First Identification and Characterization of an AdeABC-Like Efflux Pump in Acinetobacter Genomospecies 13TU

Abstract

Non-Acinetobacter baumannii spp. are emerging among clinical Acinetobacter isolates causing nosocomial infections, and some (such as genomospecies 13TU) appear to be multidrug resistant. The prevalence of non-Acinetobacter baumannii spp. in the hospital setting is likely understated due to poor identification techniques. We report the first identification of an AdeABC-type efflux pump in an Acinetobacter genomospecies 13TU clinical isolate, its contribution to multidrug resistance, and the coexistence of three Ade-type efflux pumps in this strain.

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex comprises closely related genomic species belonging to the nonfermentative Gram-negative Acinetobacter genus (7). Among them, genomospecies 2 (Acinetobacter baumannii), 3, and 13TU are rapidly emerging as opportunistic pathogens and pose an important public health issue in the clinical setting (10, 11). The treatment of infections caused by these strains can be complicated due to the increasing occurrence of multidrug-resistant (MDR) strains. The MDR phenotype in Acinetobacter spp. has been associated with transposon-based resistance islands and/or active efflux mechanisms (1, 4, 6, 9, 13, 16, 18).

To date, four main RND efflux pumps have been identified in Acinetobacter spp. The AdeABC system, only present in A. baumannii clinical isolates, provides resistance to aminoglycosides, tetracycline, erythromycin, chloramphenicol, trimethoprim, and fluoroquinolones (9). The AdeDE pump, which has been identified in genomospecies 3 and in one Acinetobacter genomospecies 13TU clinical isolate (4, 5), provides resistance to amikacin, ceftazidime, chloramphenicol, ciprofloxacin, erythromycin, meropenem, rifampin, and tetracycline. The AdeIJK efflux pump has only been found in A. baumannii and confers resistance to β-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, rifampin, and trimethoprim (6). Finally, the AdeXYZ efflux system was identified in 2006 in clinical isolates classified as genomospecies 3, but its role in antimicrobial resistance has not yet been proven (5).

A PCR screen designed to identify adeB genes in a collection of Acinetobacter calcoaceticus-Acinetobacter baumannii clinical isolates originated a positive amplicon (1.2 kb) from an Acinetobacter genomospecies 13TU strain designated strain 158029 (species was identified by amplified rRNA gene restriction analysis [ARDRA]), sequence analysis of the 16S-23S rRNA gene spacer region (internal transcribed spacer [ITS]), and recA sequencing (3, 12, 17). The amplified PCR product was sequenced and used to design an initial set of outward primers to fully sequence its flanking regions by stepwise genome walking (see Table S1 in the supplemental material). Several rounds of outward sequencing finally provided a 7,780-bp sequence containing the genes encoding a membrane fusion protein, an inner membrane efflux protein, and an outer membrane protein channel with very good similarities to the A. baumannii AYE AdeABC efflux system (96%, 99%, and 95%, respectively). A two-component regulatory system with very good similarity to AdeRS (99% and 94%, respectively) was also found upstream from adeABC in an inverted orientation.

To demonstrate the involvement of this system in multidrug resistance, an internal fragment of the adeB gene (619 bp) was amplified and cloned into the pGEM-T Easy vector (Promega) by A/T cloning and introduced into strain 158029 by electroporation. Since pGEM-T Easy is suicidal in Acinetobacter, transformants selected in LB plates containing 80 μg/ml ticarcillin should result from a single-crossover event leading to the insertional inactivation of the cognate adeB gene. adeB disruption within the resulting strain, designated JVAB02, was verified by PCR using a combination of primers matching the upstream region of the adeB gene and the pGEM-T Easy scaffold.

The antibiotic susceptibilities of 158029 and JVAB02 were studied by Etest. Strain JVAB02 proved more susceptible than 158029 to a wide range of antibiotics (Table 1), including β-lactams, chloramphenicol, tetracyclines, quinolones, trimethoprim, aminoglycosides, and the novel glycylcycline, tigecycline. The substrate specificity was similar to that of the AdeABC pump from A. baumannii (9, 15), although we found an 8-fold decrease in the MIC of ceftazidime (which is not a substrate for the AdeABC pump in A. baumannii but is for the AdeDE pump of Acinetobacter genomospecies 3 [4]), as well as a profound effect on the MIC of chloramphenicol. Although RND-type efflux pumps have been shown to extrude chloramphenicol in Acinetobacter baumannii, a novel MFS-type pump (CraA) has recently been associated with the intrinsic chloramphenicol resistance displayed by this organism (14). In this work, the use of craA-specific primers failed to detect the presence of this pump in strain 158029 (data not shown). The lack of CraA might result in a more predominant role of AdeABC in chloramphenicol resistance in Acinetobacter genomospecies 13TU.

TABLE 1.

MICs of antimicrobial agents for the investigated strains

Antimicrobial agent	MIC (μg/ml)		Difference in susceptibility (fold)
	158029	JVAB02 (adeB::Tic)
Piperacillin	6	6	1
Aztreonam	32	3	11
Ceftriaxone	16	3	5
Cefoxitin	96	4	24
Ceftazidime	4	0.5	8
Cefotaxime	12	0.5	24
Imipenem	0.5	0.25	2
Meropenem	1	0.064	16
Nalidixic Acid	12	1.5	8
Ciprofloxacin	1	0.016	63
Norfloxacin	16	0.38	42
Gentamicin	12	0.094	128
Amikacin	12	1	12
Kanamycin	6	0.75	8
Tobramycin	3	0.38	8
Tetracycline	16	0.75	21
Trimethoprim	>32	0.75	>42
Tigecycline	3	0.064	47
Chloramphenicol	>256	8	>32

We also looked for the presence of additional Ade-type efflux pumps in this strain using specific primers matching internal regions of the A. baumannii adeJ and Acinetobacter genomospecies 3 adeE and adeY genes. The use of adeE-specific primers originated an amplification product of approximately 750 bp with 90% identity to the adeE gene from Acinetobacter genomospecies 3 strain 4365 (4). Amplification with the adeJ- and adeY-specific primers also yielded amplification products of approximately 750 bp for both, with 95% and 89% identity to adeJ and adeY, respectively. It is worth mentioning that, although described as independent pumps, AdeIJK and AdeXYZ share 93% identity at the nucleotide level and 99% similarity at the protein level. Similar percentages can be found when comparing adeJ or AdeJ sequences from different A. baumannii strains.

The protein identified in this work seems to be more similar to AdeJ than to AdeY, and therefore, this would be the first time that this pump has been described in an Acinetobacter genomospecies other than A. baumannii. We believe, however, that AdeIJK and AdeXYZ represent the same pump, which is most likely present in Acinetobacter genomic DNA groups 2, 3, and 13TU, among others (2).

Overall, these results were highly unexpected, since previous studies seemed to indicate that the presence of adeABC and adeDE was species specific, with adeABC being restricted to Acinetobacter baumannii and adeDE to Acinetobacter genomospecies 3 (8). This is the first time that the AdeABC pump has been described and characterized in a non-Acinetobacter baumannii strain and that all three pumps have been shown to coexist. A larger number of strains belonging to this group should be analyzed in order to confirm whether this carriage of efflux pumps is widespread. Nonetheless, the presence of three major efflux systems in an emerging multidrug-resistant pathogen is alarming.

Nucleotide sequence accession number.

The full length of the adeSRABC sequence was submitted to GenBank and assigned accession number GU319112.