An XDR Pseudomonas aeruginosa ST463 Strain with an IncP-2 Plasmid Containing a Novel Transposon Tn6485f Encoding blaIMP-45 and blaAFM-1 and a Second Plasmid with Two Copies of blaKPC-2.

The increased carbapenem resistance among Pseudomonas aeruginosa has become a serious health issue worldwide. We reported an extensively drug-resistant (XDR) P. aeruginosa PA30 isolate which belonged to sequence type ST463 and contained an IncP-2 plasmid (pPA30_1) carrying two genes, namely, blaIMP-45 and blaAFM-1, which encoded the metallo-ß-lactamases AFM-1 and IMP-45, respectively. Additionally, the strain had a plasmid (pPA30_2) with two copies of the blaKPC-2 genes embedded. The plasmid pPA30_1 was highly similar to the previously reported plasmid pHS17-127, which has the same genetic architecture. This plasmid contained blaIMP-45, located in a second gene cassette of the integron In786, carried by a Tn1403-derivative transposon acquiring an ISCR27n3-blaAFM-1 structure. Interestingly, the transposon in pPA30_1 acquired an extra ISCR1-qnrVC6 module and formed a novel transposon, which was subsequently annotated as Tn6485f. The blaKPC-2 genes in pPA30_2 underwent duplication due to the inversion of the IS26-blaKPC-2-IS26 element, which resulted in two copies of blaKPC-2. IMPORTANCE The ST463 clone is an emerging high-risk sequence type that is spreading with blaKPC-2-containing plasmids. The core blaKPC-2 genetic platform is ISKpn27-blaKPC-2-ISKpn6 in almost all samples, and the adjacent region beyond the core platform varies by IS26-mediated inversion or duplication events, amplifying the blaKPC-2 gene copies. The ST463 P. aeruginosa strain PA30 in our study contains another two metallo-ß-lactamase genes, namely, blaIMP-45 and blaAFM-1, in a novel transposon Tn6485f that is harbored by the IncP-2 megaplasmid. The pPA30_1 carrying blaIMP-45 and blaAFM-1 is highly related to pHS17-127 from the ST369 P. aeruginosa strain, indicating the putative dissemination of the megaplasmid between different clones.

Characterization of a Conjugative Multidrug Resistance IncP-2 Megaplasmid, pPAG5, from a Clinical Pseudomonas aeruginosa Isolate.

The spread of resistance genes via horizontal plasmid transfer plays a significant role in the formation of multidrug-resistant (MDR) Pseudomonas aeruginosa strains. Here, we identified a megaplasmid (ca. 513 kb), designated pPAG5, which was recovered from a clinical multidrug-resistant P. aeruginosa PAG5 strain. The pPAG5 plasmid belonged to the IncP-2 incompatibility group. Two large multidrug resistance regions (MDR-1 and MDR-2) and two heavy metal resistance operons (merEDACPTR and terZABCDE) were identified in the pPAG5 plasmid. Genetic analysis demonstrated that the formation of MDR regions was mediated by several homologous recombination events. Further conjugation assays identified that pPAG5 could be transferred to P. aeruginosa but not Escherichia coli. Antimicrobial susceptibility testing on transconjugants demonstrated that pPAG5 was capable of transferring resistance genes to transconjugants and producing a multidrug-resistant phenotype. Comparative analysis revealed that pPAG5 and related plasmids shared an overall similar backbone, including genes essential for replication (repA), partition (par), and conjugal transfer (tra). Further phylogenetic analysis showed that pPAG5 was closely related to plasmids pOZ176 and pJB37, both of which are members of the IncP-2-type plasmid group. IMPORTANCE The emergence and spread of plasmid-associated multidrug resistance in bacterial pathogens is a key global threat to public health. It is important to understand the mechanisms of the formation and evolution of these plasmids in patients, hospitals, and the environment. In this study, we detailed the genetic characteristics of a multidrug resistance IncP-2 megaplasmid, pPAG5, and investigated the formation of its MDR regions and evolution. To the best of our knowledge, plasmid pPAG5 is the largest multidrug resistance plasmid ever sequenced in the Pseudomonas genus. Our results may provide further insight into the formation of multidrug resistance plasmids in bacteria and the molecular evolution of plasmids.

An IncP-2 plasmid sublineage associated with dissemination of blaIMP-45 among carbapenem-resistant Pseudomonas aeruginosa.

IMP-45, a variant of IMP-9, is one of the dominant metallo-ß-lactamases (MBLs) in clinical carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates in China. The aim of this study was to investigate the distribution and mechanism of dissemination of blaIMP-45. MBL genes were detected by PCR in 173 non-duplicate CRPA isolates collected from Hospital HS in Shanghai and 605 P. aeruginosa isolates from a multicenter surveillance of blaIMP-45 in China. In total, 17 IMP-45-producers (14 from Hospital HS and 3 from other hospitals) were identified. Molecular typing identified an outbreak of 11 IMP-45-producing ST508 CRPA in the ICU of Hospital HS. Conjugation assays and whole genome sequencing were conducted among IMP-45-producers. Genomic comparison revealed that 16 blaIMP-45-carrying plasmids (9 from this study and 7 from GenBank) shared a similar backbone with IncP-2 blaIMP-9-carrying plasmid pOZ176 but lacked repA-oriV-parAB region. repA2 gene was presented in pOZ176, blaIMP-45-carrying plasmids (17 from this study and 7 from GenBank) and 15 megaplasmids from GenBank. Phylogenetic analysis of repA2 showed that most blaIMP-45-carrying plasmids were clustered into a sublineage separate from the one containing pOZ176. This IncP-2 plasmid sublineage contributed to the dissemination of blaIMP-45 among genetically diverse P. aeruginosa and recruited multiple resistance genes during its evolution.