Validation of partial rpoB gene sequence analysis for the identification of clinically important and emerging Acinetobacter species

Abstract

Bacteria belonging to the genusAcinetobacterare ubiquitous in soil and water. Only a few species, includingAcinetobacter baumannii, and the unnamedAcinetobactergenomic species (gen. sp.) 3 and 13TU, which together with the soil organismAcinetobacter calcoaceticusare combined in theA. calcoaceticus–A. baumannii(Acb) complex, have been recognized as important nosocomial infectious agents. The ecology, epidemiology and pathology of most species are not yet well established. Lack of practical and accurate methods limits routine identification of clinical isolates and thus hampers precise identification of those of the Acb complex and otherAcinetobacterspecies of possible clinical significance. We previously identified a 350 bp highly variable zone on therpoBgene which appeared to be a promising target for rapid molecular identification. In the present study, we validated this method for accuracy on a collection of reference strains belonging toA. calcoaceticus(5 strains),Acinetobactergen. sp. 3 (29 strains),A.gen. sp. 13TU (18 strains),A. baumannii(30 strains) and one strain each ofA. radioresistens,A. gen. sp. 15TU,A. gen. sp. 10,A. gen. sp. 11,A. gen. sp. ‘between 1 and 3’ andA. gen. sp. 14TU=13BJ. This represents the largest analysis to date that compares a large number of well-identified strains of the Acb complex to assess the intra- and interspecies variation within this complex. All were correctly identified with 98.9–100 % intraspecies relatedness based on partialrpoBsequence analysis. We then applied this tool to identify 99Acinetobacterclinical isolates from four public hospitals in Marseille, France. All isolates could easily be identified to species as they were separated into 13 species sequence types with a sequence variance of 0–2.6 % from their respective type strains. Of these 99 isolates, 10 wereA. haemolyticus, 52 wereA. baumannii, 27 wereA.gen. sp. 3, 5 wereA. schindleri, 1 wasA. lwoffii, and 1 wasA.gen. sp. 13TU. Three were provisionally identified asA.gen. sp. 9. This is the first work to identify all specimens of a set of clinicalAcinetobacterisolates at species level usingrpoBsequence analysis. Our data emphasize the recognition ofA. schindlerias an emerging cause ofAcinetobacter-related infection and confirm thatA.gen. sp. 3 is the second most commonly isolatedAcinetobacterspecies afterA. baumanniiin patients.