College of Medicine, University of Sharjah, Sharjah, UAE
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.
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* Address correspondence to this author at the College of Medicine, University of Sharjah, Sharjah, UAE; E-mail: email@example.com
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 [1Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11(4): 589-603.[PMID: 9767057] ]. K. pneumoniae genomes have a strong virulence and a wide array of resistance factors that make them a major source of antimicrobial resistance genes [2Centres for Disease Control & Prevention. Antibiotic Resistance Threats in the United States 2013.]. 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 [3Munoz-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.[http://dx.doi.org/10.1016/S1473-3099(13)70190-7] [PMID: 23969216] ]. Colistin and polymyxin B are antimicrobial agents that, for the most part, are still active against KPC-KP [4Petrosillo 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.[http://dx.doi.org/10.1586/eri.12.162] [PMID: 23409822] ]. However, the emergence of polymyxin-resistant KPC-KP has recurrently been reported [5Bogdanovich 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.[http://dx.doi.org/10.1093/cid/cir401] [PMID: 21810751] ]. 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 [6Helander 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.[http://dx.doi.org/10.1111/j.1432-1033.1996.0272n.x] [PMID: 8620884] , 7Cheng 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.[http://dx.doi.org/10.1186/1423-0127-17-60] [PMID: 20653976] ]. The PhoQ/PhoP and PmrAB signalling systems positively regulate the pbgPE operon [7Cheng 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.[http://dx.doi.org/10.1186/1423-0127-17-60] [PMID: 20653976] ]. 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 [8Lippa AM, Goulian M. Feedback inhibition in the PhoQ/PhoP signaling system by a membrane peptide. PLoS Genet 2009; 5(12): e1000788.[http://dx.doi.org/10.1371/journal.pgen.1000788] [PMID: 20041203] ]. 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 [8Lippa AM, Goulian M. Feedback inhibition in the PhoQ/PhoP signaling system by a membrane peptide. PLoS Genet 2009; 5(12): e1000788.[http://dx.doi.org/10.1371/journal.pgen.1000788] [PMID: 20041203] ].
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 [9Liu B, Pop M. ARDB-Antibiotic resistance genes database. Nucleic Acids Res 2009; 37(Database issue): D443-7.[http://dx.doi.org/10.1093/nar/gkn656] [PMID: 18832362] ] and the Comprehensive Antimicrobial Resistance Database [10McArthur AG, Waglechner N, Nizam F, et al. The comprehensive antibiotic resistance database. Antimicrob Agents Chemother 2013; 57(7): 3348-57.[http://dx.doi.org/10.1128/AAC.00419-13] [PMID: 23650175] ] 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 [11Zowawi HM, Forde BM, Alfaresi M, et al. Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae. Sci Rep 2015; 5: 15082.[http://dx.doi.org/10.1038/srep15082] [PMID: 26478520] ].
Table 1 Resistome analysis for the SKGH01 strain of K. pneumoniae.
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 [12Cannatelli 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.[http://dx.doi.org/10.1128/AAC.02555-14] [PMID: 24841267] ].
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.
Part of the Bioinformatics work was provided by omics2view.consulting GbR, Kiel (Germany).
Supplementary material is available on the publishers Web site along with the published article.
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.[http://dx.doi.org/10.1586/eri.12.162] [PMID: 23409822]
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.[http://dx.doi.org/10.1093/cid/cir401] [PMID: 21810751]
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.[http://dx.doi.org/10.1128/AAC.02555-14] [PMID: 24841267]