Spread of Carbapenem Resistance by Transposition and Conjugation Among Pseudomonas aeruginosa

Abstract

The emergence of carbapenem-resistant Pseudomonas aeruginosa represents a worldwide problem. To understand the carbapenem-resistance mechanisms and their spreading among P. aeruginosa strains, whole genome sequences were determined of two extensively drug-resistant strains that are endemic in Dutch hospitals. Strain Carb01 63 is of O-antigen serotype O12 and of sequence type ST111, whilst S04 90 is a serotype O11 strain of ST446. Both strains carry a gene for metallo-β-lactamase VIM-2 flanked by two aacA29 genes encoding aminoglycoside acetyltransferases on a class 1 integron. The integron is located on the chromosome in strain Carb01 63 and on a plasmid in strain S04 90. The backbone of the 159-kb plasmid, designated pS04 90, is similar to a previously described plasmid, pND6-2, from Pseudomonas putida. Analysis of the context of the integron showed that it is present in both strains on a ∼30-kb mosaic DNA segment composed of four different transposons that can presumably act together as a novel, active, composite transposon. Apart from the presence of a 1237-bp insertion sequence element in the composite transposon on pS04 90, these transposons show > 99% sequence identity indicating that transposition between plasmid and chromosome could have occurred only very recently. The pS04 90 plasmid could be transferred by conjugation to a susceptible P. aeruginosa strain. A second class 1 integron containing a gene for a CARB-2 β-lactamase flanked by an aacA4'-8 and an aadA2 gene, encoding an aminoglycoside acetyltransferase and adenylyltransferase, respectively, was present only in strain Carb01 63. This integron is located also on a composite transposon that is inserted in an integrative and conjugative element on the chromosome. Additionally, this strain contains a frameshift mutation in the oprD gene encoding a porin involved in the transport of carbapenems across the outer membrane. Together, the results demonstrate that integron-encoded carbapenem and carbapenicillin resistance can easily be disseminated by transposition and conjugation among Pseudomonas aeruginosa strains.

Keywords: Pseudomonas aeruginosa; VIM-2; carbapenem resistance; conjugation; genome sequence; integrative and conjugative element; integron; transposon.

FIGURE 1

Schematic representation of the integrons of Carb01 63 and S04 90. The gene cassette promoters (Pc) are different. PcS represents a strong promoter, whereas PcW_TGN-10 represents a promoter that is considerably weaker due to nucleotide substitutions in the –10 and –35 regions, which are, however, partially compensated by a C to G substitution upstream of the –10 region, resulting in an extended –10 motif (Jové et al., 2010). These differences also affect the primary structures of the integrases encoded by the intI1 genes (dark and bright blue). Cassettes with the same nucleotide sequences are indicated with the same color. No differences were observed in the 3′ conserved sequences, except that orf5 in In99 is truncated. The integration sites attI1 and attC are indicated by ovals and circles, respectively.

FIGURE 2

Genetic context of the bla_{V IM-2} containing integron on pS04 90. (A) Composition of a mosaic 30 kb transposon containing the integron with the bla_{V IM-2} gene. Open reading frames of different transposable elements are indicated by different colors. MITE?, 332-bp inserted element possibly representing a MITE. (B) Comparison of pS04 90 (blue) with pND6-2 (sea-green). Regions of homology are indicated by color. Non-homologous regions are black and deletions are indicated by a line. The positions of the pil gene cluster (yellow) and the genes involved in the type IVB secretion system (T4BSS) (green) are indicated in both plasmids. The large insertion, detailed in (A), is indicated by a triangle at the top. The dissimilar regions (black) around 20, 90, and 105 kb in pS04 90, encode, amongst others, a toxin/antitoxin addiction module, an O-antigen transacetylase OafA, and CRISPR-related proteins, respectively. Most of the proteins putatively encoded by the dissimilar regions are hypothetical proteins and several transposases/integrases.

FIGURE 3

Genetic context of the bla_CARB-2-containing integron in Carb01 63. (A) Composition of a mosaic 18-kb transposon containing the integron with the bla_CARB-2 gene. Open reading frames of different transposable elements are indicated by different colors. (B) Composition of the ICE into which the composite transposon depicted in (A) is inserted. The ICE is bounded by 20-bp direct repeats (ICE DR). ORFs encoding proteins of different functional classes are indicated by different colors as outlined in the inset at the bottom. Inverted repeats are indicated by black boxes in between the ORFs with a colored triangle above.