title: Origin in Acinetobacter guillouiae and dissemination of the aminoglycoside-modifying enzyme Aph(3')-VI.
abstract: The amikacin resistance gene aphA6 was first detected in the nosocomial pathogen Acinetobacter baumannii and subsequently in other genera. Analysis of 133 whole-genome sequences covering the taxonomic diversity of Acinetobacter spp. detected aphA6 in the chromosome of 2 isolates of A. guillouiae, which is an environmental species, 1 of 8 A. parvus isolates, and 5 of 34 A. baumannii isolates. The gene was also present in 29 out of 36 A. guillouiae isolates screened by PCR, indicating that it is ancestral to this species. The Pnative promoter for aphA6 in A. guillouiae and A. parvus was replaced in A. baumannii by PaphA6, which was generated by use of the insertion sequence ISAba125, which brought a -35 sequence. Study of promoter strength in Escherichia coli and A. baumannii indicated that PaphA6 was four times more potent than Pnative. There was a good correlation between aminoglycoside MICs and aphA6 transcription in A. guillouiae isolates that remained susceptible to amikacin. The marked topology differences of the phylogenetic trees of aphA6 and of the hosts strongly support its recent direct transfer within Acinetobacter spp. and also to evolutionarily remote bacterial genera. Concomitant expression of aphA6 must have occurred because, contrary to the donors, it can confer resistance to the new hosts. Mobilization and expression of aphA6 via composite transposons and the upstream IS-generating hybrid PaphA6, followed by conjugation, seems the most plausible mechanism. This is in agreement with the observation that, in the recipients, aphA6 is carried by conjugative plasmids and flanked by IS that are common in Acinetobacter spp. Our data indicate that resistance genes can also be found in susceptible environmental bacteria. Importance: We speculated that the aphA6 gene for an enzyme that confers resistance to amikacin, the most active aminoglycoside for the treatment of nosocomial infections due to Acinetobacter spp., originated in this genus before disseminating to phylogenetically distant genera pathogenic for humans. Using a combination of whole-genome sequencing of a collection of Acinetobacter spp. covering the breadth of the known taxonomic diversity of the genus, gene cloning, detailed promoter analysis, study of heterologous gene expression, and comparative analysis of the phylogenetic trees of aphA6 and of the bacterial hosts, we found that aphA6 originated in Acinetobacter guillouiae, an amikacin-susceptible environmental species. The gene conferred, upon mobilization, high-level resistance to the new hosts. This work stresses that nonpathogenic bacteria can act as reservoirs of resistance determinants, and it provides an example of the use of a genomic library to study the origin and dissemination of an antibiotic resistance gene to human pathogens.
authors.affiliation: Institut Pasteur, Unité des Agents Antibactériens, Paris, France.
authors.forename: Eun-Jeong
authors.initials: EJ
authors.lastname: Yoon
authors.affiliation: Institut Pasteur, Unité des Agents Antibactériens, Paris, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Goussard
authors.forename: Marie
authors.initials: M
authors.lastname: Touchon
authors.affiliation: Laboratory of Bacterial Genetics, National Institute of Public Health, Prague, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Krizova
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Cerqueira
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, USA.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Murphy
authors.affiliation: EA4043, Faculté de Pharmacie, Université Paris Sud, Châtenay-Malabry, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Lambert
authors.affiliation: Institut Pasteur, Unité des Agents Antibactériens, Paris, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Grillot-Courvalin
authors.affiliation: Laboratory of Bacterial Genetics, National Institute of Public Health, Prague, Czech Republic.
authors.forename: Alexandr
authors.initials: A
authors.lastname: Nemec
authors.affiliation: Institut Pasteur, Unité des Agents Antibactériens, Paris, France patrice.courvalin@pasteur.fr.
authors.forename: Patrice
authors.initials: P
authors.lastname: Courvalin
mesh_terms: D000150:Acinetobacter / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000617:Aminoglycosides / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D016000:Cluster Analysis; D003227:Conjugation, Genetic; D024881:Drug Resistance, Bacterial; D004783:Environmental Microbiology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D022761:Gene Transfer, Horizontal; D020071:Interspersed Repetitive Sequences; D019868:Kanamycin Kinase / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D017385:Sequence Homology
chemical_list: D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D019868:Kanamycin Kinase