Integrated Genome-Wide Analysis of an Isogenic Pair of Pseudomonas aeruginosa Clinical Isolates with Differential Antimicrobial Resistance to Ceftolozane/Tazobactam, Ceftazidime/Avibactam, and Piperacillin/Tazobactam

Abstract

Multidrug-resistant (MDR) Pseudomonas aeruginosa is one of the main causes of morbidity and mortality in hospitalized patients and the leading cause of nosocomial infections. We investigated, here, two MDR P. aeruginosa clinical isolates from a hospitalized patient with differential antimicrobial resistance to ceftazidime/avibactam (CZA), ceftolozane/tazobactam (C/T), and piperacillin/tazobactam (P/T). Their assembled complete genomes revealed they belonged to ST235, a widespread MDR clone; and were isogenic with only a single nucleotide variant, causing G183D mutation in AmpC β-lactamase, responsible for a phenotypic change from susceptible to resistant to CZA and C/T. Further epigenomic profiling uncovered two conserved DNA methylation motifs targeted by two distinct putative methyltransferase-containing restriction-modification systems, respectively; more intriguingly, there was a significant difference between the paired isolates in the pattern of genomic DNA methylation and modifications. Moreover, genome-wide gene expression profiling demonstrated the inheritable genomic methylation and modification induced 14 genes being differentially regulated, of which only toxR (downregulated), a regulatory transcription factor, had its promoter region differentially methylate and modified. Since highly expressed opdQ encodes an OprD porin family protein, therefore, we proposed an epigenetic regulation of opdQ expression pertinent to the phenotypic change of P. aeruginosa from resistant to susceptible to P/T. The disclosed epigenetic mechanism controlling phenotypic antimicrobial resistance deserves further experimental investigation.

Keywords: Pseudomonas aeruginosa; RNA sequencing (RNAseq); antimicrobial resistance; ceftazidime/avibactam (CZA); ceftolozane/tazobactam (C/T); epigenetic profiling; piperacillin/tazobactam (P/T); whole-genome sequencing (WGS).

Figure 1

Comparative genome analysis of P. aeruginosa isolates. (A) Schematic genome of the paired P. aeruginosa isolates, PB367 and PB350. Genomic mutation C931,543T (G183D) in β-lactamase gene (bla_PDC-35) from PB367 to PB350 is indicated in the chromosome, along with other antimicrobial resistance genes (aph(3’)-IIb, fosA, catB7, and bla_OXA-50) and a gene encoding a putative Type IIG restriction-modification (RM) methyltransferase (MTase) targeting a specific motif AAG^m6AYC. Top: A unique class 1 integron in P. aeruginosa with multidrug resistance (MDR) genes, 100% identical to the ones found in plasmids of K. pneumoniae and E. coli, is located among multiple mobile genetic elements, Tn6249, resolvase, ISPa7, and tniA. Bottom: A unique Type I RM system comprised of restriction enzymes (RE, subunits R and S) and modification MTases (M1 and M2) putatively targeting a bipartite and asymmetric motif CT^m6AC(N)₅GGG/C^m4CC(N)₅GTAG; (B) Phylogeny tree of 15 P. aeruginosa isolates, mainly ST235, from kWIP hierarchical clustering using the complete linkage method. PAO1 belonged to ST549, AR_0095 belonged to ST620, and three main sub-ST235 clusters (1–3) are demonstrated; (C) Relatedness overview of 15 P. aeruginosa isolates in a plot of metric multidimensional scaling (MDS).

Figure 2

Comparative epigenome and transcriptome analysis of P. aeruginosa isolates. (A) Genome-wide differential methylation and gene expression in the paired P. aeruginosa isolates, PB367 and PB350. Top: Differentially methylated adenosine (m6A, in red) and cytosine (m4C, in blue) in PB367 and PB350, within or without sequence motif (refer to Table 2), and their distribution throughout the whole genome. Bottom: 14 genes identified with significantly differential expression (adjusted p-value < 0.05) and their particular loci at the genome. Upregulated genes in PB350 are labeled in brown, downregulated in blue, fold change less than 2 are in green; (B) Volcano plot of RNAseq for identification of 14 genes with significantly differential expression; (C) Heatmap of genes with significantly differential expression in triplicated samples. The color key represents the amount by which each gene deviates in a particular sample from the gene’s average across all the samples.

Figure 2

Comparative epigenome and transcriptome analysis of P. aeruginosa isolates. (A) Genome-wide differential methylation and gene expression in the paired P. aeruginosa isolates, PB367 and PB350. Top: Differentially methylated adenosine (m6A, in red) and cytosine (m4C, in blue) in PB367 and PB350, within or without sequence motif (refer to Table 2), and their distribution throughout the whole genome. Bottom: 14 genes identified with significantly differential expression (adjusted p-value < 0.05) and their particular loci at the genome. Upregulated genes in PB350 are labeled in brown, downregulated in blue, fold change less than 2 are in green; (B) Volcano plot of RNAseq for identification of 14 genes with significantly differential expression; (C) Heatmap of genes with significantly differential expression in triplicated samples. The color key represents the amount by which each gene deviates in a particular sample from the gene’s average across all the samples.

Figure 3

Hypothesized epigenetic regulation mechanism of P. aeruginosa antimicrobial resistance. (A) Methylation and modification in the promoter of ToxR in isolates PB367 and PB350, an extra m6A site was found in PB350 at −100 (vertical green arrow) flanking from the translation start site (horizontal blue arrows) of ToxR, in addition to differential modifications as compared with PB367; (B) Methylation and/or modification cause ToxR lower expression in PB350, which leads to higher expression of OpdQ and, furthermore, PB350 susceptible to Piperacillin/Tazobactam. +, positive transcription regulation; −, negative transcription regulation.