Characterization and PCR-Based Replicon Typing of Resistance Plasmids in Acinetobacter baumannii

Abstract

Acinetobacter baumannii is an opportunistic pathogen, especially in intensive care units, and multidrugresistant isolates have increasingly been reported during the last decade. Despite recent progress in knowledge of antibiotic resistance mechanisms in A. baumannii, little is known about the genetic factors driving isolates toward multidrug resistance. In the present study, the A. baumannii plasmids were investigated through the analysis and classification of plasmid replication systems and the identification of A. baumannii-specific mobilization and addiction systems. Twenty-two replicons were identified by in silico analysis, and five other replicons were identified and cloned from previously uncharacterized A. baumannii resistance plasmids carrying the OXA-58 carbapenem-hydrolyzing oxacillinase. Replicons were classified into homology groups on the basis of their nucleotide homology. A novel PCR-based replicon typing scheme (the A. baumannii PCR-based replicon typing [AB-PBRT] method) was devised to categorize the A. baumannii plasmids into homogeneous groups on the basis of the nucleotide homology of their respective replicase genes. The AB-PBRT technique was applied to a collection of multidrug-resistant A. baumannii clinical isolates carrying the bla OXA-58 or bla OXA-23 carbapenemase gene. A putative complete conjugative apparatus was identified on one plasmid whose selfconjugative ability was demonstrated in vitro. We showed that this conjugative plasmid type was widely diffused in our collection, likely representing the most important vehicle promoting the horizontal transmission of A. baumannii resistance plasmids.The foundation of plasmid biology was largely built on the genetic analysis of plasmid strategies for broad-host-range replication in Gram-negative bacteria. Mechanisms which guarantee the autonomous replication, addiction systems based on toxin-antitoxin factors, partitioning systems ensuring stable inheritance during cell division, and other virulence and antimicrobial resistance determinants have been described for plasmids circulating in the Enterobacteriaceae family and Pseudomonas spp. (17). Enterobacterial plasmids have also been classified into homogeneous groups on the basis of their replication controls by conjugation (plasmid incompatibility) and molecular methods (Southern blot hybridization with replicon probes and PCR-based replicon typing) (5,8,10,11). Currently, 27 incompatibility groups are recognized in the Enterobacteriaceae by the Plasmid Section of the National Collection of Type Cultures (Colindale, London, United Kingdom). In contrast, limited information is available on the plasmids circulating in Acinetobacter spp., even though Acinetobacter baumannii is an important pathogen in intensive care units (13,28). Moreover, despite recent progress in the study of antibiotic resistance mechanisms in A. baumannii, little is known about the genetic factors that have driven the recent evolution of A. baumannii toward multidrug resistance. A. baumannii may develop resistance to...

Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii

“…One of the plasmids is unusually small (less than 2 kbp) and has only three genes, a replication initiation gene and two genes of unknown function. The replication initiation gene corresponds to a previously undefined plasmid replication group (57). The other two plasmids are closely related to previously described plasmids (8,58), the larger of which was shown to be transmissible by conjugation (58).…”

Multidrug-resistant Acinetobacter baumannii strains carrying the blaOxA-23 and the blaGES-11 genes in a neonatology center in Tunisia

“…Plasmid typing was performed using the A. baumannii PCRbased replicon typing [AB-PBRT] method as described previously [19]. The PCR products fragments were purified with QIAquick column (Qiagen, Courtaboeuf, France) and sequenced with an Applied Biosystems sequencer (ABI 377).…”

Acinetobacter baumannii: biology and drug resistance — role of carbapenemases

“…Studies concerning the sequence analysis of A. baumannii plasmid replicons revealed the differences comparing to other prokaryotic species, suggesting that A. baumannii strains may carry distinct set of plasmid types [92,93]. It is believed that the most clinically relevant carbapenemases are often associated with plasmids [68].…”

“…It is believed that the most clinically relevant carbapenemases are often associated with plasmids [68]. While A. baumannii plasmids may vary in size as well as genetic content, Bertini and co-authors proposed their classification based on the replicase gene sequences [92]. Towner and co-authors analysing the group of 96 MDR A. baumannii clinical isolates from 17 European countries, confirmed the presence at least 1 (with a maximum of 4) out of 19 replicases (rep) gene groups (GR) among all clinical strains tested.…”