JAC Advance Access published online on November 19, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn481
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Original research |
ATPase genes of diverse multidrug-resistant Acinetobacter baumannii isolates frequently harbour integrated DNA
1 Department of Infection, Immunity and Inflammation, Maurice Shock Building, University of Leicester, Leicester LE1 9HN, UK 2 Department of Clinical Microbiology, Sandringham Building, Leicester Royal Infirmary, Leicester LE1 5WW, UK 3 Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust, Queens Medical Centre, Nottingham NG7 2UH, UK 4 Laboratory of Microbial Metabolism and School of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200030, China
* Corresponding author. Tel: +44-116-223-1498; Fax: +44-116-252-5030; E-mail: kr46{at}le.ac.uk
Received 19 June 2008; returned 11 August 2008; revised 28 October 2008; accepted 31 October 2008
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Abstract |
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Objectives: The aim of the study was to determine whether ATPase genes of genetically diverse Acinetobacter baumannii isolates are disrupted by potential genomic islands.
Methods: Random amplified polymorphic DNA (RAPD)-PCR, sequence grouping and PFGE were used to investigate the genetic diversity of 50 A. baumannii isolated from various clinical specimens. PCR analysis was then used to identify isolates with a potentially disrupted ATPase gene. Representative genetically distinct isolates were further characterized by PCR mapping and chromosome walking to analyse the flanking regions of the disrupted ATPase genes.
Results: Forty-one of the 50 isolates tested appeared to contain a disrupted ATPase gene. Sequence group and PFGE data for 10 ATPase PCR-negative representative isolates confirmed substantial genetic diversity. Seven isolates contained elements with ends showing high levels of sequence similarity to one or both extremities of AbaR1, the first resistance island described in A. baumannii. A further isolate, A25, possessed a highly conserved AbaR1-like 3'-end, but a divergent, though related, 5'-terminus that exhibited near identity with a distinct locus in A. baumannii ATCC 17978. A ninth isolate (A92) possessed a completely novel sequence abutting on its 5'-ATPase remnant. Three isolates appeared to lack 3'-ATPase gene segments, as was the case with the recently sequenced strain ACICU. Thus, 8 of the 10 ATPase-negative isolates investigated in detail had ATPase genes disrupted with AbaR1-like flanking regions.
Conclusions: ATPase genes of diverse A. baumannii isolates are frequently disrupted by insertions matching AbaR1-related flanking sequences. However, the ATPase gene of isolate A92 was disrupted by a DNA sequence distinct from those found in AbaR1.
Key Words: genomic island , site-specific integration , antibiotic resistance
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Introduction |
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Acinetobacter baumannii is a major cause of outbreaks of infection, especially in the hospital setting, and poses particular therapeutic problems because of intrinsic antimicrobial resistance mechanisms and an ability to rapidly acquire multiple antimicrobial resistance genes. The rapid dissemination of acquired resistance mechanisms has led to the widespread emergence of multidrug-resistant (MDR) strains associated with therapeutic failures and consequent increased patient morbidity and mortality. A. baumannii strains are notorious for their ability to exhibit decreased cell wall permeability and/or increased constitutive expression of efflux pumps. In parallel, diverse additional mechanisms conferred by numerous acquired genes borne on plasmids, transposons and integrons have resulted in strains that are resistant to almost all classes of antimicrobial agents.1
Despite extensive research on the role of integron- and transposon-mediated resistance in the evolution of MDR A. baumannii, very little was known until recently about the larger genomic context of these elements. In 2006, whole genome sequencing of the MDR strain AYE and the fully susceptible strain SDF enabled the identification of an 86 kb resistance island (AbaR1) that had integrated within the ATPase gene of strain AYE.2 AbaR1 contained 45 of the 52 recognizable resistance genes in the entire genome, many of which had probably been acquired from other bacterial genera.2 Interestingly, an entirely unrelated 20 kb genomic island (AbaG1) devoid of any antibiotic resistance genes was present at the same location in isolate SDF. Furthermore, analysis of two other complete genome sequences published during the course of the present study yielded evidence of a likely 13 kb AbaR1 prototype structure that contained only one resistance gene (strain ATCC 17978) and a further AbaR1-related truncated structure (AbaR2) that was associated with the loss of the 3'-ATPase flanking region (strain ACICU).3,4
These data suggest that the ATPase gene in A. baumannii may represent an integration hotspot potentially serving as a reservoir for foreign genomic islands that may contain large repositories of resistance genes, integrons and/or transposons. The aim of the present study was to investigate the role of the ATPase gene as a potential hotspot for genomic integration in a collection of geographically and genetically diverse MDR A. baumannii isolates.
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Materials and methods |
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Characterization of isolates
In total, 50 clinical isolates of MDR A. baumannii from 17 different countries, identified by tRNA fingerprinting5 and the presence of the blaOXA-51-like gene,6 were initially genotyped by random amplified polymorphic DNA (RAPD) analysis using primer DAF4 (5'-CGGCAGCGCC-3')5 and PuReTaq Ready-To-Go PCR Beads (GE Healthcare Life Sciences, Little Chalfont, UK). Banding patterns were analysed using BioNumerics software version 2.0 (Applied Maths, Sint-Martens-Latem, Belgium), with similarity calculated according to the unweighted pair group method using arithmetical averages (UPGMA) and the Dice coefficient. Optimization and band position tolerance settings were 0.5% and 1%, respectively. A cut-off value of >72% was used to define individual RAPD types.5
Identification of PCR-based sequence groups (SG) was carried out using two multiplex PCR assays designed to selectively amplify group 1 or group 2 alleles of the ompA, csuE and blaOXA-51-like genes.6 Each multiplex reaction used a PuReTaq Ready-To-Go PCR Bead in a final reaction volume of 25 µL. Antimicrobial susceptibilities were established using the BSAC standardized disc susceptibility testing method.7 PFGE analysis of selected isolates was performed according to the method of Turton et al.8 Gel images were analysed using Bionumerics software v.2 with the Dice coefficient and band tolerance set at 0.5%. A dendrogram was constructed using UPGMA, with a similarity threshold of >80% being used to define isolates that belonged to the same strain.
Analysis of ATPase genes and associated integrated DNA
Primers used for PCR analysis, mapping and chromosome walking were as follows: 1F (5'-CTTAATTGCCTCTGGTCAAC-3'), 1R (5'-GCGTAGCTGACCTTTAACAT-3'), 2F (5'-TCCATTTTACCGCCACTTTC-3'), 2R (5'-TTGGGGATTCTGTCCGTAAG-3'), 3F (5'-TGTACCTGCTGTCGTCTTCG-3'), 3R (5'-CTGCTACGGCTGAAACATCC-3'), 4F (5'-TATCAGCAGCAAAACGATGG-3'), 4R (5'-AATCGATGCGGTCGAGTAAC-3'), 5F (5'-CGAGTTTGACAGAAAGGTTC-3') and 5R (5'-CGCCACCAATCCATTCTACT-3'). The locations and orientations of the primers and the sizes of the expected amplicons, based on the genome sequence of strain AYE,2 are shown in Figure 1. PCR analysis to identify potentially occupied ATPase sites, based on the failure to detect an intact ATPase gene, used primers 2F and 4R (Figure 1). Selected isolates were further investigated using a single genome-specific PCR (SGSP-PCR) chromosome walking technique.9 Separate genomic libraries were generated in plasmid pBluescript II KS (+) (Fermentas, UK) using restriction enzymes EcoRI, HindIII and HincII (Roche Diagnostics Ltd, UK) and were used as a template for PCR. SGSP-PCR amplification was performed with primers 2F or 4R and the universal vector primer T7 (5'-TAATACGACTCACTATAGGG-3'). Amplicons were analysed by partial sequencing (MWG Biotech AG, Germany) and the data obtained were compared using BlastN with the NCBI DNA sequence database. Specific pair-wise comparisons were also performed against the sequences of AbaR1 and AbaG1. The divergent 5'-ATPase junction sequences from A25 and A92 have been deposited with GenBank under the accession numbers FJ406499 and FJ406500, respectively.
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Results and discussion |
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Forty-two of the 50 MDR A. baumannii isolates examined by RAPD-PCR formed three distinct clusters [1A (n = 22), 2A (n = 13) and 3A (n = 7)] based on the previously defined >72% similarity cut-off,5 while eight isolates were outliers. Negative ATPase PCR results obtained for 41 of the 50 isolates suggested the presence of ATPase-integrated elements within these isolates. In total, 10 isolates representative of the three RAPD clusters and one outlier were selected for further investigation. PFGE analysis confirmed that these isolates had <80% similarity (data not shown). The majority of these isolates were assigned to SG1 or SG2, with one isolate (A457) identified as a member of variant SG710 and one isolate (A92) assigned to a newly recognized variant designated as SG8 (Table 1). AbaR1-based PCR-mapping and SGSP-PCR chromosome walking9 identified 5'-ATPase gene segments in all 10 A. baumannii isolates, but 3'-ATPase gene segments were only detected in seven isolates (Figure 1 and Table 1). Absence of the 3'-ATPase sequence was also observed in the MDR strain ACICU,4 suggesting that this region may be prone to deletion. PCR-mapping of all 10 isolates using AbaR1-derived tniA- and sul1-specific primers suggested that four isolates had both tniA and sul1, three had tniA only and one had sul1 only (Figure 1 and Table 1)—indicating that four isolates shared a common structural organization with both extremities of AbaR1 in isolate AYE2 while four showed structural variations.
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Short-range chromosome walking data were successfully generated for eight isolates from the 5'-ATPase gene end and for seven isolates from the 3'-ATPase end (Figure 1). Isolate A457 failed to yield data using either this strategy or PCR-mapping and was not studied further. Data for the seven isolates that were PCR-positive for the 3'-ATPase gene segment demonstrated >96% sequence identity with the matching AbaR1 junction sequence (Figure 1). Chromosome walking from the 5'-ATPase remnant revealed that the identity with the AbaR1 junction was >96% in six of the eight isolates for which data were available (Figure 1). Data corresponding to the 5'-junction in isolate A25 showed only 88% sequence identity with the AbaR1 terminal sequence, but 100% identity with the corresponding ATPase segment (Figure 1). This divergent 5'-inserted sequence exhibited near identity with a second distant locus in A. baumannii strain ATCC 17978 coding for a single hypothetical protein (ABS89982 [GenBank] ) with no identifying motifs. Sequence data from the 5'-junction for isolate A92 showed an abrupt discontinuity after 105 bp, with a 97% match to the 5'-ATPase gene segment itself, followed by completely novel sequence of 588 bp that exhibited no matches in the gene and genome databases at the nucleotide or protein level. One incomplete open reading frame was identified within this short stretch of novel sequence. It coded for a helix-turn-helix motif HTH_3 (Pfam accession no. PF01381) that is frequently found in the DNA binding proteins of bacterial plasmids and bacteriophages. Thus, in addition to data concerning AbaR1-like elements and AbaG1 (strain SDF) that have been derived by whole genome sequencing,2–4 there is now evidence for a third, entirely distinct, ATPase-associated insertion in isolate A92. It also appears that the AbaR1-like 5'-ATPase extremity can show divergence as evidenced by the version present in isolate A25.
Interestingly, all these insertions are at an identical location within the ATPase genes of A. baumannii. We also observed a 5 bp direct repeat (ACCGC) flanking the inner junctions of the ATPase gene segments in isolates where chromosomal walking data flanking the ATPase gene segment were available. This indicates a likely duplication event associated with transposition of foreign DNA into the ATPase genes as observed in both AbaR1 and AbaG1 genomic islands.2 In addition, the data revealed that AbaR1-like insertions were present in 8 of the 10 A. baumannii isolates that belonged to diverse RAPD, SG and PFGE-based lineages (Table 1). Importantly, despite these AbaR1-like insertions sharing common extremities, significant diversity is likely to exist within their internal spans, as evidenced by differences between AbaR1,2 AbaR24 and the ATPase-associated island in ATCC 17978.3
The fact that 9 of the 10 representative isolates exhibiting a negative ATPase PCR possessed insertions in the ATPase gene, eight of which were AbaR1-related, strongly suggests that most of the 31 other MDR A. baumannii isolates with negative ATPase PCR results also harboured similar insertions. Indeed, the 13 kb AbaR1-like element in isolate ATCC 17978, which was isolated more than half a century ago, may represent an ancestral integrative element that has since disseminated widely within members of the A. baumannii species. Further analysis is required to determine whether the insertions detected in the present study are indicative of the presence of larger genomic islands carrying repositories of resistance genes.
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This work was supported by a British Society for Antimicrobial Chemotherapy Project Grant (GA636) to K. R., K. J. T. and K. L., a Royal Society–National Natural Science Foundation of China International Joint Project grant to K. R. and Z. D. (2007/R3) and grants from the National Science Foundation of China (30700013/C010103) and the 863 Program, Ministry of Science and Technology, China (2006AA02Z328) to H.-Y. O. F. S. was supported by a University of Leicester Open PhD studentship.
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None to declare.
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Acknowledgements |
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The laboratories supplying isolates used in this study are thanked for their cooperation. Dr Nelun Perera is thanked for useful discussions and Jon van Aartsen for assistance with GenBank submissions.
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References |
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