Characterization and PCR-Based Replicon Typing of Resistance Plasmids in Acinetobacter baumannii

Abstract

Acinetobacter baumannii is an opportunistic pathogen, especially in intensive care units, and multidrug-resistant isolates have increasingly been reported during the last decade. Despite recent progress in knowledge of antibiotic resistance mechanisms in A. baumannii, little is known about the genetic factors driving isolates toward multidrug resistance. In the present study, the A. baumannii plasmids were investigated through the analysis and classification of plasmid replication systems and the identification of A. baumannii-specific mobilization and addiction systems. Twenty-two replicons were identified by in silico analysis, and five other replicons were identified and cloned from previously uncharacterized A. baumannii resistance plasmids carrying the OXA-58 carbapenem-hydrolyzing oxacillinase. Replicons were classified into homology groups on the basis of their nucleotide homology. A novel PCR-based replicon typing scheme (the A. baumannii PCR-based replicon typing [AB-PBRT] method) was devised to categorize the A. baumannii plasmids into homogeneous groups on the basis of the nucleotide homology of their respective replicase genes. The AB-PBRT technique was applied to a collection of multidrug-resistant A. baumannii clinical isolates carrying the bla_OXA-58 or bla_OXA-23 carbapenemase gene. A putative complete conjugative apparatus was identified on one plasmid whose self-conjugative ability was demonstrated in vitro. We showed that this conjugative plasmid type was widely diffused in our collection, likely representing the most important vehicle promoting the horizontal transmission of A. baumannii resistance plasmids.

The foundation of plasmid biology was largely built on the genetic analysis of plasmid strategies for broad-host-range replication in Gram-negative bacteria. Mechanisms which guarantee the autonomous replication, addiction systems based on toxin-antitoxin factors, partitioning systems ensuring stable inheritance during cell division, and other virulence and antimicrobial resistance determinants have been described for plasmids circulating in the Enterobacteriaceae family and Pseudomonas spp. (17). Enterobacterial plasmids have also been classified into homogeneous groups on the basis of their replication controls by conjugation (plasmid incompatibility) and molecular methods (Southern blot hybridization with replicon probes and PCR-based replicon typing) (5, 8, 10, 11). Currently, 27 incompatibility groups are recognized in the Enterobacteriaceae by the Plasmid Section of the National Collection of Type Cultures (Colindale, London, United Kingdom). In contrast, limited information is available on the plasmids circulating in Acinetobacter spp., even though Acinetobacter baumannii is an important pathogen in intensive care units (13, 28). Moreover, despite recent progress in the study of antibiotic resistance mechanisms in A. baumannii, little is known about the genetic factors that have driven the recent evolution of A. baumannii toward multidrug resistance. A. baumannii may develop resistance to carbapenems through plasmid-mediated acquisition of carbapenem-hydrolyzing class D β-lactamases (CHDLs) (29). In particular, the bla_OXA-58 and bla_OXA-23 genes encoding the OXA-58 and OXA-23 CHDLs, respectively, have been reported from A. baumannii isolates collected from distant parts of the world in association with plasmids. The aim of the present study was to investigate the A. baumannii plasmids through the analysis and classification of plasmid replication systems and identification of A. baumannii-specific mobilization and addiction systems. Finally, novel tools for detecting A. baumannii resistance plasmids are proposed and the plasmids are categorized into homogeneous families on the basis of the nucleotide homologies of their respective replicase genes.

MATERIALS AND METHODS

In silico analysis of A. baumannii plasmids.

An in silico comparative analysis of fully and partially sequenced Acinetobacter plasmids was performed at GenBank using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Fifteen fully sequenced and eight partially sequenced A. baumannii plasmids available at GenBank from six completed genomes from previous studies or identified in this study were analyzed (Table 1). Multiple-sequence alignments of the replicon nucleotide sequences have been performed using DNAMAN software (Lynnon BioSoft, Vaudreuil, Quebec, Canada) set for DNA quick alignment with a gap penalty of 7, a K-tuple of 3, 5 top diagonals, and a window size of 5. Multiple-sequence alignments of the coding sequences were performed by using the DNAMAN software set for protein quick alignment with a gap penalty of 3, a K-tuple of 1, five top diagonals, and a window size of 5.

TABLE 1.

A. baumannii replicase genes analyzed in this study

Strain	Plasmid (EMBL accession no.)	Replicase name	Rep superfamily	Source of homology by BLASTp best hita	% amino acid identity (EMBL accession no.)	Iteronsb	Rep group	Reference
ACICU	pACICU1 (NC_010605)	Aci1	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	71 (YP_001338806)	Pos	GR2	19
		AciX	Rep-3 pfam01051	Neisseria lactamica	55 (ZP_05987942)	Pos	GR10
	pACICU2 (NC_010606)	Aci6	Rep pfam03090	Pseudoalteromonas sp.	41 (YP_001887739)	Neg	GR6
SDF	p1ABSDF (NC_010395)	p1ABSDF0001	Rep-3 pfam01051	Moraxella bovis	59 (YP_001966359)	Pos	GR1	34
	p2ABSDF (NC_010396)	p2ABSDF0001	Rep-3 pfam01051	Moraxella bovis	40 (YP_003289297)	Pos	GR12
		p2ABSDF0025	Rep-3 pfam01051	Moraxella bovis	50 (YP_003289297)	Pos	GR18
	p3ABSDF (NC_010398)	p3ABSDF0002	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	57 (YP_001338806)	Neg	GR7
		p3ABSDF0009	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	45 (YP_001338806)	Pos	GR9
		p3ABSDF0018	Rep-3 pfam01051	Moraxella bovis	41 (YP_003289297)	Pos	GR15
AYE	p1ABAYE (NC_010401)	p1ABAYE0001	Rep-3 pfam01051	Enhydrobacter aerosaccus	33 (ZP_05619518)	Pos	GR11	34
	p2ABAYE (NC_010402)	p2ABAYE0001	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	71 (YP_001338806)	Pos	GR2
	p3ABAYE (NC_010404)	p3ABAYE0002	Rep-3 pfam01051	Pasteurella multocida pJR2_p4	31 (NP_848174)	Neg	GR13
	p4ABAYE (NC_010403)	p4ABAYE0001	Rep-1 pfam01446	Pseudomonas putida	43 (NP_064737)	Neg	GR14
ATCC 17978	pAB1 (NC_009083)	A1S_3471	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	59 (YP_001338806)	Pos	GR17	32
	pAB2 (NC_009084)	A1S_3472	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	71 (YP_001338806)	Pos	GR2
Ab0057	pAB0057 (NC_011585)	AB57_3921	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	71 (YP_001338806)	Pos	GR2	1
Ab49	pAB49 (L77992; partial)	repApAB49	Rep-1 pfam01446	Bacillus cereus	38 (ZP_04189469)	Neg	GR16	Unpublished
AbABIR	pABIR (EU294228)	RepA_AB	Rep-3 pfam01051	Moraxella bovis	40 (YP_003289297)	Pos	GR12	35
VA-566/00	pABVA01 (NC_012813)	Aci2	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	74 (YP_001338806)	Pos	GR2	9
Ab19606	pMAC02 (AY541809)	RepM-Aci9	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	57 (YP_001338806)	Pos	GR8	12
Ab02	pAB02 (AY228470, partial)	repA_AB	Rep-3 pfam01051	Moraxella bovis	40 (YP_003289297)	Pos	GR12	Unpublished
Ab135040	p135040 (GQ861437, partial)	rep135040	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	58 (YP_001338806)	Pos	GR19	18
Ab736	p736 (GU978996; partial)	Aci7	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	92 (YP_001338806)	Pos	GR3	This study
Ab203	P203 (GU978997; partial)	Aci3	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	85 (YP_001338806)	Pos	GR3	This study
Ab844	p844 (GU978998; partial)	Aci4	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	85 (YP_001338806)	Pos	GR4	This study
Ab537	p537 (GU978999; partial)	Aci5	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	72 (YP_001338806)	Pos	GR5	This study
Ab11921	p11921 (GU979000; partial)	Aci8	Rep-3 pfam01051	Klebsiella pneumoniae pKPN5	46 (YP_001966359)	Pos	GR8	This study

^aThis comparison was performed by a BLASTP search excluding the sequence of the Acinetobacter spp.

^bPos, positive; Neg, negative.

A. baumannii PCR-based replicon typing (AB-PBRT) method.

A total of 19 PCR amplifications were devised to detect 27 replicase genes, which were grouped into 19 homology groups (GRs) on the basis of their nucleotide sequence similarities (Table 1 and Fig. 1). These groups include five novel replicase genes (aci3, aci4, aci5, aci7, and aci8 [Table 1]), cloned and sequenced as described below, from plasmids carrying the bla_OXA-58 genes from a collection of A. baumannii clinical isolates.

FIG. 1.

Multiple-sequence alignments and groups of homology of the replicase genes and their deduced amino acid protein sequences from A. baumannii plasmids.

The primers used for AB-PBRT are listed in Table 2. The PCR amplifications were organized into six multiplexes, each recognizing three or four different homology groups (Table 2). Specificity and sensitivity tests were performed for each primer pair in simplex form and in multiplex form with genomic DNA extracted from the respective reference strain (Table 2). The multiplexes were also tested using single and mixed control DNA templates. All the PCRs were highly specific on each template, as expected. The PCRs for GR2, GR3, and GR8 recognize the related replicases Aci1/Aci2, Aci3/Aci7, and Aci8/Aci9, respectively. The replicase variants belonging to GR2, GR3, and GR8 can be recognized by DNA sequencing of the respective amplicon.

TABLE 2.

Primers used to detect the replicase gene groups in the A. baumannii PCR-based replicon typing scheme

Multiplex	Group	Primer name	Primer sequence	Amplicon size (bp)	Replicase name (short name)	Reference strain/plasmid
1		gr1FW	5′-CATAGAAATACAGCCTATAAAG-3′	330	p1ABSDF001 (p1S1)	SDF-p1ABSDF
		gr1RV	5′-TTCTTCTAGCTCTACCAAAAT-3′
	GR2	gr2FW	5′-AGTAGAACAACGTTTAATTTTATTGGC-3′	851	Aci1	ACICU-pACICU1
		gr2RV	5′-CCACTTTTTTTAGGTATGGGTATAG-3′		Aci2	MAD
	GR3	gr3FW	5′-TAATTAATGCCAGTTATAACCTTG-3′	505	Aci3	Ab599
		gr3RV	5′-GTATCGAGTACACCTATTTTTTGT-3′		Aci7	Ab736
2	GR5	gr5FW	5′-AGAATGGGGAACTTTAAAGA-3′	220	Aci5	Ab537
		gr5RV	5′-GACGCTGGGCATCTGTTAAC-3′
	GR18	gr18FW	5′-TCGGGTTATCACAATAACAA-3′	676	p2ABSDF00025 (p2S25)	SDF-p2ABSDF
		gr18RV	5′-TAGAACATTGGCAATCCATA-3′
	GR7	gr7FW	5′-GAACAGTTTAGTTGTGAAAG-3′	885	p3ABSDF002 (p3S2)	SDF-p3ABSDF
		gr7RV	5′-TCTCTAAATTTTTCAGGCTC-3′
3	GR9	gr9FW	5′-GCAAGTTATACATTAAGCCT-3′	191	p3ABSDF0009 (p3S9)	SDF-p3ABSDF
		gr9RV	5′-AAAAATAAACGCTCTGATGC-3′
	GR4	gr4FW	5′-GTCCATGCTGAGAGCTATGT-3′	508	Aci4	Ab844
		gr4RV	5′-TACGTCCCTTTTTATGTTGC-3′
	GR11	gr11FW	5′-GGCTATTCAAAACAAAGTTAC-3′	852	p1ABAYE0001 (p1AYE)	AYE-p1ABAYE
		gr11RV	5′-GTTTCCTCTCTTACACTTTT-3′
4	GR12	gr12FW	5′-TCATTGGTATTCGTTTTTCAAAACC-3′	165	p2ABSDF0001 (p2S1)	SDF-p1ABSDF
		gr12RV	5′-ATTTCACGCTTACCTATTTGTC-3′
	GR10	gr10FW	5′-TTTCACTAGCTACCAACTAA-3′	371	AciX	ACICU-pACICU1
		gr10RV	5′-ACACGTTGGTTTGGAGTC-3′
	GR13	gr13FW	5′-CAAGATCGTGAAATTACAGA-3′	780	p3ABAYE0002 (p3AYE)	AYE-p3ABAYE
		gr13RV	5′-CTGTTTATAATTTGGGTCGT-3′
5	GR8	gr8FW	5′-AATTAATCGTAAAGGATAATGC-3′	233	Aci8	Ab11921
		gr8RV	5′-GACATAGCGATCAAATAAGC-3′		repM (Aci9)	pMAC02
	GR14	gr14FW	5′-TTAAATGGGTGCGGTAATTT-3′	622	p4ABAYE0001 (p4AYE)	AYE-p4ABAYE
		gr14RV	5′-GCTTACCTTTCAAAACTTTG-3′
	GR15	gr15FW	5′-GGAAATAAAAATGATGAGTCC-3′	876	p3ABSDF0018 (p3S18)	SDF-p3ABSDF
		gr15RV	5′-ATAAGTTGTTTTTGTTGTATTCG-3
6	GR16	gr16FW	5′-CTCGAGTTCAGGCTATTTTT-3′	233	repApAB49 (pAB49)	pAB49
		gr16RV	5′-GCCATTTCGAAGATCTAAAC-3′
	GR17	gr17FW	5′-AATAACACTTATAATCCTTGTA-3′	380	A1s_3471 (A1S3471)	ATCC 17978-pAB1
		gr17RV	5′-GCAAATGTGACCTCTAATATA-3′
	GR6	gr6FW	5′-AGCAAGTACGTGGGACTAAT-3′	662	Aci6	ACICU-pACICU2
		gr6RV	5′- AAGCAATGAAACAGGCTAAT-3′
	GR19	gr19FW	5′- ACGAGATACAAACATGCTCA-3′	815	rep135040	Ab135040
		gr19RV	5′- AGCTAGACATTTCAGGCATT-3′

Each multiplex reaction mixture contained (final concentrations) 1× ImmoBuffer [16 mM (NH₄)₂SO₄, 67 mM Tris-HCl, pH 8.3, 0.01% Tween 20], 4.0 mM MgCl₂, 0.4 mM deoxynucleoside triphosphate, 1.0 μM each primer, 5% dimethyl sulfoxide, 0.04 U/μl Immolase DNA polymerase (Bioline, Kondon, United Kingdom), and 200 to 400 ng of DNA template per reaction tube. Template DNA was prepared by total DNA extraction by the Wizard genomic DNA purification kit (Promega, Madison, WI), starting from 2 ml of LB broth cultures. PCR amplifications were performed with the following amplification scheme: 1 cycle of denaturation at 94°C for 7 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 52°C for 30 s, and elongation at 72°C for 1.5 min. The amplification was finished with an extension program of 1 cycle at 72°C for 5 min.

Positive controls.

The PCR amplifications were tested with the ACICU, AYE, SDF, ATCC 19798, and Ab135040 reference strains and 20 nonclonally related A. baumannii isolates known to possess plasmids carrying the CHDL gene bla_OXA-58 (n = 13, originating from France, Tunisia, Sweden, Turkey, Romania, and Belgium) or bla_OXA-23 (n = 7, originating from Belgium, Monaco, Kingdom of Bahrain, Egypt, Algeria, Libya, and Saudi Arabia). Those carbapenem-resistant A. baumannii isolates had been characterized previously (23, 25, 26, 30) and belong to the INSERM U914 strain collection (Table 3). Plasmid typing was also performed with transformants and transconjugants obtained from the bla_OXA-58- and bla_OXA-23-positive plasmids (Table 3). All the amplicons obtained with the primers listed in Table 2 were cloned into a TA cloning vector (Invitrogen-Life Technologies, Milan, Italy) and transformed into competent Escherichia coli DH5α cells (MAX Efficiency DH5α chemically competent cells; Invitrogen-Life Technologies). Selection of the transformants was performed on LB agar plates containing ampicillin (100 μg/ml). The cloned amplicons were fully sequenced and used as positive controls for the multiplex PCRs in the AB-PBRT scheme.

TABLE 3.

AB-PBRT applied to the collection of bla_OXA-58- and bla_OXA-23-positive strains and their respective transformants/transconjugants

Strain	OXA	Multiplex 1			Multiplex 2			Multiplex 3			Multiplex 4			Multiplex 5			Multiplex 6
		GR2 (851 bp)	GR3 (505 bp)	GR1 (330 bp)	GR7 (885 bp)	GR18 (676 bp)	GR5 (220 bp)	GR11 (852 bp)	GR4 (508 bp)	GR9 (191 bp)	GR13 (780 bp)	GR10 (371 bp)	GR12 (165 bp)	GR15 (876 bp)	GR14 (622 bp)	GR8 (233 bp)	GR6 (662 bp)	GR17 (425 bp)	GR16 (233 bp)	GR19 (815 bp)
ACICU	58	Aci1										AciX					Aci6
AYE		p2AYE (Aci1)						p1AYE			p3AYE				p4AYE
SDF			Aci3b	p1S1	p3S2	p2S1				p3S9			p2S25	p3S18
ATCC		A1S_3472 (Aci1)																A1S3471
135040T	143																			Rep
A21	58	Aci1											p2S25			Aci9	Aci6		pAB49
A21Ta	58						[Aci5]c		[Aci4]							Aci9
Ab11921	58	Aci1											p2S25			Aci8
11921Ta	58						[Aci5]		[Aci4]							Aci8
Ab203	58		Aci3
203Ta	58		Aci3				[Aci5]		[Aci4]
Ab844	58	Aci2							Aci4				p2S25				Aci6
844Ta	58						[Aci5]		Aci4
Ab537	58		Aci3				Aci5		Aci4
537Ta	58		Aci3				[Aci5]		[Aci4]
MAD	58	Aci2															Aci6
MADTa	58	Aci2					[Aci5]		[Aci4]
Ab736	58		Aci7														Aci6
736Ta	58		Aci7				[Aci5]		[Aci4]
Ab727	58	Aci1															Aci6		pAB49
727Ta	58	Aci1					[Aci5)]		[Aci4]
AbA22	58	Aci1															Aci6		pAB49
A22Ta	58	Aci1					[Aci5)]		[Aci4]
Ab120066	58	Aci1										AciX					Aci6
Ab587	58		Aci3
587Ta	58		Aci3				[Aci5]		[Aci4]
Ab692	58		Aci3
692Ta	58		Aci3				[Aci5]		[Aci4]
Ab599	58	Aci2	Aci3										p2S25
599Ta	58		Aci3				[Aci5]		[Aci4]
AbAS3	23												p2S25				Aci6
AS3TcJa	23						[Aci5]		[Aci4]								Aci6
Ab1190	23												p2S25				Aci6
1190Tcja	23						[Aci5]		[Aci4]								Aci6
Ab614	23												p2S25				Aci6
614Tcja	23						[Aci5]		[Aci4]								Aci6
Ab861	23	Aci1															Aci6
861Tcja	23						[Aci5]		[Aci4]								Aci6
Ab877	23								Aci4								Aci6
Ab14	23																Aci6
AbBel	23	Aci1															Aci6
Recipient							[Aci5]		[Aci4]

^abla_OXA-positive transformants (T) and transconjugants (Tcj) were obtained from the respective donor strain indicated on the line above the transformant or transconjugant designation using A. baumannii BM4547 as the recipient.

^bThe aci3 gene was not identified in the whole-genome sequencing of the SDF strain.

^cBrackets indicate that Aci4 and Aci5 replicases are present in the recipient strain.

Plasmid transfer by transformation and conjugation.

Plasmid DNAs were purified from bla_OXA-58-positive A. baumannii isolates by using an Invitrogen PureLink HiPure plasmid filter midiprep kit and electrotransformed into recipient strain A. baumannii BM4547 (22), and transformants were selected on ticarcillin-containing plates (50 μg/ml). Mating-out assays were performed by using isolates harboring bla_OXA-58 and bla_OXA-23 plasmids as donors and rifampin-resistant recipient strain A. baumannii BM4547, as described previously (26). Briefly, one colony of each of the donor and recipient strains obtained after 24 h of growth was cultured separately under weak agitation in 1 ml tryptic soy broth at 37°C, and they were then used in the mating-out assays. Conjugation was done by incubating 800 μl of the recipient strain with 200 μl of the donor strain under low agitation at 37°C for an additional 3-h step. The transconjugants were then selected by plating 200 μl of that mixture on agar plates containing ticarcillin (100 μg/ml) and rifampin (50 μg/ml).

The bla_OXA-58 and bla_OXA-23 genes were detected by PCR using previously described primer pairs (2, 7).

Identification and cloning of novel replicase genes from A. baumannii plasmids.

Plasmid DNAs were purified from the A. baumannii transformants by the Invitrogen PureLink HiPure plasmid filter midiprep kit. EcoRI-restricted fragments were separated by 0.8% agarose gel electrophoresis. Plasmid DNA was transferred to a Hybond-N⁺ membrane (Roche Diagnostics, Monza, Italy) by standard methods (31). Southern blot hybridization was carried out under low-stringency conditions (58°C) using the aci1 amplicon from the pACICU1 plasmid as the probe, and the amplicon was labeled with digoxigenin (DIG)-11-dUTP by PCR using a DIG PCR probe synthesis kit (Roche Diagnostics, Monza, Italy). After hybridization with the probe, the hybridized DNA was detected with Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolylphosphate using a DIG nucleic acid detection kit (Roche Diagnostics).

The EcoRI-restricted fragments identified by cross-reaction with the aci1 probe were separated by and eluted from the agarose gel by a Qiagen (Courtaboeuf, France) gel extraction kit and cloned into the EcoRI cloning site of the pUC18 vector, and the vector was transformed into competent E. coli DH5α cells (MAX Efficiency DH5α chemically competent cells; Invitrogen, Milan, Italy). Selection of the transformants was performed on LB agar plates containing ampicillin (100 μg/ml). The inserts were fully sequenced using standard and walking primers. The DNA sequences were determined by used of fluorescent dye-labeled dideoxynucleotides and an AB3730 automatic DNA sequencer (Perkin-Elmer, Foster City, CA).

Nucleotide sequence accession numbers.

The DNA sequences of the aci3, aci4, aci5, aci7, and aci8 replicase genes and the aci9 replicase gene from plasmid AbA21 have been deposited in the EMBL GenBank under accession numbers GU978996 to GU979001, respectively.

RESULTS AND DISCUSSION

Detailed analysis and definitions of A. baumannii replicons.

The nucleotide and deduced protein sequences of 18 A. baumannii plasmids available in GenBank were analyzed. Twenty-two intact replicons were identified in silico (Table 1). Each replicon included the origin of replication (ori) and the replicase gene (rep). A. baumannii replicons differ from all those previously described in other prokaryotic species, indicating that A. baumannii possesses its own plasmid types. For 17 out of the 22 replicons, the rep genes were preceded by four direct and perfectly conserved repeats that, in analogy with the basic replicons of plasmids, may be defined as “iterons” (Table 1 and Table 4). Iterons have been identified not only on many prokaryotic plasmids but also on chromosomes, phages, and eukaryotic ori genes (6, 27). In enterobacterial plasmids, each replicase protein binds to the reiterated iterons at the ori site and stimulates DNA replication by interacting with the host proteins (DNAK, DNAJ, RNApol) required for replication initiation. However, no iterons were identified in association with the replicase genes for five plasmids. For these plasmids, one can speculate about alternative mechanisms of replication control, presumably based on regulation of rep translation mediated by an inhibitory antisense RNA, as previously described for IncI1 and IncF plasmids (14).

TABLE 4.

Iterons in A. baumannii replicons

Replicon(s)	Iteron sequence	No. of direct repeats	Distance from iteron to rep start codon (bp)
p1ABSDF0001	5′-CAATAAGTACACCTTTATCTTG-3′	4	50
pACICU1-Aci1, p2ABAYE0001, A1S_3472 pAB0057	5′-ATATGTCCACGTTTACCTTGCA-3′	4	53
pABVA01-Aci2	5′-TTTACCTTGCAATATGACACCG-3′	3	66
Ab203-Aci3	5′-TAAAACGAGGTTTACCTTGCAT-3′	4	57
Ab736-Aci7
Ab844-Aci4	5′-ATATGACTACGTTTACCTACCA-3′	4	107
Ab537-Aci5	5′-ATATGACTACGTTTACCTACCA-3′	4	105
Ab11921-Aci8	5′-TAGGTTTATCGACCCATAAAAT-3′	4	91
pA21-Aci9	5′-TAAAACTAGGTTTATCGACCCT-3′	4	96
pMAC-Aci9	5′-ATAAAACTAGGTTTATCGACCC-3′	4	97
p3ABSDF0009	5′-TATCTATACGTTTATGCAGTCT-3′	4	60
pACICU1-AciX	5′-CATTCAATCACAGATTCCATTC-3′	4	80
p1ABAYE0001	5′-AAAGGGTACAAATAGCATGAT-3′	4a	90
p2ABSDF0001, pAB02	5′-GGATTGACTACTAACTATGAC-3′	4	41
pABIR	5′-CTAACTATGACGGATTGACTA-3′	4	55
p3ABSDF0018	5′-TATGAGGGATTGACTACTAAC-3′	4	32
pAB1	5′-ATTTCTTTGCATTTGACTACA-3′	4	10
p2ABSDF0025	5′-TAACTATGAGGGATTGACGCA-3′	5	15
p135040	5′-CATAT CTATACGTTTATCGACC-3′	4	89

^aImperfect.

Similar to plasmids described from other species (33), A. baumannii pACICU1, p2ABSDF, and p3ABSDF were multireplicon plasmids, since they carried more than one replicon (Table 1). Interestingly, the iterons and replicase genes of replicons from given plasmids showed weak sequence identities, likely to minimize the effect of competition of the replicase protein on its relative binding sites (6, 27). Plasmids pABIR and pABVA01 also showed two replicons, one that was functional, which was considered in this study, and one whose replicase gene was truncated by an insertion sequence (EMBL accession no. EU294228).

On the basis of the nucleotide sequence identities deduced from the in silico analysis, the 22 replicase genes were grouped into homology groups. Each group showed replicase genes showing less than 74% nucleotide identity (Table 1 and Fig. 1). Plasmids p2ABSDF, pABIR (carrying the bla_OXA-58 gene), and pAB02 (carrying the bla_{OXA-24/OXA-40} gene) carried highly related replicons which were included in the same homology group, designated GR12, showing conserved replicase gene and iteron sequences (>84% nucleotide identity; Fig. 1 and Table 4). This group also contains other plasmids carrying the bla_{OXA-24/OXA-40} gene that were recently identified and that showed rep gene sequences identical to the rep gene sequences of pAB02 (pMMCU1 [EMBL accession no. GQ342610], pMMCU2 [EMBL accession no. GQ476987], and pMMD [EMBL accession no. GQ904226]; the sequence of plasmid pMMCU1 is included in the tree in Fig. 1 for comparison).

Conserved replicons were observed for plasmids pACICU1, p2ABAYE, pAB2, pAB0057, pABVA01, and pMAD; and all have been included in GR2. Two variants (aci1 and aci2) showing 78% nucleotide identity were included in this group (Fig. 1). This group also contains plasmid pMMCU3 (EMBL accession no. GQ904227), carrying the bla_{OXA-24/OXA-40} gene, which had a aci2 rep gene sequence identical to that of pABVA01 (9, 24). The aci1 and aci2 replicase genes showed different iteron sequences (Table 4).

Most of the replicase proteins belonged to the Rep-3 superfamily, identified by the pfam0151 conserved domain (NCBI nonredundant Clusters of Orthologs [COG]; http://www.ncbi.nlm.nih.gov/COG/), and showed variable amino acid similarities with the replicase proteins of plasmid pKPN5, recently identified in Klebsiella pneumoniae strain MGH 78578 (GenBank accession no. CP000650.1), and with plasmids identified in Moraxella bovis, Pasteurella multocida, and Neisseria lactamica (Table 1). It may be hypothesized that these replicase genes actually derive from a common ancestor of the Rep-3 superfamily group (Table 1). Two replicase proteins from plasmids p4ABAYE and pAB49 belonged to the Rep-1 superfamily (pfam01446) and showed significant homologies with plasmids from Pseudomonas putida and Bacillus cereus. The replicase from plasmid pACICU2 was peculiar since it belonged to an undefined Rep superfamily (pfam03090) whose closest homologous plasmid was identified from a Pseudoalteromonas sp. (Table 1).

Setup of a novel A. baumannii PCR-based replicon typing scheme.

PCR amplifications were devised to recognize the replicase genes identified in silico and were successfully tested with the ACICU, AYE, SDF, ATCC 19798, and Ab135040 A. baumannii strains (Tables 2 and 3). Those PCRs were then used to test 20 clinical isolates carrying the plasmid-mediated carbapenem-hydrolyzing bla_OXA-58 and bla_OXA-23 oxacillinase genes (Table 3). Mating-out assays were initially performed with several bla_OXA-58-positive A. baumannii isolates as donors, but no transconjugants were obtained. However, all the bla_OXA-58-positive plasmids except one (from strain Ab120066) were successfully transferred by electroporation into A. baumannii BM4547 (Table 3). Transformants showed resistance to ticarcillin and reduced susceptibility to carbapenems as a result of bla_OXA-58 gene expression. Four out of seven bla_OXA-23-positive plasmids were successfully transferred by conjugation into A. baumannii BM4547 and included in this study (Table 3) (26). All the A. baumannii isolates and their respective transformants and transconjugants were previously tested by the PCR-based replicon typing method described for the Enterobacteriaceae (5), but all of them gave negative results, indicating that those plasmids were not corresponding to those known to circulate among the Enterobacteriaceae (data not shown).

Twelve strains and their respective bla_OXA-58 or bla_OXA-23 transformant or transconjugant strains were successfully typed by the PCR amplifications devised with the 22 A. baumannii replicase genes identified in silico in previously characterized plasmids (Table 1). Four bla_OXA-58-positive strains and their respective transformants (strains Ab203, Ab537, Ab587, and Ab692) were negative by all these PCRs. Furthermore, strains Ab11921, Ab844, Ab736, and Ab599 were positive by the GR12 and/or GR6 PCR, but their respective transformants, carrying the bla_OXA-58 gene, were negative for all the A. baumannii replicase genes identified in silico, suggesting that other replicons were present on these bla_OXA-58-positive plasmids.

Five novel replicase genes (aci3, aci4, aci5, aci7, and aci8 [Table 1]) were identified and subsequently cloned and sequenced from the bla_OXA-58-positive strains: the aci8 rep gene from plasmid p11921 was 74% homologous to the repM replicase gene from plasmid pMAC02 and was included in GR8; aci3 and aci7 corresponded to novel replicase genes identified in plasmids from isolates Ab203, Ab537, Ab587, Ab599 (aci3), and Ab736 (aci7). The aci3 and aci7 rep genes showed 87% nucleotide identity with each other and identical iteron sequences and were grouped into a novel homology group designated GR3; the aci4 and aci5 replicase genes from isolates Ab844 and Ab537, respectively, were classified in the novel groups GR4 and GR5, respectively, being highly divergent from all the other replicase genes (Fig. 1; Table 1). Noteworthy is the finding that the BM4547 strain used as the susceptible recipient for transformation and conjugation was positive for the aci4 and aci5 replicase genes, probably due to the integration of a multireplicon plasmid within the bacterial chromosome, since no extrachromosomal plasmids were identified for that strain (data not shown). Southern blot hybridization experiments performed with plasmid DNA purified from the 844T transformant confirmed that this bla_OXA-58 plasmid possessed the aci4 replicase gene (data not shown).

In conclusion, the AB-PBRT scheme for A. baumannii plasmid typing showed that donor strains often carried more than one plasmid type. However, each transformant or transconjugant carrying the bla_OXA-58 or bla_OXA-23 gene carried only a single replicon that was also identified from its corresponding donor strain by the AB-PBRT scheme.

AB-PBRT applied to our collection of A. baumannii strains demonstrated that the bla_OXA-58-positive plasmids differed, with six of them showing replicons belonging to GR3, including both the aci3 and aci7 replicase genes. A previously unidentified replicase of GR3 was also identified in the SDF strain. Three bla_OXA-58-positive plasmids carried the aci1 or aci2 replicase gene, belonging to GR2, and two plasmids carried the aci8 or aci9 gene, belonging to GR8. Interestingly, the AbA21 plasmid showed a replicase 99% homologous to the repM-aci9 gene of pMAC02 but carried a different iteron sequence (Table 4). All isolates carrying the bla_OXA-23 gene showed a positive PCR result for the aci6 replicase gene, which was originally identified on plasmid pACICU2.

In silico analysis of A. baumannii plasmid maintenance and inheritance.

As extrachromosomal elements, plasmids bear the burden of ensuring their own segregation at cell division and employ various strategies, such as active partition systems and postsegregational killing mechanisms. These systems have never been described for A. baumannii plasmids. A careful annotation of the coding sequences from fully sequenced plasmids allowed the identification of putative ParA and ParB (3) partitioning proteins on plasmids pACICU1, pACICU2, and p3ABAYE (Table 5).

TABLE 5.

Plasmid maintenance and addiction systems identified in silico on A. baumannii plasmids

Predicted function	Plasmid	CDSa protein identifier	Protein name, putative function	Source of homology	% best hit
Plasmid partitioning	pACICU1	P006	ParA, putative partition protein	Moraxella bovis	73
	pACICU1	P007	Probable copy no. control protein	Moraxella bovis	56
	pACICU2	P0040	ParB, involvement in plasmid partition	Collimonas fungivorans	44
	pACICU2	P0047	ParA, putative partition protein	uncultured bacterium	34
	pACICU2	P0048	ParB-like nuclease domain	Caminibacter mediatlanticus	39
	p3ABAYE	p3ABAYE0112	ParB- nuclease domain	Ralstonia eutropha	41
	p3ABAYE	p3ABAYE0113	ParA, putative partitioning protein	Chromobacterium violaceum	38
Toxin-antitoxin systems	pACICU1	P009	Antitoxin StbE, prevent-host-death protein	Burkholderia ubonensis	57
	pACICU1	P0010	Toxin RelE/StbE family	Burkholderia ubonensis	61
	p1ABAYE	p1ABAYE0004	Antitoxin, prevent-host-death protein	Burkholderia cenocepacia	56
	p1ABAYE	p1ABAYE0005	Toxin, Txe/YoeB family	Burkholderia ambifaria	70
	p2ABSDF	p2ABSDF0030	Toxin, RelE family protein	Haemophilus somnus	62
	p2ABSDF	p2ABSDF0031	Antitoxin, RelB homolog of RelB/DinJ family	Haemophilus somnus
Restriction and antirestriction systems	pACICU1	P008	Type I site-specific DNase, HsdR family	Chlorobium limicola	33
	pACICU2	P0046	Type I restriction enzyme M subunit	Haemophilus influenzae	28
	p3ABAYE	p3ABAYE0069	Type II restriction/modification enzyme	Polaromonas sp.	52
	p2ABSDF	p2ABSDF0015	HpaII restriction endonuclease	Flavobacterium psychrophilum	49
	p2ABSDF	p2ABSDF0016	HpaIIM-like cytosine-specific methyltransferase, modification enzyme	Haemophilus arainfluenzae	79
	p3ABSDF	p3ABSDF0013	Type II restriction/modification enzyme	Bacillus megaterium	55
	p3ABSDF	p3ABSDF0014	Methyltransferase cytosine, modification enzyme	Bacillus megaterium	46
	p3ABSDF	p3ABSDF0015	Bfii restriction endonuclease	Bacillus firmus	63

^aCDS, coding sequence.

Plasmids pACICU1, p1ABAYE, and p2ABSDF encoded putative postsegregational killing systems. In particular, the orthologs of the RelBE toxin-antitoxin system of plasmids from Escherichia coli (20) were identified on pAUCU1 and p2ABSDF, while the orthologs of the Txe system of plasmid pRUM of Enterococcus faecium (16) were identified on the p1ABAYE plasmid (Table 5).

Plasmids pACICU1, pACICU2, p3ABAYE, p2ABSDF, and p3ABSDF also encoded putative restriction and antirestriction systems, including type I and type II restriction/modification enzymes, the HpaII and Bfii endonucleases, and their specific antirestriction methyltransferases (Table 5).

A. baumannii plasmid transferability.

Bacterial conjugation is one of the fundamental processes used for gene dissemination in nature. A putative conjugative system was identified only for plasmid pACICU2 (Table 6). This system is homologous to a conjugative system identified for uncharacterized plasmids of Burkholderia cenocepacia and Burkholderia thailandensis, suggesting a potential common origin of ancestor plasmids among these bacteria. The conjugative system of plasmid pACICU2 also showed a protein equivalent to the relaxase-helicase (TraI) belonging to a novel clade of the MOB_F family of relaxase proteins previously described for other transmissible plasmids from the prokaryotic kingdom (15, 21).

TABLE 6.

Conjugal transfer and mobilization systems identified in silico on A. baumannii plasmids

Plasmid	CDS protein identifier	Conjugal transfer or mobilization protein name and function	Source of homology	Amino acid identity (% best hit)
pACICU2	P0058	Type IV secretory pathway, VirD4, TraD component	Burkholderia cenocepacia	40
	P0059	TraI, relaxase-helicase for conjugative transfer	Pseudomonas sp.	40
	P0070	TraA, conjugal transfer protein	Acidithiobacillus ferrooxidans	36
	P0071	TraL, putative membrane protein	Acidovorax sp.	40
	P0072	TraE, conjugative transfer protein	Burkholderia cenocepacia	30
	P0074	TraB, pilus assembly family protein	Acidovorax sp.	33
	P0075	DsbC precursor protein, disulfide isomerase	Burkholderia thailandensis	53
	P0076	TraV, membrane lipoprotein lipid attachment site	Acidovorax sp.	44
	P0077	TraC, conjugative transfer protein	Burkholderia cenocepacia	41
	P0079	TraW, conjugative transfer protein precursor	Burkholderia cenocepacia	46
	P0080	TraU, conjugative transfer protein precursor	Burkholderia cenocepacia	65
	P0081	Conjugative transfer protein	Burkholderia cenocepacia	43
	P0082	TraN, conjugal transfer mating pair stabilization	Acidovorax sp.	37
	P0083	TraF, conjugative transfer protein	Burkholderia cenocepacia	48
	P0085	TraH, conjugative transfer protein	Burkholderia cenocepacia	70
	P0086	TraG, domain containing protein	Burkholderia thailandensis	32
	P0088	DNA-directed DNA polymerase UmuC	Acinetobacter sp. strain ATCC 27244	57
	P0089	DNA-directed DNA polymerase RumB	Acinetobacter baumannii ATCC 17978	56
p1ABAYE	p1ABAYE0006	Putative mobilization protein, MobS-like	Rhizobium leguminosarum	40
	p1ABAYE0007	TraA, putative mobilization protein, MobL-like	Sinorhizobium meliloti	43
p1ABSDF	p1ABSDF0002	Putative mobilization protein, MobS-like	Psychrobacter psychrophilus	69
p2ABSDF	p2ABSDF0026	Putative mobilization protein, MobS-like	Polaromonas naphthalenivorans	46
	p2ABSDF0028	Putative mobilization protein, MobL-like	Agrobacterium tumefaciens	45
p3ABSDF	p3ABSDF0010	Putative mobilization protein, MobS-like	Psychrobacter psychrophilus	69
	p3ABSDF0011	Putative mobilization protein, MobL-like	Agrobacterium tumefaciens	46
pMAC02	pMAC_11	Putative mobilization protein, MobA-like	Escherichia coli	43
pMMCU1, pMMD	pMMCU1p5	Putative mobilization protein, MobA-like	Escherichia coli	49
pMMCU2	pMMCU2_06	Putative mobilization protein, MobA-like	Escherichia coli	49

Even if plasmid pACICU2 was a unique plasmid endowed with a conjugative apparatus, plasmids p1ABAYE, p1ABSDF, p2ABSDF, p3ABSDF pMMCU1, pMMCU2, pMAC02, and pMMD showed some orthologs of the MobS-MobL or MobA mobilization proteins that are characteristic of a number of small plasmids that are mobilizable by self-transmissible plasmids. These proteins are required for recognizing and cleaving the nic site, directing the complex to the transferosome determined by the conjugative element (14).

The transconjugants obtained from the bla_OXA-23-positive isolates harbored the aci6 replicase gene of pACICU2 that was confirmed to be located on the bla_OXA-23-positive plasmid by Southern blot hybridization (data not shown). These results clearly indicate that plasmids similar to pACICU2 are present in those isolates and are able to self-conjugate. These pACICU2-related plasmids harbored the carbapenem resistance gene bla_OXA-23, which, however, was absent from the original fully sequenced pACICU2 plasmid (19). Noteworthy is the fact that the aci6 replicase gene was also identified from 7 out of 13 bla_OXA-58-positive isolates but did not correspond to the replicon associated with this resistance gene. These findings open a new and interesting scenario describing the transmission of resistance plasmids into A. baumannii, since the pACICU2-like plasmids seem to be widely diffused and are likely responsible for both bla_OXA-58 plasmid mobilization and bla_OXA-23 plasmid self-conjugation.

Conclusion.

The present study is the first to characterize the main features of the plasmids circulating among A. baumannii strains. Through an in silico analysis complemented by several experimental cloning experiments, 27 replicase genes have been identified. Primer sequences have been defined in order to characterize those 27 replicase genes, and a PCR-based methodology has been proposed to detect them in a convenient way. A multiplex approach has been set up by defining 19 distinct groups in 6 multiplexes, each of them grouping either three or four primer pairs that may allow faster and cheaper screening. Indeed, plasmid typing is a useful tool for studying their respective circulation and spread among members of the Acinetobacter genus and eventually among isolates of other genera. Through the epidemiological survey that has been conducted here, we exemplified what kind of approach that methodology can deserve. Here, we traced the diffusion of the carbapenem-hydrolyzing oxacillinase genes bla_OXA-23 and bla_OXA-58, known to be the sources of resistance to carbapenems in A. baumannii worldwide. Interestingly, we showed that the current worldwide diffusion of the bla_OXA-23 gene was mainly related to a single plasmid type and, conversely, that the diffusion of the bla_OXA-58 gene was related to several unrelated plasmid types.

We aim to provide an easy, rapid, and reliable tool for investigating the plasmid epidemiology of A. baumannii. That kind of approach of performing plasmid typing will be useful and informative when studies focus on dissemination of specific markers only, such as a given antibiotic resistance gene, contributing to the better tracing of specific plasmids among a diversity of A. baumannii genetic backgrounds. This can be done in a way similar to that previously set up for the Enterobacteriaceae family that is now applied worldwide, and the corresponding so-called PBRT method is nowadays the main technique used to trace resistance plasmids among strains belonging to that family and improve knowledge of the evolution of drug resistance (4).