Genetic analysis of resistance and virulence characteristics of clinical multidrug-resistant Proteus mirabilis isolates

Abstract

Objective: Proteus mirabilis is the one of most important pathogens of catheter-associated urinary tract infections. The emergence of multidrug-resistant (MDR) P. mirabilis severely limits antibiotic treatments, which poses a public health risk. This study aims to investigate the resistance characteristics and virulence potential for a collection of P. mirabilis clinical isolates.

Methods and results: Antibiotic susceptibility testing revealed fourteen MDR strains, which showed high resistance to most β-lactams and trimethoprim/sulfamethoxazole, and a lesser extent to quinolones. All the MDR strains were sensitive to carbapenems (except imipenem), ceftazidime, and amikacin, and most of them were also sensitive to aminoglycosides. The obtained MDR isolates were sequenced using an Illumina HiSeq. The core genome-based phylogenetic tree reveals the high genetic diversity of these MDR P. mirabilis isolates and highlights the possibility of clonal spread of them across China. Mobile genetic elements SXT/R391 ICEs were commonly (10/14) detected in these MDR P. mirabilis strains, whereas the presence of resistance island PmGRI1 and plasmid was sporadic. All ICEs except for ICEPmiChn31006 carried abundant antimicrobial resistance genes (ARGs) in the HS4 region, including the extended-spectrum β-lactamase (ESBL) gene bla_CTX-M-65. ICEPmiChn31006 contained the sole ARG bla_CMY-2 and was nearly identical to the global epidemic ICEPmiJpn1. The findings highlight the important roles of ICEs in mediating the spread of ARGs in P. mirabilis strains. Additionally, these MDR P. mirabilis strains have great virulence potential as they exhibited significant virulence-related phenotypes including strong crystalline biofilm, hemolysis, urease production, and robust swarming motility, and harbored abundant virulence genes.

Conclusion: In conclusion, the prevalence of MDR P. mirabilis with high virulence potential poses an urgent threat to public health. Intensive monitoring is needed to reduce the incidence of infections by MDR P. mirabilis.

Keywords: ICE; PmGRI1; Proteus mirabilis; blaCTX-M-65; virulence.

Figure 1

Phylogenetic analysis of MDR P. mirabilis strains. The phylogenetic tree was constructed based on the SNPs in their core genome with the maximum-likelihood method in FastTree. Strains identified in this study are highlighted in red. The host of the isolate is indicated by triangles (animals), squares (food) or circles (humans). The tree scale indicates substitutions per site.

Figure 2

Genetic organization of plasmids or plasmid-derived region in MDR P. mirabilis strains. (A) Comparison of pCTX-M_HJP05004 with p1_FZP3115. Genes are denoted by arrows. bla_CTX-M-63, ISEcp1, repA, conjugation genes, and maintenance genes are highlighted in red, olive, green, blue, and purple, respectively. Regions of >73% identity are indicated by grey shading. (B) Organization of the plasmid-derived region on the chromosome of HJP16012, and comparisons to related regions. Genes are denoted by arrows. ARGs, replication genes, integrase genes, IS26, and other transposase genes are highlighted in red, green, blue, yellow, and olive, respectively. Regions of > 80% identity are indicated by grey shading. Δ represents truncated genes.

Figure 3

Comparative analysis of the PmGRI1 in MDR P. mirabilis strains. Genes are denoted by arrows and the direction of transcription is indicated by the arrowheads. ARGs, integrase genes, IS26, and other transposase genes are highlighted in red, blue, yellow, and olive, respectively. Regions of >90% identity are indicated by grey shading. Δ represents truncated genes.

Figure 4

Genetic features of the ICEs in MDR P. mirabilis strains. The upper line shows the backbone of SXT/R391 ICEs with conserved core genes. Genes are denoted by arrows and those involved in site-specific excision and integration (xis and int), DNA repair (rumAB), conjugative transfer (tra), and regulation (setCDR) are highlighted in orange, purple, green, and amaranth. Under the backbone, hotspots (HS1-HS5), and variable regions (II-IV) are shown, with thin arrows indicating the sites of their insertion. Other genes in the insertion regions are indicated by light blue arrows. Δ represents truncated genes.

Figure 5

Heatmap of genes related to major bacterial virulence factors. These virulence-associated genes among the isolates are denoted by filled squares (green) for presence and empty squares for absence.