Intraclonal Genome Stability of the Metallo-β-lactamase SPM-1-producing Pseudomonas aeruginosa ST277, an Endemic Clone Disseminated in Brazilian Hospitals

Abstract

Carbapenems represent the mainstay therapy for the treatment of serious P. aeruginosa infections. However, the emergence of carbapenem resistance has jeopardized the clinical use of this important class of compounds. The production of SPM-1 metallo-β-lactamase has been the most common mechanism of carbapenem resistance identified in P. aeruginosa isolated from Brazilian medical centers. Interestingly, a single SPM-1-producing P. aeruginosa clone belonging to the ST277 has been widely spread within the Brazilian territory. In the current study, we performed a next-generation sequencing of six SPM-1-producing P. aeruginosa ST277 isolates. The core genome contains 5899 coding genes relative to the reference strain P. aeruginosa PAO1. A total of 26 genomic islands were detected in these isolates. We identified remarkable elements inside these genomic islands, such as copies of the bla_SPM-1 gene conferring resistance to carbapenems and a type I-C CRISPR-Cas system, which is involved in protection of the chromosome against foreign DNA. In addition, we identified single nucleotide polymorphisms causing amino acid changes in antimicrobial resistance and virulence-related genes. Together, these factors could contribute to the marked resistance and persistence of the SPM-1-producing P. aeruginosa ST277 clone. A comparison of the SPM-1-producing P. aeruginosa ST277 genomes showed that their core genome has a high level nucleotide similarity and synteny conservation. The variability observed was mainly due to acquisition of genomic islands carrying several antibiotic resistance genes.

Keywords: Gram-negative bacilli; antimicrobial resistance; carbapenemase; comparative genomics; drug resistance; pathogenic bacteria.

Figure 1

Circular map depicting the unique regions of each SPM-1-producing P. aeruginosa isolate relative to the reference strain PAO1. The red ring represents the core genome shared by all isolates. The outermost, interspaced ring represents the localization of the predicted genomic islands found in each isolate.

Figure 2

Phylogenic tree of the six sequenced P. aeruginosa isolates, the reference genome PAO1, the 11 ST277 strains: 19BR (GCA_000223945.2), 213BR (GCA_000223965.2), 9BR (GCA_000223925.2), BWHPSA041 (GCA_000520375.1), AZPAE12409 (GCA_000797005.1), AZPAE14819 (GCA_000795205.1), AZPAE14821 (GCA_000795235.1), AZPAE14822 (GCA_000795265.1), AZPAE14853 (GCA_000789905.1), AZPAE14923 (GCA_000791205.1), CCBH4851 (GCA_000763245.1), and a single MLST locus variant strain: BWHPSA007 (GCA_000481565.1). The numbers indicate the bootstrap value associated with the nodes. (A) Consensus tree with neighbor joining method and (B) best tree drawing on scale.

Figure 3

Pairwise alignment between the P. aeruginosa chromosomes. Colors indicate conserved and highly related genomic regions, and white areas identify unique or low-identity regions. Blocks shifted below the horizontal axis indicate segments that align in the reverse orientation relative to the reference strain PAO1.

Figure 4

Schematic overview of genomic island (PAGI) distribution in the six SPM-1 isolates. Each circle represents a PAGI throughout the bacterial chromosomes. There are a total of 26 PAGIs, and each isolate carries between 16 and 21 PAGI (number at right). The circles at the top represent conserved sites (black) and variable sites (red).

Figure 5

Chromosome distribution of insertion sequences of P. aeruginosa genomes. Each circle represents an insertion sequence (IS) throughout the bacterial chromosome. In the bottom of the figure, the legend differentiates the various ISs by color. The black vertical lines are the empty sites. The small circles at the top of the figure indicate the conservative (black) and variable (red) ISs sites. The red triangles indicate the presence of spm-1 gene copies.

Figure 6

Schematic overview of PAGI-25 highlighting the region harboring genes conferring acquired antibiotic resistance.

Figure 7

Schematic representation of SNPs distribution in the six P. aeruginosa isolates using PAO1 as a reference genome. Each dot represents an SNP. The coverage counts show how many mapped reads support the SNP. (A) SNPs distribution of the six isolates relative to PAO1; the black spot refers to the SNP enriched region in PA11803. (B) A higher resolution representation of the PA11803 SNP enriched region.

Figure 8

Schematic overview of PAGI-15 region harboring the spm-1 gene. PA3448 and PA8281 carry two copies of this gene as a result of a duplication inside the island.

Figure 9

Relative transcriptional levels by qRT-PCR of (A) bla_SPM−1 gene in the SPM-1-producing P. aeruginosa isolates compared to PA1088 strain; and (B) mexB, mexD, mexF, mexY, and oprD genes in the SPM-1-producing P. aeruginosa isolates compared to PAO1 strain.