Whole Genome Sequencing of Klebsiella pneumoniae Strain Unravels a New Model for the Development of Extensive Drug Resistance in Enterobacteriaceae



Mubarak Alfaresi*
College of Medicine, University of Sharjah, Sharjah, UAE


Article Metrics

CrossRef Citations:
0
Total Statistics:

Full-Text HTML Views: 608
Abstract HTML Views: 490
PDF Downloads: 220
ePub Downloads: 164
Total Views/Downloads: 1482
Unique Statistics:

Full-Text HTML Views: 362
Abstract HTML Views: 215
PDF Downloads: 168
ePub Downloads: 120
Total Views/Downloads: 865



© 2018 Mubarak Alfaresi.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the College of Medicine, University of Sharjah, Sharjah, UAE; E-mail: uaenow@eim.ae


Abstract

Introduction:

Increased incidence of carbapenem-resistant Enterobacteriaceae (CRE) has been reported worldwide. The WHO warns about the imminent risk to global health if the spread of resistant bacteria is not contained.

Materials and Methods:

Here, single molecule real time sequencing was used to analyse the whole genome and resistome of SKGH01, a strain of Klebsiella pneumoniae.

Results and Discussions:

The data showed that SKGH01 was resistant to all commercially available antibiotics. A complete account of extensively drug-resistant (XDR) CRE at a genomic level and the entire location map of all antibiotic resistance components are here presented. Additionally, this work proposes a model of XDR acquisition in Enterobacteriaceae.

Keywords: Klebsiella pneumoniae, Extensive drug resistance (XDR), Whole genome sequencing, Antibiotics, WHO, Enterobacteriaceae.



1. INTRODUCTION

Klebsiella pneumoniae of the Enterobacteriaceae family is a non-motile, rod-shaped, Gram-negative bacterium and it is one of the primary causes of hospital-acquired infections globally [1]. K. pneumoniae genomes have a strong virulence and a wide array of resistance factors that make them a major source of antimicrobial resistance genes [2]. The K. pneumoniae that produce carbapenemase (KPC-KP) are the most challenging pathogens. They exhibit extensive drug-resistant phenotypes and high potential for rapid spread having an overwhelming impact on morbidity and mortality rates [3]. Colistin and polymyxin B are antimicrobial agents that, for the most part, are still active against KPC-KP [4]. However, the emergence of polymyxin-resistant KPC-KP has recurrently been reported [5]. In K. pneumoniae, resistance to cationic antimicrobial agents is facilitated via lipopolysaccharide (LPS) sequence alterations driven by the pbgPE operon products, which are highly conserved among Enterobacteriaceae [6, 7]. The PhoQ/PhoP and PmrAB signalling systems positively regulate the pbgPE operon [7]. Activation of the PhoQ/PhoP signalling system induces production of a transmembrane regulatory protein called MgrB. The protein acts as a negative feedback loop on this signalling system by interacting with the PhoQ sensor kinase [8]. The MgrB protein has been shown to have this regulatory function in Salmonella enterica, Escherichia coli as well as Yersinia pestis and thus might also be conserved in other species, including K. pneumoniae [8].

2. MATERIAL AND METHODS

The Hospital Medical Executive Committee approved the study. The SKGH01 strain was isolated from an 80-year-old man with urinary tract infection. The species was characterised with the VITEK II compact GN system (bioM´erieux, France). For the antimicrobial susceptibility testing the VITEK II N211 system (bioM´erieux) and the E-test method were used. Breakpoints published by the Clinical and Laboratory Standards Institute were applied to determine the susceptibility to the tested antibiotics and the European Committee for Antibiotic Susceptibility Testing breakpoints in the E-test were used to determine the minimum inhibitory concentration of colistin. K. pneumoniae SKGH01 genome was sequenced with the Pacific Biosciences (PacBio, Inc., CA) RS II Single-Molecule Real Time (SMRT) kit. Bell template libraries were prepared using the Template Preparation Kit (PacBio). A single, streamlined protocol was used to create libraries of varying insert lengths, from 250 bp to 20,000 bp. The PacBio SMRT analysis software suite (v. 3.0) and hierarchical genome assembly process were used for de novo genome assembly. For the gene calling and automatic functional annotation of SKGH01 chromosome and plasmids the Prokka v1.12b (Vicbioinformatics, Australia) software was used. ResFinder and PlasmidFinder with data from the Center for Genomic Epidemiology (CGE) were employed to analyse the antimicrobial resistance genes and plasmid types. The Antibiotic Resistance Genes Database [9] and the Comprehensive Antimicrobial Resistance Database [10] were compared to all the predicted coding regions in order to screen the outstanding antimicrobial resistance genes. The insertion sequences (IS) in the genome were identified with the online tool, ISfinder 2 (version 2016-05-27). Closely related bacterial genomes were identified with the Microbial Nucleotide BLAST program. The search set consisted of complete genomes of K. pneumoniae (taxid: 573) available in the NCBI database. The BLAST search produced 48 significant hits, with overall similarities between 95% and 99%, and coverages between 85% and 98%. A genome tree was built, which comprised SKGH01 and 40 related strains from NCBI database (accession date: 10/05/16).

3. RESULTS AND DISCUSSION

The data showed that SKGH01 is a true extensively Drug-Resistant (XDR) strain to ampicillin, ampicillin-clavulanic acid, piperacillin-tazobactam, cefotaxime, ceftazidime, cefepime, aztreonam, meropenem, cotrimoxazole, amikacin, gentamicin, and colistin. A total of 6 contigs representing 6,088,457 bases (GC content 56.54%, N50=10,230) were obtained from assembled sequences of strain SKGH01 (Table S1). 6,034 genes (total), 5,907 CDS (total), 5,777 genes (coding), and 127 tRNAs genes were annotated for final contigs. The complete genome of K. pneumoniae SKGH01 consists of a circular chromosome 5,490,611 base-pairs in length with an average G-C content of 56.4%, four circular plasmids. The complete genome of strain SKGH01 consisted of a circular chromosome (5,490,611 base-pairs long) with an average G-C content of 56.4%, and four circular plasmids. Most of the genes for acquired resistance to antibiotics were positioned on the chromosome. The complete resistomes of strain SKGH01 are presented in Table 1. The insertion sequence, ISEcp1 (synonym, ISEc9) was found in four and blaOXA-181 in three places on the SKGH01 chromosome. The search for the (partial) protein sequence encoded by mgrB was performed. The most significant tblast match was a 42-amino acid, 5’ partial sequence of mgrB, which corresponded to the first ISEcp1 position identified on the SKGH01 chromosome. The remaining 3’ partial sequence of mgrB was identified with a manual search. Another manual search identified left- and right-flanking, inverted repeats (IRL and IRR, respectively) located at the first ISEcp1 position on the chromosome. We also found two alternative IRRs (IRRalts), which produced the insertions ISEcp1-blaOXA-181-IRRalt1 and ISEcp1-blaOXA-181-IRRalt2. One of these insertions led to the inactivation of the mgrB gene (Fig. S1). 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-181 [11].

Table 1. Resistome analysis for the SKGH01 strain of K. pneumoniae.
START STOP Gene Identity %* Associated Resistance
2637986 2638846 shv-11 100 beta-lactam resistance gene
1544531 1545253 baeR 91 aminocoumarin resistance gene; aminoglycoside resistance gene;
2009887 2010294 h-ns 94 macrolide resistance gene; fluoroquinolone resistance gene; tetracycline resistance gene; beta-lactam resistance gene
253419 254558 acrE 75 beta-lactam resistance gene; fluoroquinolone resistance gene
489403 490224 bacA 89 peptide antibiotic resistance gene
1546725 1548140 mdtD 84 efflux pump conferring antibiotic resistance
95184 95666 dfrA14 99 trimethoprim resistance gene
201118 201750 crp 99 macrolide resistance gene; beta-lactam resistance gene; fluoroquinolone resistance gene
59699 60490 aadA25 99 antibiotic inactivation enzyme; aminoglycoside resistance gene
98949 99593 qnrB1 100 antibiotic target protection protein; fluoroquinolone resistance gene
2392402 2392854 arr-2 100 rifampin resistance gene
3410534 3411766 mdfA 85 efflux pump conferring antibiotic resistance
4527671 4528912 mdtM 75 efflux pump conferring antibiotic resistance
2389793 2390347 aac(6')-Ib9 99 aminoglycoside resistance gene
4436582 4437451 robA 82 chloramphenicol resistance gene; fluoroquinolone resistance gene; tetracycline resistance gene; rifampin resistance gene; beta-lactam resistance gene
1856894 1857691 oxa-181 100 beta-lactam resistance gene
3487775 3489415 pmrC 70 polymyxin resistance gene
53618 54256 cat 100 chloramphenicol resistance gene
945981 947153 emrA 81 efflux pump conferring antibiotic resistance; fluoroquinolone resistance gene
86466 88451 arnA 77 polymyxin resistance gene
1190757 1193870 mexD 91 chloramphenicol resistance gene; macrolide resistance gene; fluoroquinolone resistance gene
514487 516745 parC 94 fluoroquinolone resistance gene
5231835 5233013 mdtL 75 efflux pump conferring antibiotic resistance
4048769 4049566 oxa-181 100 beta-lactam resistance gene
250296 253406 mexD 86 chloramphenicol resistance gene; macrolide resistance gene; fluoroquinolone resistance gene
1554342 1555580 mdtA 79 aminocoumarin resistance gene
3489415 3490086 pmrA 78 polymyxin resistance gene
947279 947809 emrR 92 fluoroquinolone resistance gene;
2617505 2617882 marA 92 chloramphenicol resistance gene; fluoroquinolone resistance gene; tetracycline resistance gene; rifampin resistance gene; beta-lactam resistance gene
5125322 5126695 cpxA 94 aminocoumarin resistance gene; aminoglycoside resistance gene
3123628 3124299 phoP 91 polymyxin resistance gene; macrolide resistance gene
944427 945965 emrY 94 efflux pump conferring antibiotic resistance; tetracycline resistance gene
2219510 2220883 mdtK 87 fluoroquinolone resistance gene
5124627 5125325 cpxR 94 efflux pump conferring antibiotic resistance; aminocoumarin resistance gene; aminoglycoside resistance gene; gene modulating antibiotic efflux
3892696 3893781 acrA 86 chloramphenicol resistance gene; fluoroquinolone resistance gene; efflux pump conferring antibiotic resistance; tetracycline resistance gene; rifampin resistance gene; beta-lactam resistance gene
3308977 3309852 ctx-M-15 100 antibiotic inactivation enzyme; beta-lactam resistance gene
5075084 5075881 oxa-181 100 beta-lactam resistance gene
5278571 5279755 emrD 99 efflux pump conferring antibiotic resistance
4543523 4543945 fosA5 97 fosfomycin resistance gene
2388603 2389382 rmtF 100 aminoglycoside resistance gene
85486 86469 pmrF 83 polymyxin resistance gene; gene altering cell wall charge conferring antibiotic resistance
3757172 3757513 ramA 92 chloramphenicol resistance gene; fluoroquinolone resistance gene; tetracycline resistance gene; rifampin resistance gene; beta-lactam resistance gene
1412712 1415345 gyrA 92 fluoroquinolone resistance gene
3875438 3877114 rosB 73 polymyxin resistance gene
60898 61395 dfrA12 100 antibiotic target replacement protein; trimethoprim resistance gene
3190885 3192093 mdtH 85 efflux pump conferring antibiotic resistance
2386317 2386955 cat 100 chloramphenicol resistance gene
3124299 3125765 phoQ 81 polymyxin resistance gene; macrolide resistance gene
57095 57649 aac(6')-Ib9 99 antibiotic inactivation enzyme; aminoglycoside resistance gene
503638 505116 tolC 83 chloramphenicol resistance gene; macrolide resistance gene; fluoroquinolone resistance gene; aminocoumarin resistance gene; tetracycline resistance gene; rifampin resistance gene; beta-lactam resistance gene
3198868 3199821 mdtG 84 efflux pump conferring antibiotic resistance
1598283 1599449 pmrE 82 polymyxin resistance gene
3893804 3896950 mexD 92 efflux pump conferring antibiotic resistance; chloramphenicol resistance gene; macrolide resistance gene; fluoroquinolone resistance gene
55905 56684 rmtF 100 aminoglycoside resistance gene
1545250 1546728 baeS 77 aminocoumarin resistance gene; aminoglycoside resistance gene
987867 991019 oqxB 98.5 Quinolone resistance
991043 992218 oqxA 99.5 Quinolone resistance
* Percentage given by the Antibiotic Resistance Genes Database (ARDB) and the Comprehensive Antimicrobial Resistance Database (CARD) when compared with known resistance genes database.

CONCLUSION

Here, using the long-read sequencing technology multiple, identical, carbapenem-resistance elements in the K. pneumoniae strain SKGH01 genome were identified. Based on the data, a new model explaining how XDR in this K. pneumoniae isolate developed via colistin resistance by mgrB gene disruption by ISEcp1. In this model, new resistance was driven by the existing mobile resistance determinants. Additionally, the data showed that ISEcp1 sequence interrupted the negative feedback regulator of the PhoQ-PhoP signalling system, namely the mgrB gene. Interestingly, this disruption was previously shown to drive the KPC-KPs acquired colistin resistance. Indeed, interruption of the mgrB gene caused upregulation of PhoQ-PhoP signalling; in turn, this upregulation activated the Pmr system, which was responsible for modifying the LPS target of polymyxin [12].

NUCLEOTIDE SEQUENCE ACCESSION NUMBER

The nucleotide sequence data are available in the GenBank nucleotide database, under accession numbers CP015500.1 to CP015505.1.

AVAILABILITY OF DATA AND MATERIALS

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

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was approved by the hospital Medical executive committee.

HUMAN AND ANIMAL RIGHTS

Animals did not participate in this research. All human research procedures followed were in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national), and with the Helsinki Declaration of 1975, as revised in 2008.

CONSENT FOR PUBLICATION

Consent for publication is obtained.

CONFLICT OF INTEREST

The author declare that they have no competing interests.

ACKNOWLEDGMENTS

Part of the Bioinformatics work was provided by omics2view.consulting GbR, Kiel (Germany).

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publishers Web site along with the published article.

Download File


REFERENCES

[1] Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11(4): 589-603.
[2] Centres for Disease Control & Prevention. Antibiotic Resistance Threats in the United States 2013.
[3] Munoz-Price LS, Poirel L, Bonomo RA, et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect Dis 2013; 13(9): 785-96.
[4] Petrosillo N, Giannella M, Lewis R, Viale P. Treatment of carbapenem-resistant Klebsiella pneumoniae: the state of the art. Expert Rev Anti Infect Ther 2013; 11(2): 159-77.
[5] Bogdanovich T, Adams-Haduch JM, Tian GB, et al. Colistin-resistant, Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae belonging to the international epidemic clone ST258. Clin Infect Dis 2011; 53(4): 373-6.
[6] Helander IM, Kato Y, Kilpeläinen I, et al. Characterization of lipopolysaccharides of polymyxin-resistant and polymyxin-sensitive Klebsiella pneumoniae O3. Eur J Biochem 1996; 237(1): 272-8.
[7] 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.
[8] Lippa AM, Goulian M. Feedback inhibition in the PhoQ/PhoP signaling system by a membrane peptide. PLoS Genet 2009; 5(12): e1000788.
[9] Liu B, Pop M. ARDB-Antibiotic resistance genes database. Nucleic Acids Res 2009; 37(Database issue): D443-7.
[10] McArthur AG, Waglechner N, Nizam F, et al. The comprehensive antibiotic resistance database. Antimicrob Agents Chemother 2013; 57(7): 3348-57.
[11] Zowawi HM, Forde BM, Alfaresi M, et al. Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae. Sci Rep 2015; 5: 15082.
[12] Cannatelli A, Di Pilato V, Giani T, et al. In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment. Antimicrob Agents Chemother 2014; 58(8): 4399-403.