Genomic insights into multidrug - resistant Salmonella enterica isolates from pet dogs and cats

Abstract

Companion animals are recognized as potential reservoirs and transmitters of antimicrobial resistance (AMR) within the One Health framework. However, in-depth knowledge on AMR in pet animals remains limited. This study aimed to characterize Salmonella from companion dogs and cats using Whole Genome Sequencing (WGS). A total of 25 Salmonella obtained from clinically healthy household dogs and cats were serotyped and had their antimicrobial susceptibility tested. A discrepancy between the serovars identified by traditional slide agglutination tests and those determined by WGS analysis was observed. The isolates exhibited multidrug resistance (MDR) (n = 18) and harbored several resistance genes either chromosomally encoded or plasmid associated. Tn3 and IS26 were commonly found flanking AMR genes and class 1 integrons, while an unusual qacL-IS256-sul3 arrangement was also frequently observed. Similar AMR genes and insertion sequences were found among dogs and cats from different provinces, suggesting clonal spread and horizontal gene transfer of AMR. The similarity between plasmids (i.e., IncX1 and IncI1 plasmid) carrying AMR genes (e.g., aadA1, qacL, sul3, bla_TEM-1B, qnrS1, dfrA, tetA) in Salmonella from pets in this study and those from other sources (e.g., humans, food producing animals and environment) in different countries was revealed, suggesting that pet dogs and cats may play a significant role in the global spread of AMR. The finding underscores the role of household pets as silent reservoirs of MDR Salmonella and the need for a One Health approach to tackle the issue. Public health campaigns promoting hygiene practices among pet owners should be encouraged. Pet animals should be incorporated into AMR monitoring and surveillance programs as a component of One Health framework.

Fig. 1

Circular comparison of Salmonella Rissen SA40, SA64, SA67, SA110, SA135 and SA153. Whole chromosomal DNA sequences of Salmonella Rissen SA40, SA64, SA67, SA110, SA135 and SA153 (dark purple, blue, orange, pink, yellow, and light green circles, respectively) are compared. The outermost red arrows represent AMR genes identified using the CARD database. The locations of integration/excision genes (dark green) and the horizontal gene transfer (HGT) region (black) are marked in the next inner rings, respectively. The zoomed-in view, bordered by a black dashed line, highlights the positions of AMR genes and insertion sequences associated with AMR genes on the chromosomal DNA of SA40, SA64, SA67, SA110, SA135, and SA153. The sequencing alignment indicates the high degree similarity of the SA40, SA64, SA67, SA110, SA135 and SA153. Gaps indicate the missing regions.

Fig. 2

Circular comparison of Salmonella Rissen SA158 and Salmonella Typhimurium SA165. (A) Whole chromosomal DNA sequence of Salmonella Rissen SA158 is presented (black). The zoomed-in view shows the position and direction of AMR genes from CARD database (red arrows, integration/excision genes (blue) and horizontal gene transfer (HGT) region (light purple). (B) Whole chromosomal DNA sequence of Salmonella Typhimurium SA165 (green circle), Salmonella Typhimurium STMC172 (yellow circle) and Salmonella enterica serovar 1,4,[5],12:i:- (orange circle) are compared. A zoomed-in view highlights the position and direction of AMR genes from CARD database (red arrows), integration/excision genes (blue) and horizontal gene transfer (HGT) region (purple). The repA and repC genes indicate the position of IncQ plasmid replicon. The sequencing alignment indicates the high degree similarity of the SA165 to Salmonella Typhimurium STMC172 and Salmonella enterica serovar 1,4,[5],12:i:-. Gaps indicate the missing regions.

Fig. 3

Circular comparison of Salmonella Derby SA97 and SA99. Whole chromosomal DNA sequences of Salmonella Derby SA97 (purple) and SA99 (green) carrying aac(6’)-Iaa, aadA1, aadA2, bla_TEM-1B, cmlA1, dfrA12, floR, fosA7, qacL, qnrS1, sul2, sul3, tet(A) and tet(M) are compared. The outermost red arrows represent AMR genes identified using the CARD database. The integration/excision genes (light blue), the location of horizontal gene transfer (HGT) region (orange) and transfer genes (pink) are shown. A right zoomed-in view, bordered by gray dashed line, highlights the position of qnrS1 (red arrow) and related insertion sequences (light blue). A left zoomed-in view, bordered by black dashed line, shows the position of AMR genes (red arrows) and insertion sequences related to AMR genes (light blue arrows). The sequencing alignment indicates the high degree similarity of the SA97 to SA95. Gaps indicate the missing regions.

Fig. 4

Circular comparison between IncI1 plasmid from Salmonella Weltevreden SA5 and Salmonella Poona SA85. (A) The comparison was made between IncI1 plasmid of SA5 (red circle) and SA85 (green shades) carrying aadA1, qacL, sul3, bla_TEM−1B. AMR genes identified using CARD database (pink), insertion sequences (blue) and horizontal gene transfer (HGT) region (purple) are shown. (B) and (C) Assembly graphs of IncI1 plasmid of SA5 and SA85, respectively were proposed by Bandage with contig number and size (in parenthesis).

Fig. 5

Circular comparison of IncFIB(K)/FIA(HI1) plasmid of Salmonella Panama SA164 to pP59A-3 plasmid and p2017.15.02CC_1. The alignment was made for IncFIB(K)/FIA(HI1) plasmid of SA164 from dog in this study (light blue circle), pP59A-3 plasmid from E. coli from pork in Cambodia (green circle) and p2017.15.02CC_1 plasmid from E. coli from human in Vietnam (purple circle), carrying sul3, tetA, dfrA12, aadA2, cmlA1, aadA1, qacL, tetM and bla_TEM-1B. The AMR genes from CARD database (red vector), integration/excision genes (dark blue), transfer genes (pink) and horizontal gene transfer (HGT) region (yellow), respectively.

Fig. 6

Circular comparison between IncX1 plasmid from Salmonella Agona SA74 and SA89 to pRHBSTW-00124_2 plasmid. (A) The comparison was made between IncX1 plasmid of SA74 (green) and SA89 (light blue) and pRHBSTW-00124_2 plasmid (orange), carrying tetA, qnrS1, dfrA14, sul3, bla_TEM-1B, aph(3’’)-Ib and aph(6)-Id. SA74 and SA89 originated from dogs in Nong Khai and Bueng Kan, respectively. The aligned sequences show a significant degree of similarity of IncX1 plasmid of Salmonella Agona SA74, SA89 and pRHBSTW-00124_2 plasmid. Only the sequences of IncX1 plasmid in SA74 and SA89 that were similar to pRHBSTW-00124_2 plasmid, were shown. (B) A zoomed-in view, bordered by black dashed line, shows the comparison between IncX1 plasmid of SA74 and SA89 and pRHBSTW-00124_2 plasmid was made by Clinker. The sequence similarity and location of the genes are indicated by groups in different colors. The same color of the gene vectors indicates the best links with high identity threshold at 0.9. The position and direction of AMR genes and insertion sequences are shown.

Fig. 7

Phylogenetic tree of core genome sequences in Salmonella from dogs and cats. Chromosomal sequences of Salmonella from dogs (n = 17) and cats (n = 8) were individually aligned with those specific serovar from the human, dog isolates and specific references using SeqSero 1.2. The single nucleotide polymorphisms (SNPs) were called using Snippy. Phylogenetic trees were generated using Core SNP alignment by IQ-TREE and visualized by iTOL. The number on the branch indicates the amount of genetic change or evolutionary divergence between taxa, expressed as the number of nucleotide substitutions per site. Distinct phylogenetic trees of A), Salmonella Agona; B), Salmonella Derby; C), Salmonella Hvittingfoss; D), Salmonella Javiana; E), Salmonella Kedougou; F), Salmonella Panama; G), Salmonella Paratyphi B; H), Salmonella Poona; I), Salmonella Rissen; J), Salmonella Typhimurium; K), Salmonella Uganda; L), Salmonella Wandsworth; and M), Salmonella Weltevreden are presented.

Fig. 7

Phylogenetic tree of core genome sequences in Salmonella from dogs and cats. Chromosomal sequences of Salmonella from dogs (n = 17) and cats (n = 8) were individually aligned with those specific serovar from the human, dog isolates and specific references using SeqSero 1.2. The single nucleotide polymorphisms (SNPs) were called using Snippy. Phylogenetic trees were generated using Core SNP alignment by IQ-TREE and visualized by iTOL. The number on the branch indicates the amount of genetic change or evolutionary divergence between taxa, expressed as the number of nucleotide substitutions per site. Distinct phylogenetic trees of A), Salmonella Agona; B), Salmonella Derby; C), Salmonella Hvittingfoss; D), Salmonella Javiana; E), Salmonella Kedougou; F), Salmonella Panama; G), Salmonella Paratyphi B; H), Salmonella Poona; I), Salmonella Rissen; J), Salmonella Typhimurium; K), Salmonella Uganda; L), Salmonella Wandsworth; and M), Salmonella Weltevreden are presented.