A 10-year microbiological study of Pseudomonas aeruginosa strains revealed the circulation of populations resistant to both carbapenems and quaternary ammonium compounds

Abstract

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections. For this study, the susceptibility profiles to antipseudomonal antibiotics and a quaternary ammonium compound, didecyldimethylammonium chloride (DDAC), widely used as a disinfectant, were established for 180 selected human and environmental hospital strains isolated between 2011 and 2020. Furthermore, a genomic study determined resistome and clonal putative relatedness for 77 of them. During the ten-year study period, it was estimated that 9.5% of patients' strains were resistant to carbapenems, 11.9% were multidrug-resistant (MDR), and 0.7% were extensively drug-resistant (XDR). Decreased susceptibility (DS) to DDAC was observed for 28.0% of strains, a phenotype significantly associated with MDR/XDR profiles and from hospital environmental samples (p < 0.0001). According to genomic analyses, the P. aeruginosa population unsusceptible to carbapenems and/or to DDAC was diverse but mainly belonged to top ten high-risk clones described worldwide by del Barrio-Tofiño et al. The carbapenem resistance appeared mainly due to the production of the VIM-2 carbapenemase (39.3%) and DS to DDAC mediated by MexAB-OprM pump efflux overexpression. This study highlights the diversity of MDR/XDR populations of P. aeruginosa which are unsusceptible to compounds that are widely used in medicine and hospital disinfection and are probably distributed in hospitals worldwide.

Figure 1

Selection diagram for the study and reference strains. ¹: 6 classes tested, i.e., 16 antibiotics for hospital use; ²: didecyldimethylammonium chloride, ATCC, American Type Culture Collection; H, Strains isolated from the hospital environment; MALDI-TOF, Matrix Assisted Laser Desorption Ionisation/Time of Flight; P, Strains isolated from patients; PSAE, Pseudomonas aeruginosa; UHC, University Hospital Center; WGS, Whole Genome Sequencing.

Figure 2

Antimicrobial resistance for hospital strains (n = 4375) and study panel (n = 180). (a) Occurrence of antibiotics resistance for all P. aeruginosa strains isolated from patients (P strains) tested for antibiotic susceptibility at the University Hospital of Caen (n = 4375). Percentage of strains showing resistance (b) to 8 classes of antibiotics for P. aeruginosa P strains tested among the total panel (n = 4375), and (c) to 7 classes of antibiotics for strains of the study panel (n = 180). (c) The hatched areas represent strains that also accumulate DS to the disinfectant DDAC. Fisher’s test for populations independence for DS to DDAC and loss of susceptibility to more than three categories of antibiotics. n*: number of strains tested for the antibiotic; ***: p value ≤ 0.0001 after analysis by Fisher’s independence test. DDAC, Didecyldimethylammonium chloride; DS, Decreased susceptibility; MDR, Multidrug-resistant; PDR, Pandrug-resistant; XDR, Extensively drug-resistant.

Figure 3

Annual frequency of carbapenem (imipenem and/or meropenem) resistance phenotype for: (a) P. aeruginosa P strains tested for antibiotic susceptibility at the University Hospital of Caen (n = 4375) and (b) the study panel n = 180 (decreased susceptibility to DDAC is indicated).

Figure 4

Minimum spanning tree of 77 strains from panel for genomic characterization compared with reference strains (PAO1, PA14, ATCC15442 and ATCC27853). Core genome MLST clustering according to the cgMLST Pseudomonas aeruginosa scheme published by Tönnies et al.. The tree was surrounded by the following information: the origin of the strains (patient, P or hospital environment, H), year of sampling, sequence type (ST), carbapenem resistance phenotype, whether it was associated with a VIM-2 or GES-5 carbapenemase, and decreased susceptibility phenotype to DDAC (didecyldimethylammonium chloride). GES-5: strains producing the class A carbapenemase GES-5; VIM-2: strains producing the class B carbapenemase VIM-2.

Figure 5

Antimicrobial resistance-associated genes of 77 strains from panel for genomic characterization compared with reference strains (PAO1, PA14, ATCC15442 and ATCC27853). Strains were organized according to the cgMLST minimum distance tree. The serotype (O), the sequence type (ST), the presence of a carbapenem resistance (imipenem and/or meropenem) phenotype, whether the strain was MDR, XDR, or PDR, and the presence of a decreased susceptibility to DDAC phenotype (D) are mentioned. The sub-variants of the aph(3′) gene were grouped as well as the sub-variants of aac(6′), in case of co-occurrence of these sub-variants they were indicated and separated by a slash. C, Strains with carbapenems resistance; H, Strains isolated from the hospital environment; P, Strains isolated from patients; MDR, Multidrug-resistant; XDR, Extensively drug-resistant; PDR: pandrug-resistant.

Figure 6

Effect of efflux pumps expression and cephalosporinase activity on susceptibility to carbapenems (imipenem and meropenem) and to DDAC. For the representative short panel strains (n = 13) and 2 reference strains (PAO1 and PA14), MIC variation of the strains to (a) imipenem, (b) meropenem, and (c) DDAC in the absence or presence of PAβN, an efflux pump inhibitor, or cloxacillin, a cephalosporinase inhibitor. A distinction was made between strains with DS to DDAC (DDAC-R strains), carbapenem resistance (Carba-R strains), both phenotypes (Double-R strains), or neither phenotype (Double-S strains). The symbols represent individual MIC values, and the bars represent the average MIC for each strain type. By default, MIC values < 1 were replaced with 0.5. (d) Fold-change of expression of mexA, mexB, oprM, mexE, mexF, and oprN genes compared to PAO1 for three strains expressing VIM-2 carbapenemase.