Distribution of P. aeruginosa strains isolated from companion dogs
A total of 84 nonduplicated P. aeruginosa strains were isolated from 448 companion dogs either with or without clinical signs from 2017 to 2018. From 228 healthy dogs (healthy group), 38 strains (16.7%) were detected in the ears (14 strains, 6.1%), eyes (10 strains, 4.4%), nasal cavity (7 strains, 3.1%), or rectum (7 strains, 3.1%). However, 46 strains (17.7%) from 260 dogs with clinical signs (infected group) were isolated mostly from the ears (41 strains, 15.8%), and only a few were found in the genitalia (4 strains, 1.5%) or pus (1 strain, 0.4%) (Table 1). Among each specimen type collected from the infected group, P. aeruginosa were identified in 1.3% of isolates from genitalia (4/30), 31.5% of isolates from ears (41/130), and 1.3% of isolates from pus (1/8). Therefore, P. aeruginosa was isolated mostly from ear samples (55 strains, 11.3%) of companion dogs, which was independent of their health status.
Antimicrobial resistance profile
Strains were tested for resistance to 10 different antibiotics (Table 2, Figure 1). In the healthy group, resistance to ciprofloxacin was the most frequent (n=4, 10.5%), and resistance to ciprofloxacin-gentamicin-tobramycin, gentamicin, and tobramycin was observed for one organism. In the infected group, resistance to ciprofloxacin was the most frequent (n=7, 15.2%), followed by that to piperacillin (n=3, 6.5%), ciprofloxacin-gentamicin-tobramycin (n=2, 4.3%), gentamicin (n=2, 4.3%), tobramycin (n=2, 4.3%) and amikacin (n=1, 2.2%). No strain was resistant to cefepime, ceftazidime, colistin, imipenem, meropenem, or piperacillin-tazobactam. Additionally, resistance to piperacillin and amikacin was observed only in strains from the infected group. While resistance to ciprofloxacin was apparently higher in the infected group than in the healthy group, there was no significant difference in the frequency of resistance to ciprofloxacin between the healthy and infected groups (p-value 0.747).
The MICs for 4 fluoroquinolone antibiotics, including nalidixic acid, were evaluated (Table 3). All P. aeruginosa strains from both groups were resistant to nalidixic acid (MIC ranges: 64 to ≥ 256 µg/mL in the healthy group and 32 to ≥ 256 µg/mL in the infected group). The highest percentage of resistance among the 4 fluoroquinolone agents was seen for enrofloxacin (breakpoint, ≥ 4 µg/mL), with 15.8% (6/38) resistant strains from the healthy group and 37.0% (17/46) from the infected group. The percentage of resistance to marbofloxacin (≥ 4 µg/mL) was 13.2% (5/38) for strains from the healthy group and 26.1% (12/46) for those from the infected group, and resistance (≥ 8 µg/mL) to levofloxacin was 13.2% (5/38) for the healthy group strains and 15.2% (7/46) for those from the infected group. Moreover, resistance (≥ 4 µg/mL) to ciprofloxacin was observed for 10.5% (4/38) of strains from the healthy group and 15.2% (7/46) of strains from the infected group (Table 3). Collectively, strains from the infected group were significantly more resistant to enrofloxacin and less susceptible to marbofloxacin than those from the healthy group (p< 0.05).
Amino acid variations in the gyrA and parC gene products
Given that strains resistant to enrofloxacin among fluoroquinolone antibiotics were the most abundant, sequencing analysis of the gyrA and parC genes from the 23 enrofloxacin-resistant P. aeruginosa strains was performed (Table 4). QRDR mutations were identified in 6 (15.8%) out of 38 strains from the healthy group and in 17 (37%) out of 46 strains from the infected group; all 23 mutations belonged to the 23 enrofloxacin-resistant strains of P. aeruginosa. In addition, 11 (47.8%) of these 23 strains were resistant to ciprofloxacin. Of the amino acid substitutions found in the 23 fluoroquinolone-resistant strains, 8 were present in the gyrA gene and 4 in the parC gene. Regarding gyrA, the mutation Thr83Ile was found in 2 strains from the healthy group and in 4 strains from the infected group; in addition, other mutations were found in strains from the infected group: Asp87Gly in 2 strains, Thr83Ile-Asp87Gly in 1 strain, Leu55Gln-Asp82Asn-Thr83Ala in 1 strain, and Asp87Asn in 1 strain. Notably, the novel triple-nucleotide mutation found in gyrA, leading to codon changes Leu55→Gln, Asp82→Asn, and Thr83→Ala, corresponds to a P. aeruginosa strain from the infected group with high MIC values for the four fluoroquinolone agents tested. Moreover, the strain with two nucleotide mutations, i.e., Thr83Ile and Asp87Gly, showed identical MIC values for these four antibiotics. Mutations in gyrA were significantly more common in strains from the infected group than in those from the healthy group (p < 0.05).
Regarding the parC gene, mutations were observed in all 23 enrofloxacin-resistant P. aeruginosa strains. A novel single nucleotide mutation at parC codon 116, resulting in a Pro116→Arg mutation, was found in 21 (91.3%) of the strains, which included a double nucleotide alteration at codons 87 and 116, Ser87→Leu and Pro116→Arg, that was observed in 1 strain from the healthy group and in 2 strains from the infected group. In addition, a frameshift (fs) mutation due to a nucleotide deletion at parC codon 116, Pro116fs, CGG→CG-, was detected in 2 strains from the infected group. Therefore, mutation hotspots found for gyrA and parC were Thr83Ile (n=7) and Pro116Arg (n=21), respectively. There was no significant difference between the number of point mutations in either the gyrA or the parC genes and the MIC values for fluoroquinolone antimicrobial agents among resistant strains. In addition, QRDR mutations in the gyrA or parC genes conferring intermediate resistance to enrofloxacin (MIC: 1 to 2 µg/mL) were not found in the P. aeruginosa strains..