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Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae

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Carbapenem resistant Enterobacteriaceae (CRE) pose an urgent risk to global human health. CRE that are non-susceptible to all commercially available antibiotics threaten to return us to the pre-antibiotic era. Using Single Molecule Real Time (SMRT) sequencing we determined the complete genome of a pandrug-resistant Klebsiella pneumoniae isolate, representing the first complete genome sequence of CRE resistant to all commercially available antibiotics. The precise location of acquired antibiotic resistance elements, including mobile elements carrying genes for the OXA-181 carbapenemase, were defined. Intriguingly, we identified three chromosomal copies of an ISEcp1-blaOXA-181 mobile element, one of which has disrupted the mgrB regulatory gene, accounting for resistance to colistin. Our findings provide the first description of pandrug-resistant CRE at the genomic level, and reveal the critical role of mobile resistance elements in accelerating the emergence of resistance to other last resort antibiotics.


The “golden era” when modern medicine saved lives through antibiotic treatment is under serious threat1. In 2013, the Centers for Disease Control and Prevention (CDC) released a landmark report on “Antibiotic Resistance Threats2”. Three microorganisms were tagged as posing a threat level of urgent – Clostridium difficile, carbapenem-resistant Enterobacteriaceae (CRE) and drug-resistant Neisseria gonorrhoeae2. CRE, which include organisms such as Klebsiella pneumoniae and Escherichia coli, are resistant to almost all currently available antibiotics. Almost 50% of patients who develop bloodstream infections with these organisms die from the infection2. In healthcare settings, carbapenem resistant Enterobacteriaceae have increased sharply over the past decade3. Carbapenem resistance is typically mediated by the production of beta-lactamases4, and patients with CRE infections are treated with last-resort antibiotics such as colistin5.

The CDC and the European Centre for Disease Prevention and Control (ECDC) have jointly developed definitions for multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria6. Pandrug-resistance implies non-susceptibility to all commercially available antibiotics relevant to the treatment of a particular bacterial infection. Although there has been an anecdotal report of probable pandrug-resistance in K. pneumoniae7, no such isolates have been comprehensively analyzed.

In this manuscript, we describe the genetic basis of pandrug-resistance in a K. pneumoniae isolate using single molecule real-time (SMRT) sequencing. We show that a genetic element conferring resistance to carbapenem antibiotics has been acquired and mobilized, leading to insertional inactivation of a gene that results in resistance to colistin. Overall, our analysis provides a comprehensive description of a pandrug-resistant K. pneumoniae isolate at the whole genome level.


Case Record

An 87 year old man, hospitalized in the United Arab Emirates in April 2014, was found to be colonized with multidrug-resistant Klebsiella pneumoniae. The isolate grew from urine and a pre-sacral pressure area but blood cultures were sterile. Susceptibility testing by way of a commercial semi-automated method (Vitek, bioMérieux) showed resistance to all antibiotics tested. The urinary isolate (strain MS6671) was therefore sent to a reference laboratory for further testing. Other K. pneumoniae isolates with this antibiotic resistance phenotype were not detected at the index patient’s hospital.

Pandrug-resistant phenotype of K. pneumoniae MS6671

MS6671 was found to be non-susceptible to all antibiotics tested, which includes cephalosporins, penicillins, carbapenems, aztreonam, aminoglycosides, ciprofloxacin, colistin, tetracyclines, tigecycline, chloramphenicol, trimethoprim-sulfamethoxazole and fosfomycin (Table 1). Thus, the isolate can truly be described as pandrug-resistant6.

Table 1: Antibiotic resistance in K. pneumoniae MS6671.

K. pneumoniae MS6671 general genome features

The complete genome of K. pneumoniae MS6671 consists of a circular chromosome 5,402,900 base-pairs in length with an average G-C content of 57%, five circular plasmids and a linear plasmid prophage (Supplementary Table S1). The sequence type of the isolate was ST147. The chromosome of MS6671 is highly similar to K. pneumoniae NTUH-k20448; a hypervirulent strain associated with liver abscess and meningitis, with most variation attributable to differences in their mobile genetic element (MGE) content (Supplementary Figure 1). Further details of the complete genome are provided in the Supplementary Results.

Genetic determinants of pandrug-resistance

In order to determine the genetic basis of pandrug-resistance, we interrogated the genome to identify acquired and intrinsic resistance genes. The majority of acquired antibiotic resistance genes were located on the chromosome, with most beta-lactamase and aminoglycoside resistance genes carried within two copies of a class 1 integron or as part of mobile elements that incorporate the ISEcp1 insertion sequence (Table 1, Supplementary Table S2 and Supplementary Results). Mutations in gyrA and parC that have previously been linked with fluoroquinolone resistance (GyrA Ser83Ile and ParC Ser80Ile) were identified9,10. Fosfomycin resistance was mediated by a chromosomally encoded copy of fosA11. Mutations in chromosomal genes encoding major outer membrane porins (OmpK35 and OmpK36) were also identified. A novel variant of the ompK36 gene was encoded on the chromosome. The amino acid sequence change is located in loop 3 (L3) of the porin, which constitutes the porin channel eyelet12. L3 mutations have previously been associated with increased resistance to carbapenems13,14,15. Additionally, ompK35 has been disrupted by IS insertion. Inactivation of ompK35 has been associated with increased resistance to a number of different classes of antibiotics, including quinolones and cephalosporins16,17. Genes encoding three beta-lactamases, including an extended-spectrum beta-lactamase (ESBL) and a carbapenemase, were detected at different genomic locations – blaSHV-36, blaCTX-M-15, blaOXA-181.

Insertional inactivation of mgrB by a carbapenem-resistance element and colistin resistance

OXA-181 is an oxacillinase capable of hydrolysing carbapenems18. Three copies of an ISEcp1-blaOXA-181 transposon were identified throughout the chromosome (Fig. 1). One of these insertions has resulted in the inactivation of the mgrB gene, a negative regulator of phoPQ. Insertions in mgrB have previously been shown to cause colistin resistance in K. pneumoniae clinical isolates19,20,21. Examination of the DNA flanking the ISEcp1-blaOXA-181 transposons shows that the primary insertion site is within MS6671_10430, followed by intra-chromosomal transposition of ISEcp1-blaOXA-181 and a 37 bp fragment of MS6671_10430 to two other locations in the genome (Fig. 2). The three transposons are bracketed by imperfect 14 bp inverted repeats and flanking 5 bp direct repeats (TATCT, TGAAA and TATAA), providing direct evidence for their transposition activity (Supplementary Table S3).

Figure 1: Diagram of the pandrug-resistant K. pneumoniae MS6671 chromosome highlighting the position and context of mobile genetic elements that harbor antimicrobial resistance genes.
Figure 1

The chromosome of MS6671 is represented to scale by the black bar with ISEcp1 and integron insertion points indicated with red rectangles. Pop-outs display schematic representations of the four ISEcp1 elements which harbor beta-lactamase genes (three copies of blaOXA-181 and one copy of blaCTX-M-15) and a class 1 integron located on the chromosome of MS6671. Insertion elements are highlighted in yellow. The coordinates of each element are indicated above and below the genome bar.

Figure 2: Comparison of ISEcp1-blaOXA-181 transposons from MS6671.
Figure 2

Pairwise nucleotide comparison of ISEcp1-blaOXA-181 (OXA-181) transposons and flanking genomic regions from K. pneumonaie MS6671. ISEcp1 elements are represented by blue rectangles. Protein-coding genes are represented by coloured arrows: blaOXA-181 (green); MS6671_10430 encoding a hypothetical protein (red); other (brown). Left and right flanking inverted repeats (IRL, IRR, IRRalt1, IRRalt2) are represented by yellow bars and 5 bp direct repeat sequences created by duplication of the target sequence during transposition are given (TGAAA, TATCT or TATAA). In the primary insertion site, a single 2,855 bp transposon carrying blaOXA-181 has inserted at TGAAA (position 1152428..1155282) within MS6671_10430. This transposon, similar to Tn2013 previously described in K. pneumoniae Kp331, is flanked by 14-bp inverted repeat sequences, namely IRL and IRRalt1. IRRalt2 lies 23 bp downstream of IRRalt1 within the MS6671_10430 sequence. Mobilisation of the ISEcp1-blaOXA-181 transposon using IRRalt2 instead of IRRalt1 has resulted in a 37 bp fragment of MS6671_10430 (indicated by a small red rectangle) being packaged at the 3’ end of the other two ISEcp1-blaOXA-181 transposons inserted at TATCT and TATAA (position 126108..128999 and position 3345804..3348695, respectively). Grey shading indicates regions of homology (100% nucleotide sequence identity) between sequences.

In a similar fashion, a single copy of ISEcp1-blaCTX-M-15 has inserted into ompK35, leading to inactivation this gene (Fig. 1). The ISEcp1-mediated mobilisation and transposition of blaCTX-M-15, blaOXA-181 and other clinical relevant beta-lactamase resistance genes, including blaCMY and blaACC, has been reported previously22,23,24,25,26.

K. pneumoniae MS6671 contains two copies of a class 1 integron

A class 1 integron was identified on the chromosome encoding multiple antibiotic resistance genes (arr-3, aac(6′)-Ib-cr, rmtF, catB1) (Fig. 1). These genes result in resistance to rifampin, all aminoglycosides and chloramphenicol. A near-identical copy of this integron was also found on one of the six plasmids (Supplementary Figure S2).


This is the first genomic analysis of a pandrug-resistant CRE isolate, as defined by the rigorous CDC/ECDC assessment criteria6. With the advantage of long-reads provided by SMRT sequencing we were able to identify the genomic context of multiple resistance elements. In contrast to short-read technologies, SMRT sequencing allows complex resistance elements to be properly characterized27. This technology platform was used to investigate the German E. coli O104:H11 outbreak28 and more recently to identify plasmid-borne resistance in a large-scale study of CRE following an outbreak at the National Institute for Health Clinical Center29,30. Critically, elucidation of the complete K. pneumoniae MS6671 genome using long-read sequencing enabled the context of multiple, identical carbapenem resistance elements to be determined. Based on this analysis we propose a model for the development of pandrug-resistance in this K. pneumoniae isolate, whereby mobile resistance determinants are responsible for driving additional resistance. In this example, ISEcp1 carrying the blaOXA-181 carbapenem resistance gene has inserted three times in the chromosome, with one event causing colistin resistance by insertional inactivation of mgrB.

ISEcp1-like insertion sequences are the most common genetic element associated with blaCTX-M, blaCMY and blaACC genes and have more recently been associated with blaOXA-18122,23,24,25,26,31. By recognizing a variety of DNA sequences as right inverted repeats (IRR), ISEcp1s are capable of mobilising adjacent genes and inserting at new location32,33. Similar to previous reports on the hydrolytic activities of OXA-18118,34,35, elevated MICs for ertapenem, imipenem, meropenem and doripenem were observed for MS6671, indicating hydrolytic activity of OXA-181 towards these carbapenems and a possible blaOXA-181 copy number effect (Table 1). Notably, doripenem resistance was higher than previously reported18. The ompK36 variant encoded by MS6671 has previously been associated with increased resistance to doripenem and doripenem-colistin13, and may contribute to the elevated MIC for doripenem observed in MS6671. Porin deficient E. coli expressing OXA-48-like beta-lactamases have also been shown to have elevated MICs towards carbapenems18.

Inactivation of mgrB has recently been associated with resistance to colistin, and appears to be the most common mechanism for polymyxin resistance in K. pneumoniae19,20. Specifically, disruption of mgrB results in over expression of the phoPQ signaling system and of the pmrHFIJKLM operon which controls modification of LPS, the target of polymyxin antibiotics36. Insertional inactivation of mgrB with IS5-like or IS1 elements has been previously reported21,37, however, the present study is the first to show colistin resistance caused by insertion of a carbapenem resistance element itself. While we cannot rule out the possibility that this mechanism may have occurred in other colistin-resistant K. pneumoniae carrying ISEcp1-blaOXA-18138, the generation of a complete genome sequence of MS6671 provides unequivocal evidence for this novel insertion event.

We also found a fourth ISEcp1 element encoding an ESBL (CTX-M-15), which was inserted within the outer membrane porin gene ompK35. Disruption of ompK35 reduces the permeability of the outer membrane and mutants lacking this porin have increased resistance to quinolones, tetracyclines, beta-lactams and chloramphenicol39. Pan-aminoglycoside resistance was mediated by the rRNA methyltransferase RmtF, which was encoded on both chromosomal and plasmid copies of a class 1 integron. We did not have access to investigational antibiotics, such as plazomicin, eravacycline, ceftazidime/avibactam or ceftolozane/tazobactam, to assess their potency against MS6671.

MS6671 was defined by multi-locus sequence typing (MLST) as being ST147. K. pneumoniae ST147 was first described in Hungary in 200840. Subsequently, it has been frequently associated with carbapenem resistance, with ST147 producing KPC well described in Greece and Italy41,42. For example, epidemics of VIM-producing carbapenem resistant K. pneumoniae ST14743, and KPC-2-producing carbapenem resistant K. pneumoniae44,45 have been reported in Greece. Notably, ST147 isolates carrying both blaVIM and blaKPC-2 genes were identified41,46. Carbapenem resistant ST147 carrying the blaNDM-1 gene have been isolated in Iraq, Switzerland, Canada and the United Kingdom47,48,49. In MS6671, carbapenem resistance was most likely mediated by the beta-lactamase OXA-181, possibly in combination with permeability defects as has been reported previously in other strains18,31. ST147 K. pneumoniae producing OXA-181 have been previously reported from the Indian sub-continent18,31,50. Clearly, in addition to the KPC-producing, carbapenem-resistant K. pneumoniae ST258 clone51, ST147 also represents a clone of K. pneumoniae with a potential for global significance.

Fortunately, in six months there have been no further isolates with this resistance phenotype at the index patient’s hospital. However, the occurrence of this strain in the Arabian Gulf is of great significance. OXA-48-like-producing K. pneumoniae are frequent in this region52. It is unknown if this strain originated in the index patient, in another patient at the same hospital or was imported from another hospital, perhaps in another country. There are a large number of expatriates in the Gulf region, and travel to the Indian sub-continent, Europe and the United States is frequent53. The potential for international transfer of multidrug-resistant bacteria54,55 emphasizes the need for global surveillance efforts as one part of a strategy to control antibiotic resistance3.

In summary, we have provided the first report of a pandrug-resistant isolate of CRE using high-resolution genome data. The CDC has denoted CRE as an urgent threat. The emergence of this highly resistant strain, in a clone that has proven capable of causing outbreaks, raises this threat level even higher.


Antibiotic Resistance Phenotypic Testing

The K. pneumoniae isolate (hereafter referred to as MS6671) was sent to a reference laboratory (University of Queensland, Centre for Clinical Research) where confirmatory susceptibility testing was performed in order to determine the minimal inhibitory concentrations of all antibiotics used by the Centers for Disease Control and Prevention (CDC) and European Centre for Disease Prevention and Control (ECDC) in defining a pandrug-resistant isolate6. Susceptibility of most tested antibiotics was determined using Etests and following the breakpoints of the European Committee for Antibiotic Susceptibility Testing (EUCAST)56, except for cefazolin, cefoxitin, cefotetan, tetracycline, doxycycline, and minocycline which were determined using the breakpoints of the Clinical and Laboratory Standards Institute (CLSI)57. The minimum inhibitory concentration of colistin (sulfate, Sigma-Aldrich) was determined by broth microdilution in cation-adjusted Mueller-Hinton broth (Oxoid).

Genome sequencing

Pacific Biosciences (PacBio) RS II Single-Molecule Real Time (SMRT) sequencing of K. pneumoniae MS6671 was performed using ~4 μg of the genomic DNA sheared using g-TUBETM (Covaris®) into fragments size targeted at 10 kb. Purification of the sheared DNA was then carried out using 0.45-fold volume of washed Agencourt AMPure XP magnetic beads (Beckman Coulter Inc.). SMRTbell template libraries were subsequently prepared using the commercial Template Preparation Kit from Pacific Biosciences Inc. that involved steps of DNA end repair, adapters ligation followed by exonuclease digestion of incompletely ligated products. Next, 0.83 nM of the libraries were then annealed with sequencing primers followed by binding to 50 nM of P4 DNA polymerase, as provided in the Template Binding Kit from Pacific Biosciences Inc. For enhanced loading efficiency, 15 pM of the bound complexes were immobilized into Magbeads (Pacific Biosciences Inc.) prior to loading into the sequencing zero-mode waveguides (ZMWs). Duration for the sequence collection was set at 180 minutes with stage start option. Reads with length that were less than 50 bp were filtered off upon acquisition of the sequencing data and minimum polymerase read quality was set at 0.75.

Genome assembly

De novo genome assembly of PacBio SMRT reads from the K. pneumoniae MS6671 genome was performed using the hierarchical genome assembly process (HGAP)58 from the PacBio SMRT analysis software suit (version 2.2.0), with default parameters and a seed read length cut-off of 5 kb. Following assembly, all contigs were screened for duplicate sequences at their 3′ and 5′ ends. Overlapping sequences were manually trimmed and joined based on sequence similarity. Individual contigs with duplicate sequences on their 5′ and 3′ ends were manually trimmed and circularised. Following circularisation the chromosome and plasmid sequences were polished using quiver58 whereby the raw reads were mapped back to the chromosome and plasmid sequences to validate the assembly and resolve any remaining sequence errors. Non-circularised chromosomal and plasmid contigs were closed using primers designed on their 5′ and 3′ ends. The amplified PCR products were sequenced by the Australian Genome Research Facility and their sequences were manually integrated into the assembly.

Genome annotation

Gene calling and automatic functional annotation of the complete MS6671 chromosome and plasmids was performed using Prokka (Prokka: Prokaryotic Genome Annotation System - identifying 5,054 putative coding regions on the chromosome with an additional 644 putative coding regions distributed amongst the 5 plasmids and linear plasmid prophage. The complete annotated genome sequence has been deposited at the European Nucleotide Archive (Bioproject: PRJEB7538, Accessions: LN824133-LN824139).

Identification of antibiotic resistance genes

Initial identification of antimicrobial resistance genes from the complete PacBio assembly was performed using ResFinder (version 2.0)59. Additional screening for antimicrobial resistance genes was performed by comparison (BLASTp; sequence identity > =40%; E-value < =0.0001) of all predicted coding regions against the Antibiotic Resistance Genes Database (ARDB)60 and the Comprehensive Antimicrobial Resistance Database (CARD)61. Antimicrobial resistance genes were then subject to manual inspection to improve their functional annotation, correct start sites and identify point mutations, which may contribute to a resistant phenotype. Finally, resistance gene loci were screened for known insertion sequences and integrons by comparison against the ISFinder database62 and Integrall63, respectively.

Additional Information

How to cite this article: Zowawi, H. M. et al. Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae. Sci. Rep. 5, 15082; doi: 10.1038/srep15082 (2015).


  1. 1.

    & Antibiotic Resistance - Problems, Progress, and Prospects. The New England journal of medicine, 10.1056/NEJMp1408040 (2014).

  2. 2.

    U.S. Department of Health and Human Services, Centers for Disease Control and Prevention- Antibiotic Resistance Threats in the United States, 2013 (2013). Avaialable at: . (Accessed: 7th July 2015).

  3. 3.

    et al. Antibiotic resistance-the need for global solutions. The Lancet. Infectious diseases 13, 1057–1098, 10.1016/S1473-3099(13)70318-9 (2013).

  4. 4.

    & The new beta-lactamases. The New England journal of medicine 352, 380–391, 10.1056/NEJMra041359 (2005).

  5. 5.

    & Hospital-acquired infections due to gram-negative bacteria. The New England journal of medicine 362, 1804–1813, 10.1056/NEJMra0904124 (2010).

  6. 6.

    et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 18, 268–281, 10.1111/j.1469-0691.2011.03570.x (2012).

  7. 7.

    , , & Effectiveness of a double-carbapenem regimen for infections in humans due to carbapenemase-producing pandrug-resistant Klebsiella pneumoniae. Antimicrobial agents and chemotherapy 57, 2388–2390, 10.1128/AAC.02399-12 (2013).

  8. 8.

    et al. Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis. Journal of bacteriology 191, 4492–4501, 10.1128/JB.00315-09 (2009).

  9. 9.

    , , & The roles of mutations in gyrA, parC, and ompK35 in fluoroquinolone resistance in Klebsiella pneumoniae. Microbial drug resistance 9, 265–271, 10.1089/107662903322286472 (2003).

  10. 10.

    et al. Isolation and characterization of multidrug-resistant Klebsiella spp. isolated from shrimp imported from Thailand. International journal of food microbiology 155, 179–184, 10.1016/j.ijfoodmicro.2012.02.002 (2012).

  11. 11.

    , , & Detection of fosfomycin resistance by the polymerase chain reaction and Western blotting. The Journal of antimicrobial chemotherapy 34, 955–963 (1994).

  12. 12.

    et al. A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding. Infection and immunity 63, 903–910 (1995).

  13. 13.

    et al. Mutations of the ompK36 porin gene and promoter impact responses of sequence type 258, KPC-2-producing Klebsiella pneumoniae strains to doripenem and doripenem-colistin. Antimicrobial agents and chemotherapy 57, 5258–5265, 10.1128/AAC.01069-13 (2013).

  14. 14.

    , & Contribution of OmpK36 to carbapenem susceptibility in KPC-producing Klebsiella pneumoniae. Journal of medical microbiology 58, 1303–1308, 10.1099/jmm.0.012575-0 (2009).

  15. 15.

    et al. Klebsiella pneumoniae ST258 producing KPC-3 identified in italy carries novel plasmids and OmpK36/OmpK35 porin variants. Antimicrobial agents and chemotherapy 56, 2143–2145, 10.1128/AAC.05308-11 (2012).

  16. 16.

    et al. Roles of beta-lactamases and porins in activities of carbapenems and cephalosporins against Klebsiella pneumoniae. Antimicrobial agents and chemotherapy 43, 1669–1673 (1999).

  17. 17.

    et al. Role of Klebsiella pneumoniae OmpK35 porin in antimicrobial resistance. Antimicrobial agents and chemotherapy 47, 3332–3335 (2003).

  18. 18.

    , & Heterogeneous hydrolytic features for OXA-48-like beta-lactamases. The Journal of antimicrobial chemotherapy 70, 1059–1063, 10.1093/jac/dku524 (2015).

  19. 19.

    et al. The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae. The Journal of antimicrobial chemotherapy, 10.1093/jac/dku323 (2014).

  20. 20.

    et al. MgrB Inactivation Is a Common Mechanism of Colistin Resistance in KPC-Producing Klebsiella pneumoniae of Clinical Origin. Antimicrobial agents and chemotherapy 58, 5696–5703, 10.1128/AAC.03110-14 (2014).

  21. 21.

    et al. In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator. Antimicrobial agents and chemotherapy 57, 5521–5526, 10.1128/AAC.01480-13 (2013).

  22. 22.

    , , & ISEcp1B-mediated transposition of blaCTX-M in Escherichia coli. Antimicrobial agents and chemotherapy 49, 447–450, 10.1128/AAC.49.1.447-450.2005 (2005).

  23. 23.

    , , & Plasmid-mediated extended-spectrum beta-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS microbiology letters 201, 237–241 (2001).

  24. 24.

    et al. Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS microbiology letters 209, 161–168 (2002).

  25. 25.

    , , , & A novel type of AmpC beta-lactamase, ACC-1, produced by a Klebsiella pneumoniae strain causing nosocomial pneumonia. Antimicrobial agents and chemotherapy 43, 1924–1931 (1999).

  26. 26.

    et al. Outbreak of Klebsiella pneumoniae producing transferable AmpC-type beta-lactamase (ACC-1) originating from Hafnia alvei. FEMS microbiology letters 187, 35–40 (2000).

  27. 27.

    & Microbiology. Tracking antibiotic resistance. Science 345, 1454–1455, 10.1126/science.1260471 (2014).

  28. 28.

    et al. Origins of the E. coli strain causing an outbreak of hemolytic-uremic syndrome in Germany. The New England journal of medicine 365, 709–717, 10.1056/NEJMoa1106920 (2011).

  29. 29.

    et al. Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing. Science translational medicine 4, 148ra116, 10.1126/scitranslmed.3004129 (2012).

  30. 30.

    et al. Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae. Science translational medicine 6, 254ra126, 10.1126/scitranslmed.3009845 (2014).

  31. 31.

    et al. Characterization of OXA-181, a carbapenem-hydrolyzing class D beta-lactamase from Klebsiella pneumoniae. Antimicrobial agents and chemotherapy 55, 4896–4899, 10.1128/AAC.00481-11 (2011).

  32. 32.

    , & Insertion sequence ISEcp1B is involved in expression and mobilization of a bla(CTX-M) beta-lactamase gene. Antimicrobial agents and chemotherapy 47, 2938–2945 (2003).

  33. 33.

    , , & In vitro analysis of ISEcp1B-mediated mobilization of naturally occurring beta-lactamase gene blaCTX-M of Kluyvera ascorbata. Antimicrobial agents and chemotherapy 50, 1282–1286, 10.1128/AAC.50.4.1282-1286.2006 (2006).

  34. 34.

    , , & Emergence of oxacillinase-mediated resistance to imipenem in Klebsiella pneumoniae. Antimicrobial agents and chemotherapy 48, 15–22 (2004).

  35. 35.

    et al. Genetic and biochemical characterisation of OXA-232, a carbapenem-hydrolysing class D beta-lactamase from Enterobacteriaceae. International journal of antimicrobial agents 41, 325–329, 10.1016/j.ijantimicag.2012.11.007 (2013).

  36. 36.

    The pleiotropic two-component regulatory system PhoP-PhoQ. Journal of bacteriology 183, 1835–1842, 10.1128/JB.183.6.1835-1842.2001 (2001).

  37. 37.

    et al. Genomic analysis of the emergence and evolution of multidrug resistance during a Klebsiella pneumoniae outbreak including carbapenem and colistin resistance. The Journal of antimicrobial chemotherapy 69, 632–636, 10.1093/jac/dkt419 (2014).

  38. 38.

    , , , & OXA-181-producing Klebsiella pneumoniae establishing in Singapore. BMC infectious diseases 13, 58, 10.1186/1471-2334-13-58 (2013).

  39. 39.

    et al. Increased resistance to multiple drugs by introduction of the Enterobacter cloacae romA gene into OmpF porin-deficient mutants of Escherichia coli K-12. Antimicrobial agents and chemotherapy 35, 2155–2158 (1991).

  40. 40.

    et al. Expansion and countrywide dissemination of ST11, ST15 and ST147 ciprofloxacin-resistant CTX-M-15-type beta-lactamase-producing Klebsiella pneumoniae epidemic clones in Hungary in 2005—the new ‘MRSAs’? The Journal of antimicrobial chemotherapy 62, 978–985, 10.1093/jac/dkn287 (2008).

  41. 41.

    et al. An update of the evolving epidemic of blaKPC-2-carrying Klebsiella pneumoniae in Greece (2009-10). The Journal of antimicrobial chemotherapy 66, 1510–1513, 10.1093/jac/dkr166 (2011).

  42. 42.

    et al. KPC-mediated resistance in Klebsiella pneumoniae in two hospitals in Padua, Italy, June 2009-December 2011: massive spreading of a KPC-3-encoding plasmid and involvement of non-intensive care units. Gut Pathog 4, 7, 10.1186/1757-4749-4-7 (2012).

  43. 43.

    et al. Dissemination of blaVIM in Greece at the peak of the epidemic of 2005-2006: clonal expansion of Klebsiella pneumoniae clonal complex 147. Clin Microbiol Infect 20, 34–37, 10.1111/1469-0691.12187 (2014).

  44. 44.

    et al. Outbreak of infections due to KPC-2-producing Klebsiella pneumoniae in a hospital in Crete (Greece). The Journal of infection 58, 213–219, 10.1016/j.jinf.2009.01.010 (2009).

  45. 45.

    et al. KPC-2-producing Klebsiella pneumoniae infections in Greek hospitals are mainly due to a hyperepidemic clone. Euro surveillance: bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin 14, 69–73 (2009).

  46. 46.

    et al. The ongoing challenge of acquired carbapenemases: a hospital outbreak of Klebsiella pneumoniae simultaneously producing VIM-1 and KPC-2. International journal of antimicrobial agents 36, 190–191, 10.1016/j.ijantimicag.2010.04.002 (2010).

  47. 47.

    , , & Genetic features of blaNDM-1-positive Enterobacteriaceae. Antimicrobial agents and chemotherapy 55, 5403–5407, 10.1128/AAC.00585-11 (2011).

  48. 48.

    et al. Diverse sequence types of Klebsiella pneumoniae contribute to the dissemination of blaNDM-1 in India, Sweden, and the United Kingdom. Antimicrobial agents and chemotherapy 56, 2735–2738, 10.1128/AAC.06142-11 (2012).

  49. 49.

    , , , & The characteristics of NDM-producing Klebsiella pneumoniae from Canada. Diagnostic microbiology and infectious disease 71, 106–109, 10.1016/j.diagmicrobio.2011.06.013 (2011).

  50. 50.

    , , & Molecular mechanisms of beta-lactam resistance in carbapenemase-producing Klebsiella pneumoniae from Sri Lanka. Journal of medical microbiology 63, 1087–1092, 10.1099/jmm.0.076760-0 (2014).

  51. 51.

    , , , & Epidemic Klebsiella pneumoniae ST258 is a hybrid strain. mBio 5, e01355–01314, 10.1128/mBio.01355-14 (2014).

  52. 52.

    et al. Molecular characterization of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in the countries of the Gulf cooperation council: dominance of OXA-48 and NDM producers. Antimicrob Agents Chemother 58, 3085–3090, 10.1128/AAC.02050-13 (2014).

  53. 53.

    , , & L. beta-Lactamase production in key gram-negative pathogen isolates from the Arabian Peninsula. Clin Microbiol Rev 26, 361–380, 10.1128/CMR.00096-12 (2013).

  54. 54.

    et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. The Lancet. Infectious diseases 10, 597–602, 10.1016/S1473-3099(10)70143-2 (2010).

  55. 55.

    , , & Country-to-country transfer of patients and the risk of multi-resistant bacterial infection. Clin Infect Dis 53, 49–56, 10.1093/cid/cir273 (2011).

  56. 56.

    European Society of Clinical Microbiology and Infectious Diseases, European Committee on Antimicrobial Susceptibility Testing-Clinical Breakpoints (2014). Available at: . (Accessed: 7th July 2015).

  57. 57.

    Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement M100-S21. Wayne, PA: CLSI, 2011. Available at . (Accessed: 27th January 2011).

  58. 58.

    et al. Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data. Nat methods 10, 563–569, 10.1038/nmeth.2474 (2013).

  59. 59.

    et al. Identification of acquired antimicrobial resistance genes. The Journal of antimicrobial chemotherapy 67, 2640–2644, 10.1093/jac/dks261 (2012).

  60. 60.

    & ARDB—Antibiotic Resistance Genes Database. Nucleic Acids Res 37, D443–447, 10.1093/nar/gkn656 (2009).

  61. 61.

    et al. The comprehensive antibiotic resistance database. Antimicrobial agents and chemotherapy 57, 3348–3357, 10.1128/AAC.00419-13 (2013).

  62. 62.

    , , , & ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res 34, D32–36, 10.1093/nar/gkj014 (2006).

  63. 63.

    et al. INTEGRALL: a database and search engine for integrons, integrases and gene cassettes. Bioinformatics 25, 1096–1098, 10.1093/bioinformatics/btp105 (2009).

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We thank Robert Bonomo and Yohei Doi for their critical review of the manuscript. This work was supported by grants from the Australian National Health and Medical Research Council (APP1033799) and from the University of Malaya High Impact Research (HIR) Grants (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, Grant No. H-50001-A000027 and H00001-A000001). HMZ is academically sponsored by the government of Saudi Arabia to pursue postgraduate studies in the field of clinical microbiology and infectious diseases. SAB is supported by an NHMRC Career Development Fellowship (APP1090456) and MAS by an Australian Research Council Future Fellowship (FT100100662).

Author information

Author notes

    • Hosam M. Zowawi
    •  & Brian M. Forde

    These authors contributed equally to this work.


  1. The University of Queensland, Centre for Clinical Research (UQCCR), Herston QLD 4029, Australia

    • Hosam M. Zowawi
    •  & David L. Paterson
  2. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Australia

    • Hosam M. Zowawi
    • , Brian M. Forde
    • , Mark A. Schembri
    • , Scott A. Beatson
    •  & David L. Paterson
  3. College of Medicine, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia

    • Hosam M. Zowawi
  4. World Health Organization Collaborating Centre for Infection Prevention and Control, and the Gulf Cooperation Council Center for Infection Control, Riyadh, Saudi Arabia

    • Hosam M. Zowawi
  5. School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, QLD 4072, Australia

    • Brian M. Forde
    • , Mark A. Schembri
    •  & Scott A. Beatson
  6. Pathology and Laboratory Medicine Department at Sheikh Khalifa General Hospital, Umm Al Quwain, United Arab Emirates

    • Mubarak Alfaresi
  7. Urology Department, Sheikh Khalifa General Hospital, Umm Al Quwain, United Arab Emirates

    • Abdulqadir Alzarouni
    •  & Yasser Farahat
  8. Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

    • Teik-Min Chong
    • , Wai-Fong Yin
    •  & Kok-Gan Chan
  9. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia

    • Jian Li


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H.M.Z., B.M.F., M.A.S., S.A.B. and D.L.P. designed the study, interpreted the results and wrote the manuscript. H.M.Z., B.M.F., M.A., A.A., Y.F. and J.L. collected the data. H.M.Z., B.M.F., M.A.S., S.A.B., D.L.P., T-M.C., W-F.Y. and K-G.C. performed the analysis.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Scott A. Beatson or David L. Paterson.

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