Molecular genetic analysis of an XDR Pseudomonas aeruginosa ST664 clone carrying multiple conjugal plasmids

Abstract

Objectives A group of ST664 XDR Pseudomonas aeruginosa strains have been isolated from a burn clinic. Here we decipher their resistomes and likely mechanisms of resistance acquisition. Methods The complete nucleotide sequences of representative isolates were determined, by PacBio and Illumina MiSeq sequencing, and analysed for antimicrobial resistance (AMR) genes as well as sequence variations. S1-PFGE was used to determine the sizes and numbers of plasmids harboured by the isolates. Purified plasmid DNA was further sequenced by PacBio technology, closed manually and annotated by RAST. The mobility of plasmids was determined by conjugation assays. Results The XDR P. aeruginosa ST664 clone carries 11 AMR genes, including a blaKPC-2 gene that confers resistance to carbapenems. Most of the ST664 isolates carry three coexisting plasmids. blaKPC-2 and a cluster of three AMR genes (aadB-cmlA1-sul1) are encoded on a 475 kb megaplasmid pNK546a, which codes for an IncP-3-like replication and partitioning mechanism, but has lost the conjugative transfer system. Interestingly, however, pNK546a is mobilizable and can be transferred to P. aeruginosa PAO1 with the help of a co-residing IncP-7 conjugative plasmid. The blaKPC-2 gene is carried by an IS6100-ISKpn27-blaKPC-2-ΔISKpn6-Tn1403 mobile element, which might be brought into the ST664 clone by another co-resident IncP-1α plasmid, which is inclined to be lost. Moreover, pNK546a harbours multiple heavy metal (mercury, tellurite and silver) resistance modules. Conclusions To the best of our knowledge, pNK546a is the first fully sequenced blaKPC-2-carrying megaplasmid from P. aeruginosa. These results give new insights into bacterial adaptation and evolution during nosocomial infections.

New Sequence Type ST3449 in Multidrug-Resistant Pseudomonas aeruginosa Isolates from a Cystic Fibrosis Patient

“…No alterations were detected in AmpDh2, while mutation A219T in AmpDh3 was identified in all the isolates. This amino acid change in AmpDh3 was described before in multidrug-resistant P. aeruginosa isolates from ST175 and ST664 [ 36 , 37 ]. No mutations in the AmpC regulator dacB (PBP4) were found in our isolates.…”

Emergence and rising of ceftazidime-avibactam resistant KPC-producing Pseudomonas aeruginosa in China: a molecular epidemiology study

“…Members of this family displayed a size of 300-500 Kb and several AMR genes. However, those carrying bla KPC-2 genes were only reported in China [18, 59]. The current study showed that this megaplasmid spread widely around the country as we identified the plasmid in Eastern and Central China.…”

“…This suggested that this mobile genetic element (MGE) carrying bla KPC-2 might be able to transfer between two plasmid types, especially in case of ZPPH8 which contained both plasmids. The Type 2 plasmid containing the genetic context in which the bla KPC-2 was embedded, had been recently reported in Tianjin, China [18]. Interestingly, a sequence alignment showed that plasmid pNK546-KPC underwent large segment inversion next to the bla KPC-2 region (Figure 5).…”

“…In addition, the host range of Type 1 plasmid was comparatively small, while the Type 2 plasmid family had been found in other Pseudomonal and non-Pseudomonal genera [50]. This might be explained by the fact that the conjugal ability of Type 2 plasmid has been demonstrated experimentally [18], in contrast to Type 1 plasmids. However, the high pressure of carbapenem usage in clinics might accelerate the occurrence of P. aeruginosa harboring bla KPC-2 genes and retain such strains in the nosocomial environment, leading to dominant clones such as ST463.…”

“…Most of the investigated bla KPC genes in P. aeruginosa have been reported to be plasmid-encoded [15-18], while a small number are found on the bacterial chromosome, occasionally in prophage sequences [12, 19]. However, most of these studies are single case reports and focus on the description of the genetic structure of bla KPC and its surrounding sequences.…”