Chromosomal Amplification of the blaOXA-58 Carbapenemase Gene in a Proteus mirabilis Clinical Isolate

Abstract

Horizontal gene transfer may occur between distantly related bacteria, thus leading to genetic plasticity and in some cases to acquisition of novel resistance traits. Proteus mirabilis is an enterobacterial species responsible for human infections that may express various acquired β-lactam resistance genes, including different classes of carbapenemase genes. Here we report a Proteus mirabilis clinical isolate (strain 1091) displaying resistance to penicillin, including temocillin, together with reduced susceptibility to carbapenems and susceptibility to expanded-spectrum cephalosporins. Using biochemical tests, significant carbapenem hydrolysis was detected in P. mirabilis 1091. Since PCR failed to detect acquired carbapenemase genes commonly found in Enterobacteriaceae, we used a whole-genome sequencing approach that revealed the presence of bla_OXA-58 class D carbapenemase gene, so far identified only in Acinetobacter species. This gene was located on a 3.1-kb element coharboring a bla_AmpC-like gene. Remarkably, these two genes were bracketed by putative XerC-XerD binding sites and inserted at a XerC-XerD site located between the terminase-like small- and large-subunit genes of a bacteriophage. Increased expression of the two bla genes resulted from a 6-time tandem amplification of the element as revealed by Southern blotting. This is the first isolation of a clinical P. mirabilis strain producing OXA-58, a class D carbapenemase, and the first description of a XerC-XerD-dependent insertion of antibiotic resistance genes within a bacteriophage. This study revealed a new role for the XerC-XerD recombinase in bacteriophage biology.

Keywords: Acinetobacter baumannii; OXA-58; Proteus mirabilis; XerC-XerD recombinase; bacteriophage; carbapenems.

FIG 1

(A) Schematic representation of the chromosomal region encompassing the bla_OXA-58 and bla_AmpC genes. The two β-lactamase genes and their orientations are represented by white arrows and partial ISs are represented by striped arrows. The dashed gray line in the upper panel represents additional tandem repeats of the bla_OXA-58-ampC locus. Regions of identities with an uncultured bacterium sequence CX_IN_B_Contig_19 and A. baumannii plasmid p255n are indicated in the lower panel. Black triangles represent the repeated sequence bracketing the tandem repeats and corresponding to partial putative XerC-XerD binding sites. The four oligonucleotides used to ascertain this chromosomal organization are represented by small arrows. (B) Picture of an ethidium bromide-stained agarose gel with the three PCR products. Numbers indicate the oligonucleotides used in the reaction. (C) Phylogenetic analysis of the new AmpC identified using a maximum likelihood method. GenBank accession numbers of the sequences used for this tree are indicated. The tree is drawn to scale with branch lengths measured in number of substitutions per site (next to the branches).

FIG 2

Southern hydribization of bla_OXA-58 and LR-PCR amplification of the duplicated zone. (A) Southern experiments of P. mirabilis 1091 targeting the bla_OXA-58 gene. Left, digest of total DNA; right, Southern hybridization. Lanes: 1, gene ruler DNA ladder 1kbPlus (ThermoFisher); 2 and 3, P. mirabilis 1091; 4, P. mirabilis CIP103181; 5, A. baumannii OXA-58; 6, ladder; 7 and 8, P. mirabilis 1091; 9, P. mirabilis CIP103181; 10, A. baumannii OXA-58. Lanes 2 to 5 correspond to the digestion by EcoRI, and lanes 7 to 10 correspond to the double digestion by BglII and SacII. Digestion of total DNA from an OXA-58-producing A. baumannii strain was included as a positive control of digestion and digestion of total DNA from P. mirabilis CIP103181 as a negative control. (B) LR-PCR amplification of the tandem duplicated bla_OXA-58, bla_AmpC-like region using primers o-4 and o-3 (Fig. 1). Lanes 1 and 2 correspond to the obtained amplicons, and lane 3 corresponds to the PCR products digested by XbaI, which cleaves inside the duplicated region.

FIG 3

Prophage insertion of the bla_OXA-58-bla_AmpC element in P. mirabilis 1091. The bla_OXA-58-bla_AmpC element is inserted at a putative XerC-XerD binding site within a predicted prophage of P. mirabilis 1091. This recombination site is located in the intergenic region of two genes predicted to code for the small (S) and the large (L) subunits of the phage terminase, indicated by black arrows. (Top) Alignment of P. mirabilis 1091 partial phage sequence with the sequence of a closely related phage from strain BB2000 (GenBank CP004022). The two bla genes are indicated by light gray arrows, and the integrase gene of strain BB2000 prophage is indicated by a black arrow. The attL and attR sites are indicated by small vertical lines and the putative XerC-XerD binding sites by black triangles. Gray areas between open reading frames (ORFs) denote nucleotide identities with a gradient representing 69% (light gray) to 100% (dark gray) identity. (Bottom) Sequence alignment of the putative XerC-XerD binding sites with the predicted chromosomal dif site from P. mirabilis (33). The XerC binding sites on the left and XerD binding sites on the right are separated by six noncanonical bases. Shared nucleotides with the chromosomal dif site are indicated in black. CD-1, CD-2, and CD-3 are the predicted XerC-XerD binding sites bracketing the two bla genes in strain 1091; CD-4 was predicted in phage BB2000 between genes coding for the terminase subunits.