CHARACTERIZATION OF REPLICATION MODULES IN Acinetobacter baumannii RESISTANCE PLASMIDS

MARCHIARO P; VIALE AM; GIACONE L; MORAN BARRIO J; SANCHEZ R; LIMANSKY A

Congreso; XVIII Congreso Argentino de Microbiología General; 2022

The global spread of multidrug resistance (MDR), and in particular resistance to last-resource carbapenem β-lactams, among the clinical population of the healthcare-associated opportunistic pathogen Acinetobacter baumannii represents nowadays a major concern. The most frequent cause of carbapenem resistance among clinical A. baumannii strains is the horizontal acquisition of carbapenem-hydrolyzing class-D β-lactamases (CHDL), with the cognate blaOXA genes being most frequently carried by plasmids endowed with replication modules carrying replication initiation protein genes (repAci) of the Rep_3 (PF01051) superfamily (Rep_3 plasmids). To date, 20 to 30 different Rep_3 repAci genes have been described, largely on the basis of sequence comparisons and phylogenetic analyses. Many A. baumannii Rep_3 plasmids contain more than one replicon, posing questions on their general functionality, incompatibility, and role(s), but few functional analyses have been conducted on these matters.We have previously sequenced and characterized four different Rep_3 plasmids (pAb244_7, pAb242_9, pAb242_12 and pAb242_25) housed by three local MDR A. baumannii clinical strains of the CC15(P) clonal complex, two of them displaying additional carbapenem resistance (CRAB strains) (Cameranesi et al. 2018, 2020). pAb242_25, present only in CRAB strains, is a bi-replicon containing repAci23 and repAci22 genes and also carries a blaOXA-58- and TnaphA6-containing adaptive module conferring carbapenem and amikacin resistance. The other three plasmids contain only one replicon module each with the following repAci genes: repAci4 in both pAb244_7 and pAb242_9, and repAci21 in pAb242_12. All contain the characteristic iteron repetitive sequences upstream of the corresponding repAci genes, indicating their mode of replication. Cloning of each of these replication modules in appropriate plasmid vectors indicated that all of them are functional in an Acinetobacter host, although some differences were observed concerning stability between replicons. Moreover, transformation with different combinations of the analyzed replication modules indicated that all of them could co-exist in the same host, suggesting their belonging to different incompatibility groups. qPCR assays indicated that they were present in copy numbers that ranged between 4 and 6 per Acinetobacter cell, suggesting that they represent medium-copy number replicons. The overall observations indicate that all of the four analyzed A. baumannii replicons were functional, represent medium-copy number replicons, and could also be assigned to different incompatibility groups. Moreover, they open significant questions on their cross-regulation in the Acinetobacter cell, in particular when co-existing in a multi-replicon plasmid.