JAC Advance Access published online on September 23, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn397
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Original research |
Complete nucleotide sequence of pKP96, a 67850 bp multiresistance plasmid encoding qnrA1, aac(6')-Ib-cr and blaCTX-M-24 from Klebsiella pneumoniae
1 State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China 2 James D. Watson Institute of Genome Sciences, Zhejiang University, Hangzhou, Zhejiang 310008, China
* Corresponding author. Tel: +86-571-8723-6421; Fax: +86-571-8723-6423; E-mail: yvys119{at}163.com
Received 19 May 2008; returned 3 July 2008; revised 23 August 2008; accepted 27 August 2008
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Abstract |
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Objectives: The multiresistance plasmid pKP96 from Klebsiella pneumoniae was sequenced completely and analysed concerning its genetic environment and distributing of antimicrobial resistance genes.
Methods: The complete sequence of the plasmid was determined using a whole-genome shotgun approach. MICs of 13 antimicrobial agents were determined using Etests. A conjugation experiment was performed in liquid medium.
Results: pKP96 is a circularly closed 67 850 bp multiresistance plasmid with an IncN incompatibility group. Seventy putative genes were identified according to the annotation of the finished sequence. The backbone region of the plasmid, comprising the conjugal transfer and plasmid replication regions, showed 91% identity to the IncN plasmid R46. Several mobile elements were found to be inserted into pKP96 together with antimicrobial resistance genes, including qnrA1, aac(6')-Ib-cr and blaCTX-M-24.
Conclusions: Plasmid pKP96 is a chimera that has acquired its multiple antimicrobial resistance determinants horizontally from different sources. It may have evolved from an ancestor plasmid similar to R46 through the stepwise events of integration or recombination.
Key Words: integrons , transposons , horizontal gene transfer
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Introduction |
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Bacterial plasmids play an important role in horizontal gene transfer, especially for the spread of antimicrobial resistance genes. Genes that are often located on conjugative plasmids include those encoding for extended-spectrum β-lactamases (ESBLs) and determinants conferring resistance to aminoglycosides, tetracycline and quinolones.1,2 The capability of plasmids to spread by conjugation greatly enhances the dissemination of these resistance genes and raises a series of clinical problems.
In the past decade, CTX-M enzymes have become the most prevalent ESBLs in both nosocomial and community settings.3 To date, over 70 CTX-M-type β-lactamases have been identified, showing the intensive evolution of this group (http://www.lahey.org/studies/).
The plasmid-mediated quinolone resistance determinant qnrA was described in several reports in association with ISCR elements inserted in sul1-type class 1 integrons.4,5 Recently, another mechanism, AAC(6')-Ib-cr, a variant aminoglycoside acetyltransferase capable of modifying ciprofloxacin and reducing its activity, was recognized to be widely prevalent and circulated together with qnr genes.6,7
Plasmid-mediated quinolone resistance genes have often been reported to be co-associated with genes encoding ESBLs,5,7 especially with blaCTX-M.8 Here, we report the complete nucleotide sequence of plasmid pKP96, which carries the plasmid-mediated quinolone resistance determinants qnrA1, aac(6')-Ib-cr and the blaCTX-M-24 ESBL coding gene, from Klebsiella pneumoniae isolated in China.
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Materials and methods |
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Bacterial strain
K. pneumoniae KP96 was isolated from a sputum specimen in the First Affiliated Hospital, College of Medicine, Zhejiang University in China in 2002 and formed part of a study investigating the prevalence of qnr and aac(6')-Ib-cr in ESBL-producing K. pneumoniae and Escherichia coli.7 Species identification was confirmed using a Vitek Gram-Negative Plus Identification card (bioMérieux Vitek, Inc., Hazelwood, MO, USA).
The plasmid pKP96 was extracted from a clinical strain KP96 by using a QIAGEN Large-Construct Kit (Qiagen, Germany), according to the manufacturer's instructions. E. coli V517 was used as a size standard to evaluate the plasmid size by electrophoresis on a 0.8% agarose gel. A conjugation experiment was performed using the azide-resistant E. coli strain J53AzR as the recipient, as described previously.4 Transconjugant CKP96 was selected on Mueller–Hinton agar containing sodium azide (100 mg/L) and cefotaxime (2 mg/L).
Antimicrobial susceptibility testing
The MICs of ciprofloxacin, ofloxacin, norfloxacin, moxifloxacin, cefotaxime, ceftazidime, streptomycin, amikacin, gentamicin, ampicillin, tetracycline, fosfomycin and sulfamethoxazole for the clinical strain KP96 and the transconjugant CKP96 were determined using Etests (AB Biodisk, Solna, Sweden), following the manufacturer's instructions. E. coli ATCC 25922 was used as the quality control strain.
Shotgun sequencing, assembly and analysis of plasmid pKP96
The plasmid sequences were determined by using a whole-genome shotgun approach. DNA for plasmid pKP96 was randomly sheared by ultrasonication, and the 1.5–3.0 kb size fragments were cloned into vector pUC18 (Takara Bio, Otsu, Japan). End sequencing was performed by using BigDye Terminator 3.1 chemistry and a 3730XL sequencer (Applied Biosystems, Darmstadt, Germany). Sequence gaps were filled by primer walking on linking clones and by sequencing of the PCR products from the plasmid DNA. The resulting sequence data were assembled by using the Phred/Phrap/Consed software suite obtained from the University of Washington, Seattle, WA, USA.9–11 GLIMMER 2.13 and GeneMark.hmm 2.4 were used to identify putative open reading frames (ORFs), with those less than 30 translated amino acids abandoned.12,13 Nucleotide and amino acid sequences were analysed and compared by use of the BLAST program (National Center for Biotechnology Information).
Nucleotide sequence accession number
The annotated sequence of pKP96 has been submitted to the GenBank nucleotide sequence database under accession number EU195449.
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General features of plasmid pKP96
A single plasmid designated pKP96 harboured by the strain KP96 was experimentally evaluated to be 
60 kb in size and was transferred successfully to E. coli J53 by conjugation in liquid medium, reaching a value of 10–8 transconjugants/donor. Whole-genome sequencing revealed that the plasmid is a circularly closed 67 850 bp DNA sequence (Figure 1) with an average G + C content of 52.6%, ranging from 26% to 74% depending on the sliding window of 1000 bp and a step size of 50 bp. Annotation of the finished sequence data revealed that pKP96 contains 70 putative genes, 65 of which encode proteins with homology to known functions. Among the 65 known function genes, 19 have possible roles in transposition and recombination, 9 genes confer resistance to antibiotics including ampicillin, cefotaxime, ciprofloxacin, gentamicin, as well as tetracycline, fosfomycin and sulfamethoxazole (MICs in Table 1), 11 compose the integron-related element and the remainder were predicted to encode proteins involving replication and maintenance. More information regarding these ORFs is listed in Table S1 [available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/)].
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From positions 32 175 to 67 850, plasmid pKP96 shows 91% identity to plasmid R46 (accession no. NC_003292 [GenBank] ) from Salmonella enterica serovar Typhimurium,14 mostly in the conjugal transfer region, modified with several regions that contain many antimicrobial resistance genes or mobile genetic elements. pKP96 probably evolved from an ancestor plasmid similar to R46 through the stepwise integration or recombination of mobile genetic elements associated with antimicrobial resistance genes.
The numbering of pKP96 commences at the first nucleotide of the ATG start codon of repA (Figure 1), a gene predicted to encode a replication protein. Only one replication region was identified in the sequence of pKP96. This replication gene (positions 1–720) was confirmed to be identical to that of the IncN replicon of R46. Next to the replicon, a GC-rich segment containing a series of direct repeats (DRs) called iterons was also detected, which are common in replication origins of Gram-negative plasmids, and presumably play a role in plasmid replication and maintenance.2
oriT and conjugal transfer system
Conjugal DNA processing in a plasmid requires plasmid-encoded proteins that interact with a short DNA sequence known as the origin of transfer (oriT) to introduce a strand-specific cleavage at a unique site referred to as the nic site.14,15 A putative oriT region has been identified in pKP96, starting at position 54 738. It carries a nic site, which is common to IncN plasmids such as R46.
Upstream of the oriT region, a ca. 19.5 kb fragment contains a complete transfer region. Genes encoding for the pKP96 conjugal transfer system are organized as a conserved gene order referring to R46, merely different in an interruption of a 3 kb partial transposon Tn1721 (positions 45 793–49 206) containing tetracycline resistance genes tet(A) and tetR(A) (Figure 2).
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The qnrA1- and aac(6')-Ib-cr-containing region
Outside the common backbone, pKP96 differs from the related IncN plasmid R46 due to the presence of a considerable amount of extra DNA such as transposons and integrons. A sul1-type class 1 integron inserted with an ISCR1 element was found in pKP96 next to the replication region. The pKP96 integron possesses a complete 5' conserved sequence (5'-CS) with terminal inverted repeats (IR), whereas its 3'-CS is truncated due to the insertion of an ISCR1 element. The complex class 1 integron consists of aac(6')-Ib-cr, qacEdelta1, sul1, ISCR1, qnrA1, ampR, orf5 and orf6, which are similar to In37 of an E. coli isolate from China4 diminished by three consecutive gene cassettes, namely blaOXA-30, catB3 and arr-3, in its variable region (Figure 3). The arrangement is similar in another complex class 1 integron named In36 from the same region as In37 of China,4 which varied in its cassettes composition (Figure 3). An IS6100 transposase gene downstream of the integron region suggested that the ISCR1-associated class 1 integron ought to be inserted by this IS element. Upon insertion, a 5 bp DR (CTGTT) was duplicated (Figure 2).
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The 14 kb blaCTX-M-24-containing region
The other segment outside the common backbone from R46 is a 14 kb (positions 17 755–32 176) blaCTX-M-24-containing region (Figure 2). It is flanked by two 9 bp DRs (CGTTTAGCC), indicating the occurrence of a transposition event. Two copies of IS10R are found at the left and right ends of the region, respectively, and the IS10R left inverted repeat (IRL) and the right inverted repeat (IRR) are present at the boundary with other regions. A putative fosfomycin-resistance gene fosA downstream of the left copy of IS10R suggests that a stepwise integration and deletion of Tn10 elements have occurred, for transposon Tn10 often carries tetracycline resistance genes but not the fosA gene. Between the fosA gene and the right copy of IS10R, a part of transposon Tn1721 with DRs (TAACC) and complete IRs is inserted (positions 20 256–30 698). Tn1721 is a member of the Tn501 subfamily of Tn3 family transposons with the tet(A) tetracycline resistance determinant. The 5' segment of Tn1721 inserted downstream of fosA consists of transposase, resolvase and putative methyl-accepting chemotaxis gene. It is interesting that the 3' segment of Tn1721 including tet(A) and tetR(A) genes (positions 45 793–49 206) inserted into the conjugal transfer region of pKP96 with DRs (AGCAA) and IRs differed from the 5' segment of Tn1721. Moreover, the 5' segment of Tn1721 is disrupted by another mobile element (positions 21 490–26 289). The element containing ISEcp1B, blaCTX-M-24 and IS903D interrupts the methyl-accepting chemotaxis gene in Tn1721 with 5 bp DRs (TAACA). A similar structure described by Poirel et al.16 suggests that mobilization and expression of blaCTX-M-24 in this case are linked to the ISEcp1B-associated transposition element.
The mosaic structure of the 14 kb region clearly suggests that it arose from multiple insertions. These events caused the acquisition of several mobile elements, some of which were acquired together with other resistance genes.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsFundingTransparency declarationsSupplementary dataReferences |
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Complete nucleotide sequencing of plasmid pKP96 from K. pneumoniae has investigated the detailed genomic structure of some important resistance genes such as qnrA1, aac(6')-Ib-cr and the blaCTX-M-24. It has provided insight into the mechanisms of dissemination of multiple antimicrobial resistance determinants in both nosocomial and community settings.
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Funding |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsFundingTransparency declarationsSupplementary dataReferences |
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This work was supported in part by the National Basic Research Programme 973 of China (no. 2005CB523101) and the Natural Science Foundation of Zhejiang Province (no. Y206123).
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Transparency declarations |
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None to declare.
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Supplementary data |
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Table S1 is available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
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Acknowledgements |
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We are grateful to Minggui Wang, PhD, Huashan Hospital, Fudan University, for kindly providing the E. coli strain J53AzR.
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References |
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1 . Golebiewski M, Kern-Zdanowicz I, Zienkiewicz M, et al. Complete nucleotide sequence of the pCTX-M3 plasmid and its involvement in spread of the extended-spectrum β-lactamase gene blaCTX-M-3. Antimicrob Agents Chemother (2007) 51:3789–95.
2
.
Chen YT, Shu HY, Li LH, et al. Complete nucleotide sequence of pK245, a 98-kilobase plasmid conferring quinolone resistance and extended-spectrum-β-lactamase activity in a clinical Klebsiella pneumoniae isolate. Antimicrob Agents Chemother (2006) 50:3861–6.
3 . Canton R, Coque TM. The CTX-M β-lactamase pandemic. Curr Opin Microbiol (2006) 9:466–75.[CrossRef][Web of Science][Medline]
4
.
Wang M, Tran JH, Jacoby GA, et al. Plasmid-mediated quinolone resistance in clinical isolates of Escherichia coli from Shanghai, China. Antimicrob Agents Chemother (2003) 47:2242–8.
5
.
Wu JJ, Ko WC, Tsai SH, et al. Prevalence of plasmid-mediated quinolone resistance determinants QnrA, QnrB, and QnrS among clinical isolates of Enterobacter cloacae in a Taiwanese hospital. Antimicrob Agents Chemother (2007) 51:1223–7.
6 . Robicsek A, Strahilevitz J, Jacoby GA, et al. Fluoroquinolone- modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med (2006) 12:83–8.[CrossRef][Web of Science][Medline]
7
.
Jiang Y, Zhou Z, Qian Y, et al. Plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in extended-spectrum β-lactamase-producing Escherichia coli and Klebsiella pneumoniae in China. J Antimicrob Chemother (2008) 61:1003–6.
8
.
Lavigne JP, Marchandin H, Delmas J, et al. qnrA in CTX-M-producing Escherichia coli isolates from France. Antimicrob Agents Chemother (2006) 50:4224–8.
9
.
Ewing B, Hillier L, Wendl MC, et al. Base-calling of automated sequencer traces using Phred. I. Accuracy assessment. Genome Res (1998) 8:175–85.
10
.
Ewing B, Green P. Base-calling of automated sequencer traces using Phred. II. Error probabilities. Genome Res (1998) 8:186–94.
11
.
Gordon D, Abajian C, Green P. Consed: a graphical tool for sequence finishing. Genome Res (1998) 8:195–202.
12
.
Delcher AL, Harmon D, Kasif S, et al. Improved microbial gene identification with GLIMMER. Nucleic Acids Res (1999) 27:4636–41.
13
.
Lukashin AV, Borodovsky M. GeneMark.hmm: new solutions for gene finding. Nucleic Acids Res (1998) 26:1107–15.
14
.
Paterson ES, More MI, Pillay G, et al. Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1. J Bacteriol (1999) 181:2572–83.
15
.
Inamoto S, Yoshioka Y, Ohtsubo E. Site- and strand-specific nicking in vitro at oriT by the TraY–TraI endonuclease of plasmid R100. J Biol Chem (1991) 266:10086–92.
16
.
Poirel L, Lartigue MF, Decousser JW, et al. ISEcp1B-mediated transposition of blaCTX-M in Escherichia coli. Antimicrob Agents Chemother (2005) 49:447–50.
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