Programme Hospitalier Recherche Clinique, Institut Pasteur, Inserm, French Public Health Agency.

Multidrug-resistant strain Acinetobacter baumannii BM4454 was isolated from a patient with a urinary tract infection. The adeB gene, which encodes a resistance-nodulation-cell division (RND) protein, was detected in this strain by PCR with two degenerate oligodeoxynucleotides. Insertional inactivation of adeB in BM4454, which generated BM4454-1, showed that the corresponding protein was responsible for aminoglycoside resistance and was involved in the level of susceptibility to other drugs including fluoroquinolones, tetracyclines, chloramphenicol, erythromycin, trimethoprim, and ethidium bromide. Study of ethidium bromide accumulation in BM4454 and BM4454-1, in the presence or in the absence of carbonyl cyanide m-chlorophenylhydrazone, demonstrated that AdeB was responsible for the decrease in intracellular ethidium bromide levels in a proton motive force-dependent manner. The adeB gene was part of a cluster that included adeA and adeC which encodes proteins homologous to membrane fusion and outer membrane proteins of RND-type three-component efflux systems, respectively. The products of two upstream open reading frames encoding a putative twocomponent regulatory system might be involved in the regulation of expression of the adeABC gene cluster.

The AdeABC pump of Acinetobacter baumannii BM4454, which confers resistance to various antibiotic classes including aminoglycosides, is composed of the AdeA, AdeB, and AdeC proteins; AdeB is a member of the RND superfamily. The adeA, adeB, and adeC genes are contiguous and adjacent to adeS and adeR, which are transcribed in the opposite direction and which specify proteins homologous to sensors and regulators of two-component systems, respectively (S. Magnet, P. Courvalin, and T. Lambert, Antimicrob. Agents Chemother. 45:3375-3380, 2001). Analysis by Northern hybridization indicated that the three genes were cotranscribed, although mRNAs corresponding to adeAB and adeC were also present. Cotranscription of the two regulatory genes was demonstrated by reverse transcription-PCR. Inactivation of adeS led to aminoglycoside susceptibility. Transcripts corresponding to adeAB were not detected in susceptible A. baumannii CIP 70-10 but were present in spontaneous gentamicin-resistant mutants obtained in vitro. Analysis of these mutants revealed the substitutions Thr1533Met in AdeS downstream from the putative His-149 site of autophosphorylation, which is presumably responsible for the loss of phosphorylase activity by the sensor, and Pro1163Leu in AdeR at the first residue of the ␣ 5 helix of the receiver domain, which is involved in interactions that control the output domain of response regulators. These mutations led to constitutive expression of the pump and, thus, to antibiotic resistance. These data indicate that the AdeABC pump is cryptic in wild A. baumannii due to stringent control by the AdeRS two-component system.

We have identified a second resistance-nodulation-cell division (RND)-type efflux pump, AdeIJK, in clinical isolate Acinetobacter baumannii BM4454. The adeI, adeJ, and adeK genes encode, respectively, the membrane fusion, RND, and outer membrane components of the pump. AdeJ belongs to the AcrB protein family (57% identity with AcrB from Escherichia coli). mRNA analysis by Northern blotting and reverse transcription-PCR indicated that the genes were cotranscribed. Overexpression of the cloned adeIJK operon was toxic in both E. coli and Acinetobacter. The adeIJK genes were detected in all of the 60 strains of A. baumannii tested. The two latter observations suggest that the AdeIJK complex might contribute to intrinsic but not to acquired antibiotic resistance in Acinetobacter. To characterize the substrate specificity of the pump, we have constructed derivatives of BM4454 in which adeIJK (strain BM4579), adeABC (strain BM4561), or both groups of genes (strain BM4652) were inactivated by deletion-insertion. Determination of the antibiotic susceptibility of these strains and of BM4652 and BM4579, in which the adeIJK operon was provided in trans, indicated that the AdeIJK pump contributes to resistance to ␤-lactams, chloramphenicol, tetracycline, erythromycin, lincosamides, fluoroquinolones, fusidic acid, novobiocin, rifampin, trimethoprim, acridine, safranin, pyronine, and sodium dodecyl sulfate. The chemical structure of these molecules suggests that amphiphilic compounds are the preferred substrates. The AdeABC and AdeIJK efflux systems contributed in a more than additive fashion to tigecycline resistance.

Acinetobacter baumannii is a major nosocomial pathogen which frequently develops multidrug resistance by acquisition of antibiotic resistance genes and overexpression of intrinsic efflux systems, such as the RND efflux pumps AdeABC and AdeIJK. A third RND system was characterized by studying spontaneous mutants BM4663 and BM4664, which were selected in the presence of chloramphenicol and norfloxacin, respectively, from the AdeABC-and AdeIJK-defective derivative A. baumannii BM4652. They exhibited enhanced resistance to fluoroquinolones, tetracycline-tigecycline, chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, sodium dodecyl sulfate, and dyes such as ethidium bromide, safranin O, and acridine orange. Comparison of transcriptomes of mutants with that of their parental strain, using a microarray technology, demonstrated the overexpression of three genes that encoded an RND efflux system, named AdeFGH. Inactivation of AdeFGH in BM4664 restored an antibiotic susceptibility profile identical to that of BM4652, indicating that AdeFGH was cryptic in BM4652 and responsible for multidrug resistance in its mutants. RNA analysis demonstrated that the three genes were cotranscribed. The adeFGH operon was found in 36 out of 40 A. baumannii clinical isolates, but none of the 22 isolates tested overexpressed the pump genes. Spontaneous MDR mutant BM4684, overexpressing adeFGH, was obtained from clinical isolate BM4587, indicating that adeFGH can be overexpressed in a strain harboring adeABC-adeIJK. An open reading frame, coding a LysR-type transcriptional regulator, named adeL, was located upstream from the adeFGH operon and transcribed in the opposite direction. Mutations in adeL were found in the three adeFGH-overexpressing mutants, suggesting that they were responsible for overexpression of AdeFGH.

No abstract

Bacterial genomics has greatly expanded our understanding of microdiversification patterns within a species, but analyses at higher taxonomical levels are necessary to understand and predict the independent rise of pathogens in a genus. We have sampled, sequenced, and assessed the diversity of genomes of validly named and tentative species of the Acinetobacter genus, a clade including major nosocomial pathogens and biotechnologically important species. We inferred a robust global phylogeny and delimited several new putative species. The genus is very ancient and extremely diverse: Genomes of highly divergent species share more orthologs than certain strains within a species. We systematically characterized elements and mechanisms driving genome diversification, such as conjugative elements, insertion sequences, and natural transformation. We found many error-prone polymerases that may play a role in resistance to toxins, antibiotics, and in the generation of genetic variation. Surprisingly, temperate phages, poorly studied in Acinetobacter, were found to account for a significant fraction of most genomes. Accordingly, many genomes encode clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems with some of the largest CRISPR-arrays found so far in bacteria. Integrons are strongly overrepresented in Acinetobacter baumannii, which correlates with its frequent resistance to antibiotics. Our data suggest that A. baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection. The outstanding diversification of the species occurred largely by horizontal transfer, including some allelic recombination, at specific hotspots preferentially located close to the replication terminus. Our work sets a quantitative basis to understand the diversification of Acinetobacter into emerging resistant and versatile pathogens.

A self-transferable plasmid of ca. 80 kb, pIP1204, conferred multiple-antibiotic resistance to Klebsiella pneumoniae BM4536, which was isolated from a urinary tract infection. Resistance to ␤-lactams was due to the bla TEM1 and bla CTX-M genes, resistance to trimethroprim was due to the dhfrXII gene, resistance to sulfonamides was due to the sul1 gene, resistance to streptomycin-spectinomycin was due to the ant3؆9 gene, and resistance to nearly all remaining aminoglycosides was due to the aac3-II gene and a new gene designated armA (aminoglycoside resistance methylase). The cloning of armA into a plasmid in Escherichia coli conferred to the new host high-level resistance to 4,6-disubstituted deoxystreptamines and fortimicin. The deduced sequence of ArmA displayed from 37 to 47% similarity to those of 16S rRNA m 7 G methyltransferases from various actinomycetes, which confer resistance to aminoglycoside-producing strains. However, the low guanine-pluscytosine content of armA (30%) does not favor an actinomycete origin for the gene. It therefore appears that posttranscriptional modification of 16S rRNA can confer high-level broad-range resistance to aminoglycosides in gram-negative human pathogens.