Characteristics of MDR E. coli strains isolated from Pet Dogs with clinic diarrhea: A pool of antibiotic resistance genes and virulence-associated genes

Abstract

The increasing number of multi-drug resistant (MDR) bacteria in companion animals poses a threat to both pet treatment and public health. To investigate the characteristics of MDR Escherichia coli (E. coli) from dogs, we detected the antimicrobial resistance (AMR) of 135 E. coli isolates from diarrheal pet dogs by disc diffusion method (K-B method), and screened antibiotic resistance genes (ARGs), virulence-associated genes (VAGs), and population structure (phylogenetic groups and MLST) by polymerase chain reaction (PCR) for 74 MDR strains, then further analyzed the association between AMRs and ARGs or VAGs. Our results showed that 135 isolates exhibited high resistance to AMP (71.11%, 96/135), TET (62.22%, 84/135), and SXT (59.26%, 80/135). Additionally, 54.81% (74/135) of the isolates were identified as MDR E. coli. In 74 MDR strains, a total of 12 ARGs in 6 categories and 14 VAGs in 4 categories were observed, of which tetA (95.95%, 71/74) and fimC (100%, 74/74) were the most prevalent. Further analysis of associations between ARGs and AMRs or VAGs in MDR strains revealed 23 significant positive associated pairs were observed between ARGs and AMRs, while only 5 associated pairs were observed between ARGs and VAGs (3 positive associated pairs and 2 negative associated pairs). Results of population structure analysis showed that B2 and D groups were the prevalent phylogroups (90.54%, 67/74), and 74 MDR strains belonged to 42 STs (6 clonal complexes and 23 singletons), of which ST10 was the dominant lineage. Our findings indicated that MDR E. coli from pet dogs carry a high diversity of ARGs and VAGs, and were mostly belong to B2/D groups and ST10. Measures should be taken to prevent the transmission of MDR E. coli between companion animals and humans, as the fecal shedding of MDR E. coli from pet dogs may pose a threat to humans.

Fig 1. Antibiotic resistance patterns of E. coli isolates from pet dogs.

(A) The abscissa 0R represents the strains that were sensitive to all antibiotics, and 1–6 R represents strains that were resistant to 1–6 antibiotic categories, respectively. Seventy-four E. coli isolates are MDR, of which 17 strains were resistant to 6 antibiotic categories; (B) Color bars demonstrate the distribution of phenotypic resistance patterns in 74 MDR E. coli isolates, and the Arabic number represent the number of strains. A total of 56 resistance patterns were observed by using disc diffusion assay. The red boxes highlight the prevalent resistant-phenotypes patterns (occurring three times), the other combinations (without red box) occurred only once or twice.

Fig 2. Distribution of antibiotic resistance genes (ARGs) and virulence-associated genes (VAGs) in 74 MDR E. coli strains from pet dogs.

(A) The bar graphs show the detection rates of ARGs and VAGs. A total of 12 ARGs and 14 VAGs were detected, of which tetA (95.95%) and fimC (100%) were the most prevalent; (B) The abscissa represents the ID of MDR isolates and ordinate represents ARGs and VAGs. The red and blue regions represent the presence or absence of corresponding ordinate genes in an isolate, respectively. A high diversity of ARGs and VAGs was detected among MDR strains.

Fig 3. Heatmap of the correlation-coefficient (r) between ARGs and AMR or VAGs in 74 MDR E. coli strains from pet dogs.

Blue indicates positive association (r > 0, P < 0.05) and red indicates negative association (r < 0, P < 0.05). The color scale on the right of figure indicates the r-valve: (A) Heatmap of the correlation coefficient between ARGs and AMRs. The color scale and corresponding r-valve indicate the association between corresponding abscissa AMRs and ordinate ARGs. Twenty-three positive association pairs were observed, of which the strongest association was found between flor and C; (B) Heatmap of correlation coefficient between ARGs and VAGs. The color scale and corresponding r-valve indicate the association between corresponding VAGs (abscissa) and ARGs (ordinate). Five association pairs were observed (3 association pairs were positive and 2 association pairs were negative), of which the strongest positive association was found between ompT and sul2, the strongest negative association was found between bla_CTX-M and tsh.

Fig 4. Distribution of phylogenetic groups and STs in 74 MDR E. coli strains from pet dogs.

(A) The distribution of phylogroups in MDR strains. Commensal groups included groups A and B1, and virulent extraintestinal-related groups included groups B2 and D. Marking * represents a significant difference, *, P < 0.05; **, P < 0.01; ***, P < 0.001. Significantly, the virulent groups were the most prevalent; (B) The average number of VAGs per isolate in each phylogroup. Marking * represents significant difference, *, P < 0.05; **, P < 0.01; ***, P < 0.001. Obviously, the highest value observed was 8.28 in group B2.; (C) Minimum spanning tree of MLST types in MDR strains. The circle size indicates the proportion of isolates belonging to the ST. The color within each circle represents phylogroups and indicates the proportion of isolates belonging to different phylogroups. Each link between circles indicates one mutational event and the distance is scaled as the number of allele differences between STs. The yellow-green outlines of the circles represent the ST is the founder ST of one clonal complex (CC), and the other STs (with purple outlines of the circles) of the CC are derived from the founder ST with two allelic differences. The 74 MDR strains exhibited a high diversity of STs (42 STs were identified) in our present study and ST10 was the most prevalent; (D) The clonal complexes (CCs) among the 42 STs. Forty-two STs were clustered into 6 clonal complexes, and the remaining 23 STs were single. ST-10CC was the most prevalent lineage containing 6 STs; (E) Heatmap demonstrates the distribution of STs and phylogroups. The color scale and corresponding value indicate the number of E. coli isolates belonging to the corresponding phylogroups (abscissa) and STs (ordinate). Blue indicates a low number of strains, white indicates an intermediate value, and red indicates a high number of strains. As the heatmap shows, B2-ST10 was the most prevalent clone.