Origin in Acinetobacter gyllenbergii and dissemination of aminoglycoside-modifying enzyme AAC(6′)-Ih

Abstract

Objectives

The aac(6′)-Ih gene encoding aminoglycoside 6′-N-acetyltransferase type I subtype h [AAC(6′)-Ih] is plasmid-borne in Acinetobacter baumannii where it confers high-level amikacin resistance, but its origin remains unknown. We searched for the gene in the genomes of a collection of 133 Acinetobacter spp. and studied its species specificity, expression and dissemination.

Methods

Gene copy number was determined by quantitative PCR, expression by quantitative RT–PCR, MIC by microdilution and transfer by plasmid mobilization.

Results

The aac(6′)-Ih gene was present in the chromosome of the two Acinetobacter gyllenbergii of the collection and was detected in all seven A. gyllenbergii clinical isolates. They had indistinguishable flanking regions indicating that the gene was intrinsic to this species. A. baumannii P_ISAba23 promoters were provided by insertion of ISAba23, which disrupted the P_native promoter in A. gyllenbergii. Both types of promoters were similarly potent in Escherichia coli and A. baumannii. Aminoglycoside MICs for A. baumannii harbouring pIP1858 were higher than for A. gyllenbergii due to gene dosage. The non-self-transferable plasmid could be mobilized to other A. baumannii cells by the broad host range plasmid RP4.

Conclusions

We have found the origin of aac(6′)-Ih in A. gyllenbergii, a species isolated, although rarely, in humans, and documented that dissemination of this gene is restricted to the Acinetobacter genus.

Introduction

Acinetobacter baumannii is an opportunistic human pathogen increasingly responsible for epidemics of nosocomial infections, which are difficult to treat, at least in part, because the strains are often MDR.¹ Resistance can be intrinsic or acquired following transfer of mobile genetic elements^2–4 or occurrence of mutations in regulatory genes.⁵

A. baumannii is predominant in clinical settings, but other Acinetobacter species are increasingly found associated with human infections.⁶ Aminoglycosides, in particular amikacin, constitute with β-lactams and quinolones the most useful drug classes for the treatment of infections due to Acinetobacter. However, aminoglycoside resistance, primarily caused by enzymatic modification of the drugs, is increasingly reported in hospital isolates worldwide.⁷

In Acinetobacter spp., amikacin resistance is mainly due to the production of either 6′-N-acetyltransferase type I [AAC(6′)-I] or 3′-O-phosphotransferase type VI [APH(3′)-VI].⁸ The AAC(6′) family of enzymes is of particular significance because it modifies aminoglycosides of therapeutic importance such as amikacin, gentamicin, netilmicin and tobramycin. Based on enzymatic substrate profiles, two subclasses of AAC(6′) have been distinguished: type I confers resistance to amikacin, but not to gentamicin, whereas type II confers resistance to gentamicin, but not to amikacin. A minimum of 50 AAC(6′)-I isozymes have been reported.⁹ Many aac(6′)-I structural genes are chromosomally located, in particular in Acinetobacter spp.,¹⁰ where they are most often cryptic or weakly expressed; this is also the case, for example, for aac(6′)-Ic in Serratia marcescens,¹¹ and aac(6′)-Iy in Salmonella enterica.¹² In contrast, aac(6′)-Ib, which is the most clinically relevant amikacin resistance determinant in Gram-negative bacteria, such as Pseudomonas aeruginosa and Vibrio cholerae, because of its ubiquity and the high level of resistance it confers, is usually found as a gene cassette in various integrons and in transposons.¹³ In A. baumannii, the aac(6′)-Ib gene was detected in 6 of 97 A. baumannii clinical isolates¹⁴ and 4 of 32 A. baumannii of our collection of fully sequenced Acinetobacter spp.¹⁵ (E.-J. Yoon, P. Courvalin and C. Grillot-Courvalin, unpublished results). Another gene, aac(6′)-Ih, is plasmid-borne in A. baumannii and associated with high-level resistance.¹⁶ The gene was first found in 1989 as part of the non-conjugative plasmid pIP1858 in an amikacin-resistant clinical A. baumannii.¹⁶ It is closely related to the other Acinetobacter aac(6′)-I genes and we proposed that this may be the result of a translocation event from the chromosome of an Acinetobacter sp. to a plasmid indigenous to this bacterial genus.¹⁰ However, its putative species of origin remains unknown.

Recently, analysis of the genomes of a unique collection of 133 strains of Acinetobacter spp. covering the taxonomic diversity of the genus enabled us to build a robust phylogeny of the genus.¹⁵ Screening these genomes for the presence of resistance genes, we have shown that the aphA6 gene for an APH(3′)-VI originated in the chromosome of Acinetobacter guillouiae before dissemination to A. baumannii and to phylogenetically distant genera pathogenic for humans.¹⁷

By genome analysis of this extended panel of Acinetobacter species, we have searched for genes encoding AAC(6′)-I enzymes and studied their putative dissemination to other intrageneric species. We report the origin of aac(6′)-Ih in Acinetobacter gyllenbergii, a recently identified species¹⁸ that has been isolated, although rarely, from human clinical specimens.⁶ Our data also account for the observation that dissemination of aac(6′)-Ih is limited to the Acinetobacter genus.

Materials and methods

Bacterial strains

The whole-genome sequences of 133 Acinetobacter¹⁵ were studied. In addition, seven strains of A. gyllenbergii isolated from human specimens were screened for the presence of the aac(6′)-Ih gene (Table S1, available as Supplementary data at JAC Online). Six of these seven strains were from the nomenclatural study of Nemec et al.¹⁸ while the remaining isolate, ANC 4712, was identified as A. gyllenbergii based on metabolic testing, rpoB gene comparative analysis and whole-cell MALDI-TOF MS profiling (A. Nemec and L. Krizova, unpublished results). Five A. baumannii clinical isolates from our laboratory collection known to harbour aac(6′)-Ih were also studied (Table S1). They were collected from 1984 to 1990 at the Hôpital Saint Michel in Paris, France. Escherichia coli One Shot^® TOP10 (Invitrogen) and A. baumannii BM4587¹⁹ were used as recipients for cloning. Bacteria were grown at 30–37°C in brain heart infusion broth and agar (Difco Laboratories).

Identification of AAC(6′)-Ih

The presence of AAC(6′)-Ih protein was determined with Blastp²⁰ using the AAC(6′)-Ih prototype (GenBank Accession ID, AAA21889)¹⁶ against the 133 whole-genome sequences of Acinetobacter spp.¹⁵ The presence of aac(6′)-Ih in the additional A. gyllenbergii was searched for by PCR using primer pair qRT-Ih F/qRT-Ih R (Table S2) and the flanking regions were analysed by PCR, sequencing and PCR mapping using the primer pairs indicated in Table S2 and Figure S1.

Antimicrobial susceptibility testing

Antibiotic susceptibility testing was by disc diffusion on Mueller–Hinton (MH) agar²¹ and the MICs were determined by microdilution in cation-adjusted MH broth according to the CLSI guideline.²²

DNA manipulation

Genomic DNA was extracted as described previously.¹⁷ DNA amplification was performed in a GeneAmp PCR system 9700 (Perkin Elmer Cetus) with Phusion high-fidelity DNA polymerase (Thermo Scientific). Plasmid DNA was purified with a Nucleospin plasmid miniprep kit (Macherey-Nagel). Nucleotide sequencing was carried out with a CEQ 8000 DNA analysis system automatic sequencer (Beckman Instruments).

Determination of gene copy number by quantitative PCR (qPCR)

DNA collected using a modified rapid extraction method²³ was used for qPCR with primer pair qRT-Ih F/qRT-Ih R targeting aac(6′)-Ih (Table S2). Standard curves were generated using serial dilutions of DNA from 10⁴ to 10⁸ genome copies. Gene copy number was normalized to that of the single copy genes rpoB, gyrA and secE using primer pairs, qRT-Aba-rpoB-F/-R, qRT-Aba-gyrA-F/-R and qRT-Aba-secE-F/-R, respectively (Table S2). Each experiment was performed in duplicate at least twice independently.

RNA isolation and quantitative RT–PCR (qRT–PCR)

Total RNA was extracted from exponentially grown bacterial cells (OD₆₀₀ ≈ 0.8) by using TRIzol^® reagent (Invitrogen). RNA samples were treated with a Turbo DNA-free™ kit (Applied Biosystems). Expression of aac(6′)-Ih and rpoB genes was quantified by qRT–PCR using a LightCycler^® RNA amplification kit with SYBR Green I (Roche Diagnostics). The primers used are listed in Table S2. The transcriptome of aac(6′)-Ih was normalized to that of rpoB and each experiment was performed in duplicate at least twice independently.

Cloning of the aac(6′)-Ih gene

The 746 bp fragment that included 159 bp upstream from the −35 motif of the aac(6′)-Ih promoter was amplified from A. gyllenbergii NIPH 230 and A. baumannii BM2686 with primers aac6′Ih-305F_PciI and aac6′Ih-R_XbaI, and ISAba23-F_PciI and aac6′Ih-R_XbaI, respectively (Table S2). The amplicons were ligated to the pCR^®-Blunt vector (Invitrogen), the hybrid plasmids were digested with PciI and XbaI, and the inserts ligated to PciI-XbaI-linearized E. coli–Acinetobacter shuttle vector pAT747.¹⁷ The plasmids were introduced by transformation into E. coli TOP10 with selection on medium containing 100 mg/L ampicillin and 8 mg/L kanamycin, and by electrotransformation into A. baumannii BM4587 with selection on medium containing 30 mg/L ticarcillin and 8 mg/L kanamycin. The orientations and sequences of all the inserts were verified with forward and reverse universal primers.

Plasmid transfer

For plasmid mobilization A. baumannii A.62 having acquired plasmid RP4²⁴ by conjugation was used as a donor. Equal amounts of donor and of BM4587 Rif^R25 recipient exponential cultures in LB were harvested and mixed on a mating filter (Millipore) placed on LB agar. After overnight mating at 37°C, cells were recovered and placed on LB agar with antibiotics selective for the donor (tobramycin 15 mg/L), recipient (rifampicin 50 mg/L) and transconjugants (tobramycin and rifampicin). Transfer of plasmid RP4 was verified on tobramycin and rifampicin.

GenBank accession numbers

The sequence of plasmid pIP1858 was deposited in GenBank under accession number KP890934. The accession numbers of the sequences of aac(6′)-Ih in A. gyllenbergii are KT778787 (ANC 4712), KT778788 (NIPH 802), KT778789 (NIPH 822), KT778790 (NIPH 975), KT778791 (NIPH 1773), KT778792 (NIPH 2021) and KT778793 (NIPH 2353).

Results and discussion

AAC(6′)-Ih in Acinetobacter

The aac(6′)-Ih gene, originally reported in amikacin-resistant A. baumannii BM2686, was located on pIP1858, an ∼10 kb non-conjugative plasmid.¹⁶ The gene was also detected by PCR with specific primers in 32 of 51 A. baumannii clinical isolates collected at the Hôpital Saint Michel (Paris, France) at the time of a plasmid epidemic.²⁶ Five of these strains (Table S1) harbouring the plasmid were studied and found to belong to ST 2, 250 and 644, as determined by MLST (Pasteur Scheme).²⁷

Analysis of the whole-genome sequences of 133 strains of Acinetobacter enabled us to detect aac(6′)-Ih in the two A. gyllenbergii strains of this collection, and only in this species. The gene in these strains was identical to that of plasmid pIP1858. It was located in large contigs, 145 kb for CIP 110306^T and 189 kb for NIPH 230, that both also encoded a member of the RsmD family of RNA methyltransferases indicating a chromosomal location for the gene in A. gyllenbergii. Seven additional A. gyllenbergii obtained from human clinical specimens were screened for the presence of aac(6′)-Ih by PCR and were all found to harbour the gene (Table S1). The sequences of 682 bp PCR products comprising the aac(6′)-Ih gene, 150 bp upstream, including the promoter, and 92 bp downstream from the seven strains were identical except for NIPH 802 and NIPH 1773, which had seven identical SNPs in the gene resulting in amino acid substitutions R₄₂C and F₈₂Y. The genomic environment of aac(6′)-Ih was found to be indistinguishable over 7 kbp by PCR mapping in the seven strains and in the two entirely sequenced A. gyllenbergii. Taken together these data can be taken as evidence that the gene is intrinsic to the species.

Genomic environment of aac(6′)-Ih in plasmid pIP1858

The 10 389 bp plasmid pIP1858 was fully characterized (Figure 1; GenBank accession number KP890934). In the plasmid, aac(6′)-Ih had 47 bp upstream and 1104 bp downstream flanking regions identical to those in the chromosomes of the two entirely sequenced A. gyllenbergii strains (Figure 2). This identity was interrupted in the plasmid by insertion of ISAba23 47 bp upstream from the gene and by a new IS, designated ISAcsp5, 1104 bp downstream from the gene resulting in a truncated lipoprotein-releasing system ATP-binding protein LolD (Figure 2). The aac(6′)-Ih gene and both transposases were coded for on opposite strands and, since the 5′- and 3′-terminal sequences differed, the structure did not appear to form a functional composite transposon.

Figure 1.

Genetic organization of plasmid pIP1858. The portion nearly identical to the A. gyllenbergii chromosome is indicated by a blue line and that to plasmid p1ABAYE by an orange line; purple arrow, replication origin; yellow arrows, mob genes; red arrow, aac(6′)-Ih; green arrow, tesA; striped brown arrow, ΔlolD; open arrows, ORFs; grey open box, ISAba23; grey arrow, transposase; grey boxes, inverted repeats; black open box, ISAcsp5; black arrows, transposases; black boxes, inverted repeats.

Figure 2.

Genomic environment and promoters of aac(6′)-Ih. Top. Arrows indicate ORFs and sense of transcription. Red arrow, aac(6′)-Ih; blue arrow, cynT; green arrow, tesA; brown arrow, lolD; striped brown arrow, ΔlolD; open arrows, ORFs; grey open box, ISAba23; grey arrow, transposase; grey boxes, inverted repeats; black open box, ISAcsp5; black arrows, transposases; black boxes, inverted repeats. Bottom. Sequence alignment of aac(6′)-Ih promoters from two A. gyllenbergii isolates and from pIP1858. Grey arrow, portion of ISAba23; blue arrow, portion of cynT gene. The P_native and P_ISAba23 promoters are underlined; the putative −35 and −10 sequences determined by BPROM³⁰ of P_native are in red characters and those of P_ISAba23 in green or yellow boxes. Red box, start codon of aac(6′)-Ih.

In pIP1858, ISAba23 insertion disrupted the P_native promoter from A. gyllenbergii located from bp 61 to bp 31 upstream from the start codon of the gene. However, the left end of ISAba23 provided two P_ISAba23 portable promoters, located from bp 120 to bp 91 and from bp 109 to bp 80 upstream from P_native (Figure 2). The −35 and −10 sequences of the two promoters were separated by 17 bp, 1 bp less than in P_native.

Expression of aac(6′)-Ih in A. gyllenbergii and A. baumannii

AAC(6′)-I enzymes confer resistance to netilmicin, amikacin and tobramycin, but not to gentamicin. They also confer resistance to 2′-N-ethylnetilmicin, but not to 6′-N-ethylnetilmicin, and, since these compounds have similar activity against aminoglycoside-susceptible strains, this difference can be taken as evidence for the production of a 6′-N-acetyltransferase.¹¹

The A. gyllenbergii isolates harbouring aac(6′)-Ih were resistant to low levels of netilmicin and tobramycin, and had diminished susceptibility to amikacin, whereas, as expected, gentamicin remained active (Table 1). The strains were also resistant to 2′-N-ethylnetilmicin, but not to 6′-N-ethylnetilmicin (Figure S2). Plasmid pIP1858 conferred resistance to netilmicin, tobramycin and amikacin to A. baumannii BM2686 (Table 1).¹⁶ The MICs of the three aminoglycosides were always higher for BM2686/pIP1858 and the five A. baumannii clinical isolates harbouring this plasmid than for A. gyllenbergii (Table 1 and Table S1).

Table 1.

Susceptibility of strains to aminoglycosides

Strain/plasmid	MIC (mg/L)
	amikacin	tobramycin	gentamicin	netilmicin
E. coli TOP10	2	0.5	2	0.25
E. coli TOP10/pAT747 (pUC18Ωori pWH1266)	2	0.5	2	0.25
A. baumannii BM4587	2	2	1	1
A. baumannii BM4587/pAT747 (pUC18Ωori pWH1266)	2	2	1	1
A. gyllenbergii CIP 110306T	1	1	0.5	8
A. gyllenbergii NIPH 230	4	4	0.5	8
A. baumannii BM2686/pIP1858	32	32	1	128
E. coli TOP10/pAT747ΩPnativeaac(6′)-Ih	16	16	2	8
E. coli TOP10/pAT747ΩPISAba23aac(6′)-Ih	64	32	2	32
A. baumannii BM4587/pAT747ΩPnativeaac(6′)-Ih	128	128	2	512
A. baumannii BM4587/pAT747ΩPISAba23aac(6′)-Ih	128	128	2	512

P_native, native promoter in A. gyllenbergii; P_ISAba23, promoter of ISAba23; ^T, type strain.

MICs were determined by microdilution.²²

The level of aac(6′)-Ih expression in A. gyllenbergii and A. baumannii was determined by qRT–PCR normalized to that of rpoB (Table S1). The five A. baumannii strains had aac(6′)-Ih mRNA levels of ∼20 to 1000 times higher than in the nine A. gyllenbergii isolates, which were consistent with the aminoglycoside MICs.

To compare the strength of the P_native and P_ISAba23 promoters, the genes from A. gyllenbergii CIP 110306^T and from pIP1858 were cloned under the control of their respective promoters in shuttle vector pAT747 (pUC18ΩoripWH1266)¹⁷ and introduced into E. coli TOP10 by transformation and into aminoglycoside-susceptible A. baumannii BM4587 by electrotransformation (Table 1).

The MICs of amikacin, tobramycin and netilmicin were always higher for A. baumannii BM4587/pAT747Ωaac(6′)-Ih than for BM2686/pIP1858 (Table 1), both strains harbouring plasmids that carry the aac(6′)-Ih gene. This can be accounted for by a gene dosage effect; the pIP1858 copy number in BM2686 being ∼8 per genome equivalent, whereas it was 20 for pAT747 in BM4587, as determined by qPCR.

There were no changes in aminoglycoside resistance of E. coli TOP10 or A. baumannii BM4587 after acquisition of pAT747 (Table 1). The aac(6′)-Ih gene was expressed and functional under the control of the P_native promoter in both E. coli and A. baumannii. It was responsible for slightly higher levels of resistance (2 to 4 times) when expressed from the P_ISAba23 promoter in E. coli. The MICs of aminoglycosides were always higher for the A. baumannii than the E. coli hosts (from 8 to 64 times under the control of P_native, and from 2 to 16 times under that of P_ISAba23) despite the fact that the plasmid copy number is ∼12 times more elevated in E. coli than in A. baumannii.¹⁷ This expands our previous observation of the importance of the bacterial host background for expression of resistance determinants,¹⁷ and is consistent with an origin in A. gyllenbergii of aac(6′)-Ih. In A. baumannii BM4587, the P_native and P_ISAba23 promoters were similarly potent.

Dissemination of aac(6′)-Ih

Searches in databases, such as those of non-redundant GenBank CDS translations and SwissProt, found aac(6′)-Ih only in the A. baumannii collected at the time of the plasmid outbreak and in members of the A. gyllenbergii sp., indicating limited dissemination. The backbone of pIP1858 was that of Tra⁻ Mob⁺ 5.6 kb p1ABAYE (Figure 1), a cryptic plasmid from A. baumannii AYE,⁴ which provided a replication origin found so far only in Acinetobacter spp. The two plasmids shared a nearly identical 4.1 kb region, including genes for mobilization proteins MobS/L and the replication protein.

The five additional A. baumannii from the plasmid epidemic were studied. Restriction by HindIII and PCR mapping (Figure S3) confirmed the plasmid outbreak. Plasmid pIP1858 was not transferable by conjugation from these strains to A. baumannii BM4587 Rif^R,²⁵ as already reported.¹⁶ Since the plasmid had an Acinetobacter-specific replication origin dissemination of aac(6′)-Ih remained intrageneric.

Additional A. baumannii A.62 (Table S1) was selected to study mobilization of pIP1858 by RP4²⁴ to A. baumannii BM4587 Rif^R. Plasmid RP4 conjugated at a frequency of 10⁻³ per donor, whereas pIP1858 could be mobilized at a frequency of ∼10⁻⁷.

Conclusions

To the best of our knowledge, the aac(6′)-Ih gene has not been reported in other Gram-negative bacteria. This is consistent with the observation that the gene was: (i) not part of a functional transposon; (ii) carried by a non-conjugative plasmid with a replication origin specific to Acinetobacter; and (iii) could be mobilized to A. baumannii by the broad host range plasmid RP4 from P. aeruginosa.

The paradigm stipulating that aminoglycoside-modifying enzymes originate from antibiotic producers is based on horizontal gene transfer between soil microorganisms and pathogenic bacteria.²⁸ However, in other instances, a functional role such as acetylation of proteins involved in transcriptional regulation is consistent with aac(6′)-I genes being housekeeping genes.²⁹ Taking into account the clearance of unnecessary DNA sequences, the maintenance of these genes during evolution favours a physiological role, a hypothesis that requires additional investigations.

In summary, this work provides an example of the evolution of housekeeping genes into antibiotic resistance determinants, which, when moved into the appropriate background, contribute to the resistance phenotype of the new bacterial hosts.

Funding

This project was funded in part with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. HHSN272200900018C. E.-J. Y. was supported by an unrestricted grant from Reckitt-Benckiser, and A. N. was supported by grant NT14466-3/2013 of the Internal Grant Agency of the Ministry of Health of the Czech Republic.

Transparency declarations

None to declare.

Supplementary data

Tables S1 and S2 and Figures S1 to S3 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).