholar 

  8. Falagas ME, Rafailidis PI, Matthaiou DK. Resistance to polymyxins: mechanisms, frequency and treatment options. Drug Resist Updat. 2010;13(4–5):132–8.

    Article  CAS  Google Scholar 

  9. Velkov T, Deris ZZ, Huang JX, Azad MA, Butler M, Sivanesan S, et al. Surface changes and polymyxin interactions with a resistant strain of Klebsiella pneumoniae. Innate Immun. 2014;20(4):350–63.

    Article  Google Scholar 

  10. Cheng HY, Chen YF, Peng HL. Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43. J Biomed Sci. 2010;17:60.

    Article  Google Scholar 

  11. Poirel L, Jayol A, Bontron S, Villegas MV, Ozdamar M, Turkoglu S, et al. The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae. J Antimicrob Chemother. 2015;70(1):75–80.

    Article  CAS  Google Scholar 

  12. Wright MS, Suzuki Y, Jones MB, Marshall SH, Rudin SD, van Duin D, et al. Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance. Antimicrob Agents Chemother. 2015;59(1):536–43.

    Article  Google Scholar 

  13. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. CLSI supplement M100. Wayne, PA: Clinical and Laboratory Standards Institute; 2020.

  14. Wayne P. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. CLSI document. Wayne: Clinical and Laboratory Standards Institute; 2019. p. M07-A10.

    Google Scholar 

  15. Sato T, Harada K, Usui M, Tsuyuki Y, Shiraishi T, Tamura Y, et al. Tigecycline susceptibility of Klebsiella pneumoniae complex and Escherichia coli isolates from companion animals: the prevalence of tigecycline-nonsusceptible K. pneumoniae complex, including internationally expanding human pathogenic lineages. Microb Drug Resist. 2018;24(6):860–7.

    Article  CAS  Google Scholar 

  16. Chabou S, Leangapichart T, Okdah L, Le Page S, Hadjadj L, Rolain JM. Real-time quantitative PCR assay with Taqman® probe for rapid detection of mcr-1 plasmid-mediated colistin resistance. New Microbes New Infect. 2016;13:71–4.

    Article  CAS  Google Scholar 

  17. Gomes AEI, Stuchi LP, Siqueira NMG, Henrique JB, Vicentini R, Ribeiro ML, et al. Selection and validation of reference genes for gene expression studies in Klebsiella pneumoniae using reverse transcription quantitative real-time PCR. Sci Rep. 2018;8(1):9001.

    Article  Google Scholar 

  18. Diancourt L, Passet V, Verhoef J, Grimont PA, Brisse S. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol. 2005;43(8):4178–82.

    Article  CAS  Google Scholar 

  19. Ahmadi M, Ranjbar R, Behzadi P, Mohammadian T. Virulence factors, antibiotic resistance patterns, and molecular types of clinical isolates of Klebsiella Pneumoniae. Expert Rev Anti Infect Ther. 2021.

    Article  PubMed  Google Scholar 

  20. Le Trong N, Viet Ho D, Quoc Doan T, Le Tuan A, Raal A, Usai D, et al. Biological activities of essential oils from leaves of Paramignya trimera (Oliv.) Guillaum and Limnocitrus littoralis (Miq.) Swingle. Antibiotics. 2020.

    Article  Google Scholar 

  21. Donadu MG, Le Trong N, Viet Ho D, Quoc Doan T, Le Tuan A, Raal A, et al. Phytochemical compositions and biological activities of essential oils from the leaves, rhizomes and whole plant of Hornstedtia bella Skornick. Antibiotics. 2020.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Bua A, Usai D, Donadu MG, Delgado Ospina J, Paparella A, Chaves-Lopez C, et al. Antimicrobial activity of Austroeupatorium inulaefolium (H.B.K.) against intracellular and extracellular organisms. Nat Prod Res. 2018;32(23):2869–71.

    Article  CAS  Google Scholar 

  23. Cannas S, Usai D, Pinna A, Benvenuti S, Tardugno R, Donadu M, et al. Essential oils in ocular pathology: an experimental study. J Infect Dev Ctries. 2015;9(6):650–4.

    Article  CAS  Google Scholar 

  24. Hameed F, Khan MA, Bilal H, Muhammad H, Tayyab Ur R. Detection of mcr-1 gene in multiple drug resistant Escherichia coli and Klebsiella pneumoniae in human clinical samples from Peshawar, Pakistan. Comb Chem High Throughput Screen. 2020.

    Article  Google Scholar 

  25. Lomonaco S, Crawford MA, Lascols C, Timme RE, Anderson K, Hodge DR, et al. Resistome of carbapenem- and colistin-resistant Klebsiella pneumoniae clinical isolates. PLoS ONE. 2018;13(6):e0198526.

    Article  Google Scholar 

  26. Reza A, Sutton JM, Rahman KM. Effectiveness of efflux pump inhibitors as biofilm disruptors and resistance breakers in gram-negative (ESKAPEE) bacteria. Antibiotics. 2019.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Usai D, Donadu M, Bua A, Molicotti P, Zanetti S, Piras S, et al. Enhancement of antimicrobial activity of pump inhibitors associating drugs. J Infect Dev Ctries. 2019;13(2):162–4.

    Article  CAS  Google Scholar 

  28. Mathur P, Veeraraghavan B, Devanga Ragupathi NK, Inbanathan FY, Khurana S, Bhardwaj N, et al. Multiple mutations in lipid-A modification pathway and novel fosA variants in colistin-resistant Klebsiella pneumoniae. Future Sci OA. 2018;4(7):FSO319.

    Article  CAS  Google Scholar 

  29. Sun L, Rasmussen PK, Bai Y, Chen X, Cai T, Wang J, et al. Proteomic changes of Klebsiella pneumoniae in response to colistin treatment and crrB mutation-mediated colistin resistance. Antimicrob Agents Chemother. 2020.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Bialek-Davenet S, Mayer N, Vergalli J, Duprilot M, Brisse S, Pages JM, et al. In-vivo loss of carbapenem resistance by extensively drug-resistant Klebsiella pneumoniae during treatment via porin expression modification. Sci Rep. 2017;7(1):6722.

    Article  Google Scholar 

  31. Breazeale SD, Ribeiro AA, Raetz CR. Oxidative decarboxylation of UDP-glucuronic acid in extracts of polymyxin-resistant Escherichia coli. Origin of lipid a species modified with 4-amino-4-deoxy-L-arabinose. J Biol Chem. 2002;277(4):2886–96.

    Article  CAS  Google Scholar 

  32. King JD, Kocincova D, Westman EL, Lam JS. Review: lipopolysaccharide biosynthesis in Pseudomonas aeruginosa. Innate Immun. 2009;15(5):261–312.

    Article  CAS  Google Scholar 

  33. Yan A, Guan Z, Raetz CR. An undecaprenyl phosphate-aminoarabinose flippase required for polymyxin resistance in Escherichia coli. J Biol Chem. 2007;282(49):36077–89.

    Article  CAS  Google Scholar 

  34. Choi M, Ko KS. Identification of genetic alterations associated with acquired colistin resistance in Klebsiella pneumoniae isogenic strains by whole-genome sequencing. Antibiotics. 2020.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Jaidane N, Bonnin RA, Mansour W, Girlich D, Creton E, Cotellon G, et al. Genomic insights into colistin-resistant Klebsiella pneumoniae from a Tunisian teaching hospital. Antimicrob Agents Chemother. 2018.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Moubareck CA, Mouftah SF, Pal T, Ghazawi A, Halat DH, Nabi A, et al. Clonal emergence of Klebsiella pneumoniae ST14 co-producing OXA-48-type and NDM carbapenemases with high rate of colistin resistance in Dubai, United Arab Emirates. Int J Antimicrob Agents. 2018;52(1):90–5.

    Article  CAS  Google Scholar 

  37. Shankar C, Venkatesan M, Rajan R, Mani D, Lal B, Prakash JAJ, et al. Molecular characterization of colistin-resistant Klebsiella pneumoniae and its clonal relationship among Indian isolates. Indian J Med Res. 2019;149(2):199–207.

    Article  CAS  Google Scholar 

  38. Giske CG, Froding I, Hasan CM, Turlej-Rogacka A, Toleman M, Livermore D, et al. Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of blaNDM-1 in India, Sweden, and the United Kingdom. Antimicrob Agents Chemother. 2012;56(5):2735–8.

    Article  CAS  Google Scholar 

  39. Navon-Venezia S, Kondratyeva K, Carattoli A. Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol Rev. 2017;41(3):252–75.

    Article  CAS  Google Scholar 

  40. Sonnevend A, Ghazawi A, Hashmey R, Haidermota A, Girgis S, Alfaresi M, et al. Multihospital occurrence of pan-resistant Klebsiella pneumoniae sequence type 147 with an ISEcp1-directed blaOXA-181 insertion in the mgrB gene in the United Arab Emirates. Antimicrob Agents Chemother. 2017.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Mathers AJ, Stoesser N, Chai W, Carroll J, Barry K, Cherunvanky A, et al. Chromosomal integration of the Klebsiella pneumoniae carbapenemase gene, blaKPC, in Klebsiella species is elusive but not rare. Antimicrob Agents Chemother. 2017.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Belbel Z, Chettibi H, Dekhil M, Ladjama A, Nedjai S, Rolain JM. Outbreak of an armA methyltransferase-producing ST39 Klebsiella pneumoniae clone in a pediatric Algerian Hospital. Microb Drug Resist. 2014;20(4):310–5.

    Article  CAS  Google Scholar 

Download references


We would like to thank Health Security Partners (HSP)-USA for funding this project. We would also like to thank the staff of Department of Pathology and Laboratory Medicine for providing help during the project.


This study was supported by the Health Security Partners (HSP)-USA, as part of their collaboration with the Department of Pathology and Laboratory Medicine, Aga Khan University, for laboratory capacity building and strengthening for antimicrobial resistance surveillance. We declare that there was no competing interest by the funding agency in determining or influencing the study at any point.

Author information

Authors and Affiliations



Conception and design: RH, KI, SU, JF, ZH, SS, JR; analysis and interpretation: KI, SU, ZH, SAR, NJ; drafting the manuscript for important intellectual content: RH, KI, SU, JF, ZH. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Rumina Hasan.

Ethics declarations

Ethics approval and consent to participate

The clinical isolates from the Aga Khan University Clinical Laboratory strain bank stored at − 80 °C were used anonymously. No samples from human subjects were directly used. Hence, the need from obtaining informed consent from human subjects was waived and this study received exemption from full ethical review by The Aga Khan University’s Institutional Ethical Review Committee (ERC number: 2018-0424-813).

Consent for publication

Not applicable.

Competing interests

Nothing to disclose.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1: Table S1.

Primers and probe sequences to target the plasmid-mediated colistin resistance (mcr-1) and housekeeping gene rho. Figure S1. Amplification of mcr-1 gene in a positive control NCTC 13846 E. coli strain. DNA was extracted from the pure culture of NCTC 13846 E. coli strain using DNA extraction kit (Qiagen) as per manufacturers instruction. Extracted DNA was subjected to PCR using sequence specific primers of mcr-1 gene. The figure shows the amplification of mcr-1 gene in the positive control used in the experiment. PC denotes positive control and NTC denotes non-template control.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Masood, K.I., Umar, S., Hasan, Z. et al. Lipid A-Ara4N as an alternate pathway for (colistin) resistance in Klebsiella pneumonia isolates in Pakistan. BMC Res Notes 14, 449 (2021).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: