Antimicrobial Resistance and Genetic Diversity of Pseudomonas aeruginosa Strains Isolated from Equine and Other Veterinary Samples

Abstract

Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections in humans. This bacterium is less represented in veterinary medicine, despite causing difficult-to-treat infections due to its capacity to acquire antimicrobial resistance, produce biofilms, and persist in the environment, along with its limited number of veterinary antibiotic therapies. Here, we explored susceptibility profiles to antibiotics and to didecyldimethylammonium chloride (DDAC), a quaternary ammonium widely used as a disinfectant, in 168 P. aeruginosa strains isolated from animals, mainly Equidae. A genomic study was performed on 41 of these strains to determine their serotype, sequence type (ST), relatedness, and resistome. Overall, 7.7% of animal strains were resistant to carbapenems, 10.1% presented a multidrug-resistant (MDR) profile, and 11.3% showed decreased susceptibility (DS) to DDAC. Genomic analyses revealed that the study population was diverse, and 4.9% were ST235, which is considered the most relevant human high-risk clone worldwide. This study found P. aeruginosa populations with carbapenem resistance, multidrug resistance, and DS to DDAC in equine and canine isolates. These strains, which are not susceptible to antibiotics used in veterinary and human medicine, warrant close the setting up of a clone monitoring, based on that already in place in human medicine, in a one-health approach.

Keywords: Pseudomonas aeruginosa; antimicrobial susceptibility; equine; resistome; whole-genome sequencing and typing.

Figure 1

Selection diagram for the strains of the study. 1: six classes tested by disc diffusion method, i.e., 16 antibiotics for human and/or veterinary use according to the CASFM/EUCAST 2021 V1.0 list for Pseudomonas spp. and 4 antibiotics for veterinary use according to the CASFM 2013; 2: didecyldimethylammonium chloride tested by the broth dilution method; 3: in total 21 antibiotics for hospital or veterinary use. Anses: Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail, B: bovine, C: canine, DDAC: didecyldimethylammonium chloride, E: equine, F: feline, MALDI-TOF: matrix-assisted laser desorption/ionization–time-of-flight, MIC: minimum inhibitory concentration, P. aeruginosa: Pseudomonas aeruginosa, WGS: whole-genome sequencing.

Figure A1

Bioinformatics pipeline for whole-genome sequencing and sequence analysis. [50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74]. cg: core genome; MLST: multilocus sequence typing; WGS: whole-genome sequencing.

Figure 2

Antimicrobial resistance for veterinary strains. Frequency of resistance in P. aeruginosa strains tested for antibiotic susceptibility and antimicrobial resistance for the study panel (n = 168) (A) and for equine strains (n = 135) (B). Frequency histogram of the number of strains (in percent) showing at least one resistance for each different antibiotic class for the study panel (n = 168) (C) and for equine strains (n = 135) (D). Except for netilmicin, ceftiofur, cefquinome, marbofloxacin, and enrofloxacin, tested only for the panel for genomic characterization (n = 41), all other antimicrobials were tested for the study panel (n = 168). The four veterinary antibiotics (ceftiofur, cefquinome, marbofloxacin, and enrofloxacin) were excluded from the class-based resistance analysis for figure (B) Populations were tested for independence for DS to DDAC and loss of susceptibility to more than three categories of antibiotics using Fisher’s exact test. Ns: p-value > 0.05. DDAC: didecyldimethylammonium chloride; DS: decreased susceptibility; MDR: multidrug-resistant.

Figure 3

Antimicrobial-resistance-associated genes of strains from the panel for genomic characterization and for reference strains (n = 41 + 4). Strains were organized according to the cgMLST minimum distance tree. The figure lists serotype, sequence type, presence of a carbapenem resistance (imipenem and/or meropenem) phenotype, whether the strain was multidrug-resistant or not, and presence of decreased susceptibility to DDAC phenotype. The sub-variants of the aph(3′) gene were grouped, as well as the sub-variants of aac(6′). Co-occurrences of these sub-variants are indicated and separated by a backslash. The “-like genes” were variants not referenced on AMRFinder that had less than 100% shared identity. C: canine, B: bovine, DDAC: didecyldimethylammonium chloride, E: equine, F: feline, MDR: multidrug-resistant, O: serotype, ST: sequence type.

Figure 4

Multilocus sequence typing (MLST) of 41 sequenced strains. Representation of (A) the whole panel for genomic characterization, (B) equine strains, and (C) strains from other animal species (i.e., canine, feline, and bovine). The sequence types written in bold were found to be associated with several animal species. In figures (A,B), sequence types represented only once were grouped together. Figure (A) contained ST27, ST155, ST162, ST170, ST179, ST200, ST233, ST252, ST260, ST261, ST483, ST840, ST1007, ST1238, ST1239, ST1337, ST2123, ST2683, ST3016, ST3244, ST3314, and ST3719; figure (B) contained ST27, ST155, ST170, ST179, ST200, ST233, ST253, ST483, ST840, ST1238, ST1239, ST1337, ST2123, ST3016, ST3244, and ST3719. ST: Sequence type.

Figure 5

Minimum spanning tree of the strains from the panel for genomic characterization and reference strains (n = 41 + 4). Core genome MLST clustering according to the cgMLST Pseudomonas aeruginosa scheme previously published [71] and based on 3076 genes. The reference strains are ATCC15442, ATCC27853, PA14, and PAO1. The tree charts: origin of the strains (equine, canine, feline, bovine, human or environmental), serotype, sequence type, carbapenem resistance phenotype, DS to DDAC phenotype, and whether or not the strain was multidrug-resistant. C: canine, B: bovine, DDAC: didecyldimethylammonium chloride, E: equine, F: feline, MDR: multidrug-resistant, O: serotype, ST: sequence type, XDR: extensively drug-resistant.