Antimicrobial resistance and virulence of Pseudomonas spp. among healthy animals: concern about exolysin ExlA detection

Abstract

Pseudomonas is a ubiquitous genus that also causes human, animal and plant diseases. Most studies have focused on clinical P. aeruginosa strains from humans, but they are scarce on animal strains. This study was aimed to determine the occurrence of Pseudomonas spp. among faecal samples of healthy animals, and to analyse their antimicrobial resistance, and pathogenicity. Among 704 animal faecal samples analysed, 133 Pseudomonas spp. isolates (23 species) were recovered from 46 samples (6.5%), and classified in 75 different PFGE patterns. Low antimicrobial resistance levels were found, being the highest to aztreonam (50.3%). Five sequence-types (ST1648, ST1711, ST2096, ST2194, ST2252), two serotypes (O:3, O:6), and three virulotypes (analysing 15 virulence and quorum-sensing genes) were observed among the 9 P. aeruginosa strains. Type-3-Secretion System genes were absent in the six O:3-serotype strains that additionally showed high cytotoxicity and produced higher biofilm biomass, phenazine pigments and motility than PAO1 control strain. In these six strains, the exlAB locus, and other virulence genotypes (e.g. RGP69 pathogenicity island) exclusive of PA7 outliers were detected by whole genome sequencing. This is the first description of the presence of the ExlA exolysin in P. aeruginosa from healthy animals, highlighting their pathological importance.

Figure 1

Phenotypic assays of virulence of the 9 P. aeruginosa strains. (a) Biofilm biomass production determined by staining with crystal violet (CV); (b) metabolic activity within biofilm determined by staining with fluorescein diacetate (FDA); (c) Pyocyanin production assay; (d) Pyorubin production assay, and (e) Elastase assay. Dotted line (PAO1 value = 100%). Data expressed as mean ± SD. Virulotype III-strains (dark grey bar); virulotype II-strains (light grey bar); virulotype I-strains (grey striped bar). Two-tailed t test *p < 0.05, ***p < 0.001, ns no significant differences.

Figure 2

Phylogenetic tree of the exlA-positive sequenced P. aeruginosa strains. The dendrogram was constructed using a Neighbour-Joining algorithm, using ExlA amino acid sequence as an alignment. Bootstrap values for 10,000 replicates. P. aeruginosa of this study (Ps533, Ps616 and Ps633) are marked with bold letters. Amino acid sequences of the remaining exlA-positive strains were obtained from the Pseudomonas Genome Database. Two clades (1 and 2) and subclades (2a and 2b) are highlighted with square brackets.

Figure 3

Cytotoxicity levels of P. aeruginosa strains studied by LDH release in THP-1 monocytes (a), and in A549 epithelial cells (b). Data expressed as mean ± SEM (n = 4). One-way Anova test *p < 0.05, **p < 0.01, ***p < 0.001, ns no significant differences. P. aeruginosa PA7 (exlA-positive), PAO1 (exoS-positive) and PA14 (exoU-positive) strains (black bars) were used as reference strains. For the A549 test, PAO1 is used as negative control, and PA14 as positive control. Virulotype III-strains (dark grey bar); virulotype II-strains (light grey bar); virulotype I-strains (grey striped bar).