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

Yu Yuan; Yan Hu; Xiaoli Zhang; Wenhao Zhong; Shulei Pan; Liqin Wang; Ziyao Zhou; Haifeng Liu; Shaqiu Zhang; Guangneng Peng; Ya Wang; Qigui Yan; Yan Luo; Keyun Shi; Zhijun Zhong

doi:10.1371/journal.pone.0298053

. 2024 Feb 28;19(2):e0298053. doi: 10.1371/journal.pone.0298053

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

Yu Yuan ^1,^#, Yan Hu ^1,^#, Xiaoli Zhang ^2,^#, Wenhao Zhong ^1,^#, Shulei Pan ¹, Liqin Wang ³, Ziyao Zhou ¹, Haifeng Liu ¹, Shaqiu Zhang ¹, Guangneng Peng ¹, Ya Wang ¹, Qigui Yan ¹, Yan Luo ¹, Keyun Shi ^2,^*, Zhijun Zhong ^1,^*

Editor: Md Tanvir Rahman⁴

PMCID: PMC10901357 PMID: 38416699

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.

1. Introduction

Escherichia coli (E. coli) is one of commensal microbiota in the gut of humans and animals [1]. With the widespread use of antimicrobials, the occurrence of multi-drug resistance (MDR) E. coli in humans and animals has posed a major threat to public health [2,3]. The presence of MDR E. coli in companion animals, such as pet dogs, undoubtedly raises concerns for both dogs and humans due to the high antimicrobial resistance (AMR) and the capability of carrying various antibiotic resistance genes (ARGs) of MDR strains [4,5]. Furthermore, previous studies have shown that there were associations between ARGs and virulence-associated genes (VAGs) existed in E. coli, and ARG-carrying strains may increase the likelihood of carrying VAGs [6]. However, only limited studies focused on MDR strains from pets in China, especially in Sichuan province which is considered as a major province for keeping pets in Southwest China [7].

In addition, recent studies have found that E. coli clones can be extensively shared between humans and household animals [8,9]. The transmission of high-risk E. coli clones between animals and humans has been recognized as a major public health issue [10,11]. For the population structure of E. coli clones, phylogenetic studies have shown that the B2 and D groups were more prevalent than the commensal groups (A and B1 groups) in E. coli isolates from diarrheic pet dogs [12,13]. Moreover, the multilocus sequence typing (MLST) was also used to analyze the population structure of E. coli clones [14]. Recent studies have identified a high population diversity of STs which related to zoonotic or pathotype in E. coli isolates from pet dogs, such as ST354, ST393, and ST457 E. coli were observed in companion animals from Australia [15]. And the high-risk ExPEC clones associated with humans and multidrug resistance, including sequence type (ST) 38, ST131, ST224, ST167, ST354, ST410, ST617 and ST648, have been identified in cats and dogs in Thailand which suggested there may be clonal dissemination between pets and humans [16].

In 2023, the number of domestic pets has exceeded 100 million in China [7]. Moreover, the latest annual report shows that the use of veterinary antibiotics for animals in China has exceeded 30,000 tons in 2020, tetracyclines, β-lactamases, and sulfonamides antimicrobial agents were widely used (Veterinary Bulletin of the Ministry of Agriculture and Rural People’s Republic of China, 2020) [17].With the widespread use of antimicrobials in animals, the high prevalence of MDR E. coli isolates from pet dogs has raised health concerns for both companion animals and humans [18,19]. To better understand the characteristics of MDR E. coli from pet dogs, we analyzed the antimicrobial resistance of E. coli isolates from diarrheal dogs, focusing on ARGs, VAGs, and population structure (ST and phylogenetic groups) for MDR E. coli to evaluate the potential threat.

2. Materials and methods

2.1 Sample collection

A total of 185 fresh feces samples were collected from pet dogs with clinical diarrhea during August 2021 to June 2022. Samples were excluded if the pet dogs were prescribed antimicrobial therapy or veterinary admission within the previous 3 months. The study was permitted by committee of Sichuan agricultural university (Permission number: DYY-2020303164) and the Sichuan Agricultural University Animal Ethical and Welfare Committee (Permission number: 20210268).

All samples were collected by professional veterinarians in Veterinary Teaching Hospital of Sichuan Agricultural University. Disinfection of hands and changing of disposable gloves were mandatory before sample collection. Samples were taken from the center of fresh feces by using sterile cotton swabs as soon as the pet dogs defecated to avoid cross-contamination from environmental bacteria on the ground. Then, samples were collected in sterile micro centrifuge tubes (Eppendorf) and were sent for processing to the laboratory within 12 h after collection.

2.2 Strains isolation

The isolation and identification of E. coli was performed as previous studies described [20,21]. Fecal samples were enriched in Luria-Bertani (LB) broth at 37°C, 120 r/min in a shaking incubator for 24 h. All isolates were identified presumptively by using phenotypic methods, including Gram staining, MacConkey agar growth and Eosin Methylene Blue agar growth. We further used 16S rDNA sequences (Primer: 5’-GAGTTTGATCCTGGCTCAG-3’; 5’-AGAAAGGAGGTGATCCAGCC-3’) [22] for final identification of E. coli. The confirmed isolates were stored in Luria-Bertani (LB) broth containing 50% glycerol at −20°C for further analysis.

2.3 Screening of MDR strains by antimicrobial susceptibility test

The antimicrobial susceptibilities of all isolates were tested using the standard disk diffusion method recommended by the Clinical and Laboratory Standards Institute (CLSI). A total of 16 antimicrobial agents in 6 categories as below were tested. aminoglycosides (gentamicin, CN, 10 μg; tobramycin, TOB, 10 μg), tetracyclines (tetracycline, TET, 30 μg; doxycycline, DOX, 30 μg), amide alcohols (chloramphenicol, C, 30 μg), quinolones (ciprofloxacin, CIP, 5 μg), sulfonamides (trimethoprim-sulfamethoxazole, SXT, 10 μg), β-lactams (ampicillin, AMP, 10 μg; cefazolin, KZ, 30 μg; cefuroxime, CXM, 30 μg; cefotaxime, CTX, 30 μg; cefepime, FEP, 30 μg; cefoxitin, FOX, 30 μg; aztreonam, ATM, 30 μg; imipenem, IPM, 10 μg; amoxicillin/clavulanic acid 2:1, AMC, 20 μg). For the 16 antimicrobial agents, the CN and TOB in aminoglycosides, DOX in tetracyclines, KZ, CTX, AMP, AMC in β-lactams and CIP in quinolones were used in pet animals at the location of our present study. The other antimicrobial agents (TET, C, CXM, FEP, ATM, IPM, FOX and SXT) have been found with E. coli resistance in dogs according to previous studies [23–25]. Results were interpreted in accordance with CLSI criteria (CLSI, 2023) [26]. E. coli ATCC25922 was used as a control. MDR strain was defined as being resistant to three or more antimicrobial categories [20].

2.4 DNA extraction and detection of ARGs, VAGs

Total genomic DNA of strains was extracted from isolates by using TIANamp Bacteria DNA kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. DNA samples were stored at -20°C for subsequent polymerase chain reaction (PCR) detection.

Primers of 23 ARGs in 6 categories (including 5 bla_CTX-M alleles for group1, 2, 8, 9, and 25) and 25 VAGs in 5 categories were synthesized by Huada Gene Technology Co. Ltd (Shenzhen, China). The PCR primer sequences and conditions for the ARGs and VAGs were showed in S1 and S2 Tables, respectively. PCR products were separated by gel electrophoresis in a 1.0% agarose gel in 1 × TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.3), stained with GoldView^TM (Sangon Biotech, Shanghai, China), and photographed under ultraviolet light using the Bio-Rad ChemiDoc MP omnipotent imager (Bole, USA). All positive PCR products were sequenced with Sanger sequencing in both directions by Sangon Biotech (Shanghai, China). Sequences of ARGs and VAGs were analyzed online using the BLAST function of NCBI (http://blast.ncbi.nlm.nih.gov).

2.5 Phylogenetic grouping and MLST

Phylogenetic grouping (A/B1/B2/D) was determined for MDR E. coli isolates using PCR targeting chuA, yjaA, and TSPE4.C2 following the protocol of Clermont et al. [27]. MLST was based on the sequencing results of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA) in each strain to obtain a numerical allelic profile which is abbreviated to a unique identifier of each strain: sequence type (ST) [28]. Allelic types of all seven housekeeping genes and ST of strains were determined following the protocol of the E. coli MLST database (https://pubmlst.org) [28,29]. The primer sequences of genes for MLST and phylogroups were shown in S3 Table. PCR products were separated by gel electrophoresis in a 1.0% agarose gel stained with GoldViewTM and photographed under ultraviolet light. All positive PCR products were sequenced with Sanger sequencing in both directions by Sangon Biotech (Shanghai, China). The sequences of housekeeping gene for MLST were analyzed online using pubMLST database (https://pubmlst.org).

The goeBURST algorithm in phyloviz 2.0 was used to clustering analysis of STs for MDR E. coli isolates, which divided the STs into several clusters, named as clonal complexes (CC), which consist of closely related STs with two allelic differences [30]. A clonal complex is typically composed of a single predominant genotype and closely relatives genotype [31].

2.6 Association analysis between ARGs and Antimicrobial resistance phenotypes (AMRs) or VAGs

The statistically analysis of data of AMR, ARGs and VAGs was conducted by using SPSS Statistics (version 26.0). P-value < 0.05 was considered to be statistically significant. Association analysis between ARGs and AMR or VAGs was performed by ggplot2 in RStudio (version 4.2.2; http://www.r-project.org) [32].

3. Results

3.1 AMRs of 135 isolates and Screening of MDR strains

A total of 135 E. coli strains were successfully isolated from 185 fecal samples of diarrheal dogs. Among the 135 E. coli isolates, 118 strains (87.41%, 118/135) were resistant to at least one antimicrobial agent, while only 17 (12.59%, 17/135) strains were sensitive to all antimicrobial agents (Fig 1A). Among the 6 antibiotic categories, the resistance rate for β-lactam antibiotics had the highest resistance rats (76.30%, 103/135), followed by tetracyclines (64.44%, 87/135) and sulfonamides (59.26%, 80/135). The resistance rate to quinolones antibiotics was the lowest (25.93%, 35/135). Moreover, the resistance rates to AMP (71.11%, 96/135), TET (62.22%, 84/135), and SXT (59.62%, 80/135) were the top 3 in 16 antimicrobial agents. The lowest resistance rate was observed for FOX (3.70%, 5/135), and the resistance rates for remaining 12 antibiotics ranged from 4.44% (IPM) to 45.19% (DOX) (Table 1). We further analyzed the resistant-phenotype patterns of the 135 E. coli isolates, revealing that 54.81% (74/135) of isolates were identified as MDR E. coli (Fig 1A). Among the 74 MDR strains, 56 types of resistance phenotypic patterns were observed. The more common patterns were TET/SXT/AMP, TET/DOX/C/SXT/AMP/AMC, TET/DOX/C/SXT/AMP, TET/C/SXT/AMP/AMC, and CN/TOB/TET/DOX/C/KZ/CXM/CTX/FEP/CIP/SXT/ATM/AMP (Fig 1B).

Fig 1 — (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.

Table 1. Antimicrobial resistance (AMR) detected in E. coli strains isolated from pet dogs (n = 135).

Category of antimicrobial	No. of Resistant Isolates (%)	Antibiotic		No. of Resistant Isolates (%)
β-lactams	103(76.30)	Penicillins	Ampicillin (AMP)	96 (71.11)
		1rd/2nd generation Cephalosporins	Cefazolin (KZ)	48 (35.56)
		1rd/2nd generation Cephalosporins	Cefuroxime (CXM)	44 (32.59)
		3rd/4th generation Cephalosporins	Cefotaxime (CTX)	45 (33.33)
		3rd/4th generation Cephalosporins	Cefepime (FEP)	21 (15.56)
		β-lactam compound	Amoxicillin/clavulanic acid (AMC)	32 (23.70)
		Monobactams	Aztreonam (ATM)	25 (18.52)
		Carbapenems	Imipenem (IPM)	6 (4.44)
		Cephamicins	Cefoxitin (FOX)	5 (3.70)
Tetracyclines	87(64.44)	Tetracycline (TET)		84 (62.22)
Tetracyclines	87(64.44)	Doxycycline (DOX)		61 (45.19)
Sulfonamides	80(59.26)	Trimethoprim-sulfamethoxazole (SXT)		80 (59.26)
Aminoglycosides	45(33.33)	Gentamicin (CN)		39 (28.89)
Aminoglycosides	45(33.33)	Tobramycin (TOB)		28 (20.74)
Amide alcohols	41(30.37)	Chloramphenicol (C)		41 (30.37)
Quinolones	35(25.93)	Ciprofloxacin (CIP)		35 (25.93)

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3.2 Distribution of ARGs and VAGs in MDR E. coli strains

Twelve out of 18 ARGs in 6 categories were detected in our present study (Table 2 and Fig 2). The detection rate of tetA (95.95%, 71/74) was the highest, followed by bla_TEM (93.24%, 69/74) and bla_CTX-M (90.54%, 67/74). Moreover, detection rates of flor, qnrS, and sul2 were all over 70% (74.32%, 74.32% and 70.27%, respectively). The detection rates of the remaining ARGs ranged from 16.22% (oqxAB) to 44.59% (sul1).

Table 2. Details of antimicrobial resistance (AMR) and antibiotic resistance genes (ARGs) detected in MDR E. coli strains isolated from pet dogs (n = 74).

Category of antimicrobial	No. of Resistant Isolates (%)	Antibiotics	No. of Resistant Isolates (%)	ARGs	No. of Positive Isolates (%)	Alleles of bla_TEM/bla_CTX-M (%)
β-lactams	71 (95.95)	AMP	71 (95.95)	bla _TEM	69(93.24)	bla_TEM-1, 81.16 (56/69)
		KZ	38 (51.35)			bla_TEM-135, 15.94 (11/69)
		CTX	38 (51.35)			bla_TEM-176, 2.90 (2/69)
		CXM	38 (51.35)	bla _CTX-M	67(90.54)	bla_CTX-M-14, 79.10 (53/67)
		ATM	25 (33.78)			bla_CTX-M-14, 79.10 (53/67)
		FEP	21 (28.38)			bla_CTX-M-65, 4.45 (3/67)
		AMC	19 (25.67)			bla_CTX-M-55, 31.34 (21/67)
		IPM	6 (8.11)			bla_CTX-M-64, 7.46 (5/67)
		FOX	5 (6.76)			bla_CTX-M-15, 5.97 (4/67)
Tetracyclines	71 (95.95)	TET	69 (93.24)	tetA	71(95.95)	--
Tetracyclines	71 (95.95)	DOX	49 (66.22)	tetA	71(95.95)	--
Sulfonamides	64 (90.54)	SXT	64 (90.54)	sul2	52(70.27)	--
				sul1	33(44.59)	--
				sul3	13(17.57)	--
Aminoglycosides	2 (56.76)	CN	37 (50.00)	aacC2	52(70.27)	--
Aminoglycosides	2 (56.76)	TOB	28 (37.84)	aacC4	31(41.89)	--
Amide alcohols	40 (54.05)	C	40 (54.05)	flor	55(74.32)	--
Amide alcohols	40 (54.05)	C	40 (54.05)	cmlA	19(25.68)	--
Quinolones	34 (45.95)	CIP	34 (45.95)	qnrS	55(74.32)	--
Quinolones	34 (45.95)	CIP	34 (45.95)	oqxAB	12(16.22)	--

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We further analyzed the subtypes of bla_TEM and bla_CTX-M, 3 variants of the bla_TEM gene and 5 variants of the bla_CTX-M gene were detected. Among the 3 variants of the bla_TEM gene, bla_TEM-1 was the most frequent (81.16%, 56/69), followed by bla_TEM-135 (15.94%, 11/69) and bla_TEM-176 (2.90%, 2/69). For the bla_CTX-M gene, 3 variants in bla_CTX-M-1 group (bla_CTX-M-55, bla_CTX-M-15 and bla_CTX-M-64) and 2 variants in bla_CTX-M-9 group (bla_CTX-M-65 and bla_CTX-M-14) were detected, bla_CTX-M-14 (79.10%, 53/67) and bla_CTX-M-55 (31.34%, 21/67) were more prevalent among the 5 variants observed, followed by bla_CTX-M-64 (7.46%, 5/67), bla_CTX-M-15 (5.97%, 4/67) and bla_CTX-M-65 (4.45%, 3/67).

Fourteen out of 25 VAGs in 4 categories were detected in our study (Table 3 and Fig 2). The detection rate of fimC (100%, 74/74) was the highest, followed by iucD (90.54%, 67/74) and sitA (85.14%, 63/74). The detection rates of iss (72.97%, 54/74), ompT (71.62%, 53/74) and fyuA (71.62%, 53/74) were all above 70%. Detection rates of the remaining VAGs were ranged from 4.05% (aggR) to 54.05% (eae). Further analysis of the virulence determinants linked with different pathotypes revealed 3 DEC-related VAGs (eae, aggR, astA) and 11 ExPEC-related VAGs were detected. Notably, all MDR E. coli isolates carried at least one ExPEC-related VAG, 75.68% (56/74) of strains carried at least one DEC-related VAG, which also carried at least one ExPEC-related VAG. The average number of VAGs carried by per DEC-related strain (8.36) was significantly higher than that of DEC-unrelated VAGs strains (5.56) (P < 0.01).

Table 3. Detection rate of virulence-associated genes (VAGs) in MDR E. coli isolated from pet dogs (n = 74).

Category of virulence	VAGs	No. of Resistant Isolates (%)	Related pathotypes
Iron transport-related	iucD	67 (90.54)	ExPEC
	sitA	63 (85.14)	ExPEC
	fyuA	53 (71.62)	ExPEC
	Irp2	39 (52.70)	ExPEC
	iroN	23 (31.08)	ExPEC
Adhesion-related	fimC	74 (100.00)	ExPEC
	eae	40 (54.05)	DEC
	tsh	14 (18.91)	ExPEC
Invasion-and-toxin related	vat	33 (44.59)	ExPEC
	astA	36 (48.65)	DEC
	aggR	3 (4.05)	DEC
Antiserum survival factors-related	iss	54 (72.97)	ExPEC
	ompT	53 (71.62)	ExPEC
	cvaC	17 (22.97)	ExPEC

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3.3 Associations between ARGs and AMRs or VAGs in MDR E. coli strains

Details of the detection rates of ARGs and AMR in 74 MDR E. coli strains were showed in Table 2. High prevalence of the β-lactam antibiotics genes (bla_TEM and bla_CTX-M, 93.24% and 90.54%, respectively) were detected, and resistance rate of β-lactams antibiotic (AMP) was higher than 90% (71/74, 95.95%). For tetracyclines, tetA (71/74, 95.95%) was detected in MDR strains, and the proportions of strains with tetracyclines-resistant phenotypes were 95.95%. For sulfonamides, the resistant rate of SXT was 90.54% (64/74) in MDR strains, while detection rates of three related resistance genes (sul1, sul2 and sul3) ranged from 17.57% (13/74) to 70.27% (52/74). For aminoglycosides, only two resistance genes (aacC2 and aacC4) were detected with the detection rates ranging from 41.89% (31/74) to 70.27% (52/74), while the detection rate of MDR strains with aminoglycosides-resistant phenotype (CN and TOB) ranged from 37.87% (28/74) to 50% (37/74). Similarly, the detection rates of remaining antibiotic resistance phenotypes and related ARGs were fluctuated. Above results showed that only β-lactams and tetracyclines antibiotic-resistant phenotypes were generally matched with related ARGs in detection rates, but the others were not completely consistent.

We further analyzed the associations between ARGs and AMR in 74 MDR strains. As shown in Fig 3A, a total of 23 positive association pairs (r > 0, P < 0.05) were observed between ARGs and AMR, of which the association between amide alcohols antibiotic resistance gene flor and amide alcohols-resistant phenotype (C) was the strongest. Moreover, the associations between 12 ARGs and 14 VAGs were further analyzed and the results were showed in Fig 3B, only 5 association pairs were observed, of which 3 pairs (sul2/ompT, aacC2/ompT, aacC4/astA) showed positive association (r > 0, P < 0.05), and the strongest association pair was observed between sul2 and ompT. The other 2 pairs (bla_CTX-M/tsh, bla_CTX-M/cvaC) were negative associations (r < 0, P < 0.05), and the strongest negative association pair was observed between bla_CTX-M and tsh.

3.4 Phylogenetic Grouping and MLST of 74 MDR E. coli isolates

As shown in the Fig 4A, group B2 (71.62%, 53/74) was the most prevalent in 74 MDR E. coli strains, followed by group D (18.92%, 14/74) and group B1 (9.46%, 7/74). 90.54% (67/74) of the MDR E. coli strains belonged to virulent extraintestinal-related group (B2 and D) and only 9.46% strains belonged to commensal group (B1) (P < 0.001). Furthermore, we analyzed the average number of VAGs carried by per strain in different groups, of which the highest observed was 8.28 in group B2, followed by 7.29 in group B1 and 5.57 in group D (P < 0.01). And no significant difference (P > 0.05) was observed in the average number of VAGs carried by per strain between commensal group (B1) and virulent extraintestinal-related groups (B2/D) (Fig 4B).

In addition, 42 different sequence types (STs) were identified in 74 MDR E. coli isolates (Fig 4C). Eleven strains belonged to ST10, followed by ST155 (4 strains), ST162 (4 strains), ST457 (4 strains), ST127 (3 strains) and ST410 (3 strains), the remaining 36 STs contained only one strains, respectively. Moreover, two new sequence types (nST1, nST2) were identified in our present study (S4 Table). By using goeBURST algorithm, 42 STs were clustered into 6 clonal complexes (CCs) and 23 singletons. Among the 6 clonal complexes, ST44, ST175, ST744, ST1415 and ST2197 were included in clonal complex 10CC with the founder ST10 (ST-10CC), and ST-10CC was the predominant lineage containing 17 MDR strains. ST345, ST410 and ST423 belonged to 90CC with the founder ST90 (ST-90CC); ST58, ST155 and nST1 belonged to 155CC with founder ST155 (ST-155CC); ST6209 and ST156 belonged to one clonal complex with the founder ST156 (ST-156CC), ST9580 and ST206 belonged to one clonal complex with the founder ST206 (ST-206CC), respectively. However, the CC contained ST359 and ST101 without any founder ST (Fig 4D). The heatmap of phylogroups and MLST distribution showed that 11 (14.86%, 11/74) strains simultaneously belonged to B2 group and ST10, which were the most prevalent clones in our present study (Fig 4E).

4. Discussion

The widespread use of antibiotics has significantly caused the increase of MDR E. coli which were isolated from companion animals [33]. To understand the characteristics of MDR strains and evaluate the potential threat, we detected the AMR of 135 E. coli isolates from pet dogs and further screened ARGs, VAGs, phylogenetic grouping and MLST for 74 MDR strains.

One hundred and thirty-five E. coli isolates from pet dogs showed high resistance to TET, AMP and SXT, which was consistent with another study in China [34]. Notably, TET has been used for treating animal infection disease and has also been used as “growth promoters” or “feed efficiency products” for a long time [35,36], which may activate the resistance of TET in E. coli. Once the antimicrobial-resistant E. coli occurs, it exists for a long time due to hard elimination [37]. In addition, TET resistance has been widely observed in E. coli from animals, such as dogs [13,24], yaks [38] and pigs [39], and the antibiotic resistance genes encoding AMR can be transmitted through environment and mobile genetic elements [40–42], which may be one of the reasons for high TET-resistant E. coli detected in our present study, even though TET has not been used in the area recently. The similar phenomenon was also found in the resistant strains to 3rd/4th generation cephalosporins, fluoroquinolones and carbapenems antibiotics in our present study, which have been advocated to avoid or restrict use in veterinary antimicrobial selection in China [34,43]. The resistance rates of AMP and SXT were consistent with the results from pet dogs in Nigeria, Australia and Brazil [19,23,44]. According to previous studies, frequent use of antimicrobial agents in clinics may be responsible for the high resistance to AMP and SXT [45,46], the β-lactam antibiotics have also been widely used in the location of the present study, and SXT is one of the most animal-used antimicrobials in China [45]. In our study, we further screened 74 MDR strains from 135 E. coli isolates. Compared with previous studies in Sichuan, the detection rate of MDR strains among E. coli isolates from pet dogs decreased from 100% in 2017 [47] and 70% in 2018 [48] to 54.81% in our study during 2021–2022, which was similar to the trend observed in Northeast China (which decreased from 76.92% in 2012–2013 to 62.42% in 2021) [43]. The decrease in the MDR detection rate may be due to that the relevant authorities of China have released several policies to curb the increase in antimicrobial resistance in animal-derived bacteria [43]. Although the detection rate of MDR E. coli from pet dogs in Sichuan has decreased, it is still higher than that detected in other countries [44,49]. Overall, resistant strains may have been circulating in the dog population long before the antibiotic usage restriction were introduced, and AMR strains are currently difficult to eliminate. The high detection rates of MDR E. coli in our study implied that more effective measures should be taken to control the occurrence and spread of MDR E. coli from companion animals.

It is well-known that the antibiotic resistant phenotype of bacteria is related to ARGs [50]. Therefore, we further analyzed the distribution of ARGs in 74 MDR strains. In our study, 12 out of 18 ARGs were detected in 74 MDR strains (detection rates ranged from 16.22% to 95.95%), of which tetA, bla_TEM and bla_CTX-M were the dominant ARGs. Notably, the detection rate of tetA in our study was higher than previous studies from dogs in China which ranged from 28% to 85.08% [43,51,52]. The bla_TEM and bla_CTX-M were the prevalent β-lactam resistance genes in our study which were consistent with previous study for E. coli from dogs in China [43]. We further analyzed the prevalent subtypes of bla_CTX-M genes, bla_CTX-M-55 (in bla_CTX-M-1 group) and bla_CTX-M-14 (in bla_CTX-M-9 group) were more prevalent. Among variants of bla_CTX-M-9 group, bla_CTX-M-14 has been considered as a common CTX-M variant worldwide, especially in China, Korea and Japan [53]. A high prevalence of bla_CTX-M-14 was also observed in our present study (79.10%). Notably, bla_CTX-M-14 was usually found on plasmid (such as IncF and IncK) according to previous studies [54,55]. The high prevalence of bla_CTX-M-14-positive strains observed in our present study indicated a high risk of potential transmission. For variants of the bla_CTX-M-1 group, bla_CTX-M-55 has become the second most common CTX-M subtype in Chinese clinical E. coli isolates after bla_CTX-M-14 [54], which was also dominant in our present study. Furthermore, Cottell et al. has observed the frequent clonal transmission of bla_CTX-M-55-positive E. coli between different hosts (humans and animals such as duck, chicken, and swine) [54]. The high prevalence of bla_CTX-M-14-positive and bla_CTX-M-55-positive strains observed in our present study suggests more studies should focus on the capability of clonal transmission between different hosts in the future.

Increasing studies proposed that the relationship between AMRs and ARGs is not completely consistent [22,56]. The detection rates of β-lactam and tetracycline antibiotics resistant phenotypes were generally consistent with the related ARGs. However, for sulfonamides, aminoglycosides, amide alcohols, and quinolones, the detection rates of related ARGs were fluctuated, e.g., the resistant rate of sulfonamides was 90.54% in MDR strains, while the detection rates of sul ranged from 17.57% to 70.27%. Moreover, we further analyzed the statistical associations between ARGs and AMRs, 23 positive association pairs were observed in MDR strains. Only flor and C, bla_CTX-M and CTX/CXM/KZ, oqxAB and CIP, aacC2 and TOB/CN showed consistency in ARG and related AMR. The other 16 association pairs were not completely consistent (Fig 3A). The similar phenomenon was also found in E. coli from captive non-human primates [22] and waterfowl [56] in China which indicated the different expression of ARGs, such as the abnormal expression of ARGs and the expression of ARGs has not reached that level which can activate the antibiotic resistance, may be a possible reason the incomplete consistency between AMR and ARGs [56].

Recent studies have shown that there was an association between ARGs and VAGs, and have proposed that they were linked and interacted with each other [6,56]. In order to explore the virulence traits and the association of ARGs and VAGs in MDR E. coli, we analyzed the distribution of VAGs in MDR strains. In our present study, 25 VAGs were selected for detection according to previous studies, among which 12 VAGs (eae, papA, bfpA, aggR, pic, astA, ipaH, stx1, stx2, elt, esta and estb) are related to Diarrhoeagenic Escherichia coli (DEC) [57–59], the remaining 13 VAGs (fyuA, iroN, sitA, iucD, Irp2, fimC, tsh, vat, ompT, cvaC, iss, hlyF, hlyA) are related to Extraintestinal Escherichia coli (ExPEC) [60–62]. Eleven ExPEC-related VAGs and 3 DEC-related VAGs (eae, astA, aggR) were detected in our present study. And the detection rates of VAGs in our study, which ranged from 4.99% to 100%, were higher than previous studies in dogs from Shaanxi and Shandong, China (ranging from 0.6% to 81.6% in Shaanxi and 2.53% to 87.34% in Shandong, respectively) [34,63]. Moreover, 75.68% (56/74) of the MDR strains simultaneously combined at least one ExPEC-related and DEC-related VAG, indicating the MDR strains in our present study harbored VAGs related to different pathotypes and could be considered as potentially virulent hybrid pathogenic strains. MDR strains with potential hybrid pathogen detected in pet dogs will pose a threat to other companion animals and their owners.

In addition, the association between VAGs and ARGs among MDR strains was further analyzed. Five associated pairs were observed, of which 3 pairs were positive and 2 pairs were negative. Compared with other studies of E. coli from giant pandas (46 associated pairs, of which 45 pairs were positive and 1 pair was negative) [6] and waterfowl (43 associated pairs, of which 36 pairs were positive and 7 pairs were negative) [56], the associations observed between ARGs and VAGs in MDR strains from pet dogs were not common in our present study. The possible reasons for drawing associations between VAGs and ARGs may be related to the co-location on the same mobile genetic elements, while negative associations indicated gene incompatibilities [64]. Recent studies also showed that many ARGs were inserted into conjugative plasmids carrying VAGs, which may provide conditions for drawing positive associations between ARGs and VAGs [65]. In our study, the strongest positive association was observed between ompT and sul2, which is usually detected in plasmids, indicating the positive association between ompT and sul2 may be related to the plasmid [40,66]. Similarly, the location of aacC, which usually was detected in the variable region of integron, may be related to the other two positive associations (aacC2/ompT, aacC4/astA) observed in our present study [5]. The positive association between ARGs and VAGs implied the high risk of plasmid-mediated co-transmission of ARGs and VAGs in E. coli, which could accelerate the spread of VAGs and ARGs within E. coli populations and enhance the emergence possibility of new pathogens with increased virulence and resistance potential. Negative associations observed in our present study may also be related to mobile genetic elements. For example, the VAG cvaC is usually found in virulent IncF plasmids [66], while bla_CTX-M is usually found in integron [67], the different locations on mobile genetic elements may be related to the negative associations between bla_CTX-M and cvaC in our present study. While the specific mechanism for the associations observed in our study required more comprehensive studies to validate.

Previous studies have shown that groups A and B1 are considered to be commensal groups, and groups B2 and D are considered to be virulent extraintestinal-related groups [27], which have been classified as potentially pathogenic [5,6]. A study from Iran showed that resistant E. coli strains mostly belonged to groups B2 and D [68]. Similarly, our present results showed a high prevalence of strains belonging to virulent extraintestinal-related groups (B2 and D) were detected among MDR E. coli from pet dogs. According to previous study, phylogenetic grouping is closely related to virulence genes (group A and B1 strains carry fewer virulence genes, while group B2 and D strains carry more virulence genes) [6], whereas in our present study, no significant difference was observed in the average number of VAGs carried by per strains between commensal groups and virulent extraintestinal-related groups. These phenomena suggest that antimicrobial resistance may increase the possibility of carrying more virulent factors (for example, the number of VAGs carried) in E. coli strains [69], implying that highly antibiotics resistant intestinal E. coli strains may also lead to extraintestinal infections which could be an emergent public health issue for humans, animals and the environment [70,71].

At last, we used MLST to analyze the dominant lineage of MDR strains, and our results showed that 74 MDR strains exhibited a high population diversity of STs (42 STs were identified), of which ST10 was the most prevalent. Another study of E. coli from dogs in China has found that the pandemic clone was ST131 (accounting for 9.8%) [63], which was identified as the most common ST in ExPEC, covering all geographical regions [72]. Moreover, studies from Australia and Thailand showed that ST354 and ST410 were the dominant clones in E. coli isolates from dogs, respectively [15,16]. In our study, the most common ST was ST10 (occurring in 14.86% of strains) which was different from the above studies, indicating that the prevalent ST lineage of E. coli isolates from pet dogs may vary in different geographical regions. ST10 was widely detected in E. coli from other animals in China, such as pigs [73] and chickens [74], indicating that ST10 may be the prevalent ST in E. coli strains from animals in China. Moreover, ST10 strains mostly belonged to B2 groups in our present study, and this phenomenon was also observed in E. coli isolates from chickens [74] and in the ExPEC isolates from humans [75]. In other studies, ST10 strains identified from pigs [73] and humans [76] mostly belonged to group A or D. This phenomenon indicated that the relationship between ST and phylogroups may vary in E. coli isolated from different hosts.

5. Conclusions and limitations

Seventy-four MDR strains were observed among 135 E. coli isolates from pet dogs, of which 12 ARGs and 14 VAGs were detected, implying that the E. coli isolates can be considered as a pool of ARGs and VAGs. Moreover, groups B2 and D, which are classified as potentially pathogenic, were predominant in the MDR isolates, and ST10 was the prevalent clonal lineage. Our findings have important implications for a preliminary understanding of the characteristics of MDR E. coli from diarrheal dogs and evaluating the potential risk of resistance and virulence in canine E. coli.

In our present study, the number of samples included was limited and the sampling site was only at our Veterinary Teaching Hospital. Future investigations should include more samples and expand sampling areas. Moreover, the use of High-throughput Sequencing or Whole Genome Sequencing could provide more comprehensive information on MDR E. coli strains from diarrheal dogs.

Supporting information

S1 Table. PCR primer and conditions for antibiotic resistance genes (ARGs) used in this study.

(XLSX)

pone.0298053.s001.xlsx^{(12.4KB, xlsx)}

S2 Table. PCR primer and conditions for virulence-associated genes (VAGs) used in this study.

(XLSX)

pone.0298053.s002.xlsx^{(12.2KB, xlsx)}

S3 Table. PCR primer and conditions of 7 housekeeping genes for MLST and 3 related genes for phylogroups used in this study.

(XLSX)

pone.0298053.s003.xlsx^{(10.4KB, xlsx)}

S4 Table. Distribution of sequence types (STs) in MDR E. coli strains from pet dogs (n = 74).

(XLSX)

pone.0298053.s004.xlsx^{(10.2KB, xlsx)}

S5 Table. Details of STs, phylogroups, AMR, ARGs and VAGs pattern for MDR E. coli isolated from pet dogs.

(XLSX)

pone.0298053.s005.xlsx^{(16.7KB, xlsx)}

S6 Table. The performance Standards for Antimicrobial Susceptibility testing in the present study.

(XLSX)

pone.0298053.s006.xlsx^{(10.3KB, xlsx)}

S7 Table. Clinical history of the pet dogs which successfully isolated E. coli strains in our present study.

(XLSX)

pone.0298053.s007.xlsx^{(16.2KB, xlsx)}

Acknowledgments

We thank Dr Ziyao Zhou, Junai Gan, and Lei Deng for the English language revision.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This research was funded by the National Key Research and Development Program of China (2018YFD0500900, 2016YFD0501009), the Chengdu Giant Panda Breeding Research Foundation (CPF2017-05, CPF2015-4) and the Science and Technology Achievements Transfer Project in Sichuan province (2022JDZH0026). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0298053.r001

Decision Letter 0

Md Tanvir Rahman

16 Nov 2023

PONE-D-23-30998Characteristics of MDR E. coli strains isolated from Pet Dogs with clinic diarrheal: A Pool of Antibiotic Resistance Genes and Virulence-Associated GenesPLOS ONE

Dear Dr. Zhong,

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Reviewer #1: Partly

Reviewer #2: No

Reviewer #3: No

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Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: General:

The study by Yuan et al. did screen 135 E. coli obtained from the feces of diarrheic pet dogs for their antimicrobial resistance, the antibiotic resistance genes und virulence associated genes, conducted phylogenetic grouping and multi locus sequence typing.

As the study serves the purpose characterising E. coli strains in order to monitor its prevalence and distribution, I strongly advise the authors to work with a writing coach or copyeditor to improve the language and readability.

The authors need to rerun the phylogenetic grouping and/or MLST, as it is highly unlikable that ST10 belongs to phylogroup B2. Please recheck!

The authors should reexamine the references cited in the discussion to determine whether the comparisons thus drawn are indeed based on comparable study conditions.

The authors should reconsider, why they are drawing associations between VAGs and ARGs and explain the importance of these results.

Please add limitations of the study.

Specifics:

Line 52-53: The authors should clarify the statement. Reference 11 was unable to find link between the examined resistance and virulence genes.

Line 58: The authors should revise the use of the term „virulent groups“ (B2 and D), as Clermont et al (Reference 19) refer to virulent extra-intestinal strains belonging to groups B2 and D. Please check throughout the manuscript.

Line 58, 216, 224, 304, 342, 360: Italics in the scientific name

Line 61-63: The authors should clarify the sentence to avoid confusion.

Line 70-71: Bulletin of the Ministry of Agriculture and Rural People’s Republic of China, 2020). (27). Please check punctuation.

Line 76: Please change diarrheal in diarrheal disease or diarrhea.

Line 99: Please change 16 S rRNA into 16S rRNA and Primer:5’-G to Primer: 5’-G

Line 114, 173, 332, 347, 349: Please use the correct abbreviation for SXT.

Line 116: Since there are no breakpoints for tobramycin, ciprofloxacin, cefuroxime,

cefotaxime, cefepime, cefoxitin, aztreonam in CLSI Vet06 2023, the authors should clarify how they obtained the breakpoints.

Line 138-140: The authors should explain why they used the protocol of Clermont from 2000 which distinguishes phylogroups A, B1, B2, and D instead of the new protocol from 2013 which enables an E. coli isolate to be assigned to one of the eight phylogroups (A, B1, B2, C, D, E, F and Escherichia cryptic clade I).

Line 177-178: Please change Results s howed to Results showed

Line 179-180: Please change were o bserved to were observed

Figure 1.B: Please change the image in favor of clarity and quality. For example, summarize combinations that occur only once and enlarge the remaining that occur multiple times.

Line 186: Please change 0 R to 0R according to figure 1.A. Please revise the language in the following sentences

Table 1: Please change ”Antibiotics” in the top heading to “Antibiotic”. Please insert a space between number of isolates and percentage in the first column. Please change 1rd/2th to 1st/2nd. As the interpretation of zone diameter or MIC changes with revised breakpoints every few years, a table with the results of the zone diameters should be added as a supplementary figure.

Line 201, 212, 206, 207, 373: Please check gene names.

Line 2018-220: The authors should clarify the sentence to avoid confusion.

Table 2: Please remove the % from the percentage of resistance to beta-lactams. Your results of tetracyclines suggest you found strains which exhibited resistance to doxycycline and at the same time susceptibility to tetracycline. Did you recheck these?

Line 261: You write “The other 2 pairs (blaCTX-M/tsh, blaCTX-M/iss) were negative association (r < 0, P < 0.05)”. In Figure 3 it shows a negative association with cvaC instead of iss. Please check.

Line 284: Please check sentence with keeping in mind that no strains belonged to phylogroup A.

Line 286-288: Please explain the marking representing significance in Figure 1.B.

Figure 4.C and 4.D: Please clarify the meaning of the different coloured outlines of the circles.

Figure 4.E: Please clarify on which data the tree is based.

Line 368-369: You stated you found blaTEM-1, blaTEM-135, and blaTEM-176. Neither of these is a ESBL-encoding gene. Please rephrase the sentence accordingly.

Line379-380: Please add in Material and Methods, how you located the blaCTX-M-14 on the plasmid and present the data in results.

Line 383-388: The authors use very general terms in the two sentences. Please clarify, what “paying more attention” means. Please explain in more detail what you mean by “diversity and evolution” and the implications of that, or refrain from using the sentence.

Line 399-400: The authors are repeating the sentence from 391-392. Please amend.

Line 405-406: The authors speak in very general terms. The manuscript would improve by the use of examples.

Line 412: Please cross-reference the percentages presented with the sources.

Line 412-414: As in the cited study a different set of virulence genes was tested, a comparison with the results of the study appears difficult. Please revise or delete.

Line 416-419: Since the study does not provide data on the prevalence of combining VAGs separated into categories of ExPEC-related and DEC-related VAGs, it is difficult to follow the argument of the sentence.

Line: 428-429: The authors repeat the results already given in discussion. Please delete.

Line 431-434: The authors explain the possible reason for the positive correlation between sul2 and ompT very well. Is there a similar rationale for the other two positive pairs? Is anything known about possible reasons for the negative association pairs?

Line 442-443: Please clarify the sentence to avoid the reader’s confusion.

Line 448-449: Please clarify the sentence to avoid the reader’s confusion.

Line 452: According to the reference, ST131 has been identified the most common sequence type in ExPEC. Please rephrase.

Line 455: The reader might wonder as ST10 is the most prevalent sequence type in the study, if there is anything known about its distribution?

Line 462-463: Please specify.

Reviewer #2: (1) Abstract: So far I know, any abstract has four essential parts with few additional or optional parts, such as objective(s), methodology, findings, and concluding remarks as essential parts and background, limitations, future indication etc could be present as additional portion(s). In this abstract, the objectives and methodology were not properly written, even absent. The abstract should be re-written properly, concisely and with proper structures.

(2) Introduction: In my opinion, research gaps, justification and novelty of the present research were not mentioned or presented properly.

(3) Methodology:

(A) Sample size could be larger, and could be determined using formula. However, the less number of samples could be explained properly in the 2.1 section.

(B) Line 77: why this findings unnecessarily came here?

Line 80-81: Could you please provide the permission number?

(D) Line 113-115: these information could be added in discussion.

(E) Line 124-125: please rephrase the sentence.

(F) Line 127-130: Could be added to the discussion.

(G) The 2.4 section was not described properly and scientifically. Supplementary Table information should be mentioned.

(4) Results: poorly represented.

(A) for determination of ARGs and AMR or VAGs association, in my opinion, all the E. coli isolates should be taken into consideration. Only MDR or resistant isolates can not give any conclusive data. Moreover, for this purpose, the sample size should be larger than the present research work.

(B) Phylogroup and ST distribution was not properly depicted.

(5) Discussion: poorly written. repetition of results came many times. Exact platform was not made.

(6) Conclusions: did not reflect the research outcome properly.

(7) References: huge reference list. should be restricted within 50-55. better less than 40.

(8) Others: Grammatical errors and poor English in many places of the manuscript.

Moreover, I could not find any new information from this research works. Though have few new ideas. However, following published articles could be mentioned as justification or research gaps...

https://doi.org/10.1128%2FJCM.40.10.3586-3595.2002

Sanchez S, McCrackin Stevenson MA, Hudson CR, Maier M, Buffington T, Dam Q, Maurer JJ. Characterization of multidrug-resistant Escherichia coli isolates associated with nosocomial infections in dogs. Journal of Clinical Microbiology. 2002 Oct;40(10):3586-95.

https://doi.org/10.5455/javar.2020.g466

Deb, P., Das, T., Nath, C., Ahad, A., & Chakraborty, P. (2020). Isolation of multidrug-resistant Escherichia coli, Staphylococcus spp., and Streptococcus spp. from dogs in Chattogram Metropolitan Area, Bangladesh. Journal of Advanced Veterinary and Animal Research, 7(4), 669-677

https://doi.org/10.1128/AEM.00599-11

Karczmarczyk M, Abbott Y, Walsh C, Leonard N, Fanning S. Characterization of multidrug-resistant Escherichia coli isolates from animals presenting at a university veterinary hospital. Applied and Environmental Microbiology. 2011 Oct 15;77(20):7104-12.

https://doi.org/10.3389/fvets.2023.1104812

Tong YC, Zhang YN, Li PC, Cao YL, Ding DZ, Yang Y, Lin QY, Gao YN, Sun SQ, Fan YP, Liu YQ. Detection of antibiotic-resistant canine origin Escherichia coli and the synergistic effect of magnolol in reducing the resistance of multidrug-resistant Escherichia coli. Frontiers in Veterinary Science. 2023 Mar 15;10:1104812.

https://doi.org/10.1016/j.prevetmed.2022.105767

Osman M, Albarracin B, Altier C, Gröhn YT, Cazer C. Antimicrobial resistance trends among canine Escherichia coli isolated at a New York veterinary diagnostic laboratory between 2007 and 2020. Preventive Veterinary Medicine. 2022 Nov 1;208:105767.

Reviewer #3: The manuscript described isolation of MDR E. coli strains from pet dogs carrying different antimicrobial resistance genes. The isolates were mostly associated with virulent phylogroups (B2 and D) and ST10 clonal lineage. The objectives are interesting and the methodology is standard. Language is poor and the manuscript is full of typing, grammatical and syntax errors which should be rectified by a well-versed English speaker. Most of the findings lack proper justification in the discussion section. Consequently, the conclusions are not supported with the findings.

Following issues should be replied point-wise.

Line 28. blaTEM-1 cannot be considered as ESBL…if it is necessary to describe TEM-1, please replace the ‘ESBL’ with ‘beta-lactamase’ throughout the manuscript

Line 76. ‘clinical diarrheal’ should be replaced with ‘clinical diarrhea’

Line 81. ‘animal ethics committee’ should be added

Line 82. ‘professional veterinaries’ should be replaced with ‘professional veterinarians’

Line 87. ‘Eppendorf (EP) tubes’ should be replaced with ‘micro centrifuge tubes (Eppendorf)’

Line 87. Clinical history of the pet dogs is missing, age/breed/sex of the diarrhoeic dogs, types of diarrhoea (mucoid/ bloody etc.), description of other clinical symptoms like fever, anorexia, vomition etc. should be added

Line 91. ‘Fecal samples were enriched in Luria-Bertani (LB) broth at 37 °C, 120 r/min for 24 h’…if the authors mean to describe about incubation in shaking incubator, that should be indicated

Line 94. ‘singe clone’ ??

Line 113. I agree with the authors that the canine E. coli isolates earlier showed resistance against the antibiotic discs selected in the present study. However, are these antibiotics commonly used in pet/companion animals in the study location? Did you consider to go through the prescriptions or did you conduct any qualitative survey to know the antibiotic usage pattern in local canine population?

Line 135. After BLASTing did you submit the sequences into genbank? If so, please indicate the accession numbers

Line 334. Did you find use of tetracycline as growth promoter in the study location? Moreover, how did you correlate growth promotional use of tetracyclines with higher occurrence of TET-resistant isolates in canine population?

Line 345. ‘Those phenomena indicated that AMR sustain for a long time and not easy to eliminate, once AMR is activated’ ….I found the sentence ambiguous which should be modified or deleted. It seems that implementation of antibiotic use restriction in the study location is not very effective or there are resistant strains circulating in the canine population since long before the imposition of antibiotic usage restriction and the strains are difficult to eliminate currently.

Line 348. ‘Frequent use of β-lactams and sulfonamides antimicrobial agents in clinic…’ another speculation, it should be justified with the screening of prescriptions in the clinics used in the present study

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

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PLoS One. 2024 Feb 28;19(2):e0298053. doi: 10.1371/journal.pone.0298053.r002

Author response to Decision Letter 0

15 Jan 2024

Dear Editor:

Thank you very much for your letter and the reviewers’ comments regarding our manuscript submitted to “PLOS ONE” (Manuscript ID: PONE-D-23-30998). We have checked the article and revised it according to the comments, and carefully proof-read the manuscript. The language presentation was improved with assistance from English speakers with appropriate research background. All revisions were marked in revised manuscript with track changes.

We hope, with these modifications and improvements based on your suggestion and the reviewer’s comments, the quality of our manuscript would meet the publication standard of “PLOS ONE”. Once again, we acknowledge your comments and constructive suggestions very much, which are valuable in improving the quality of our manuscript.

We submit here the revised manuscript with track changes as well as a list of changes. Thanks again for your reconsideration of our manuscript for publication in your journal. If you have any question about this paper, please don’t hesitate to let me know.

Best wishes for you!

Sincerely yours,

Zhijun Zhong

College of Veterinary Medicine

Key Laboratory of Animal Disease and Human Health of Sichuan Province

Sichuan Agricultural University

Chengdu 611130, P. R. China

E-mail: zhongzhijun488@126.com

Response to Reviewer 1 Comments

Response to Reviewer:

Thank you very much for your time and thoughtful comments, many of which have been incorporated into the revised manuscript (Tracked Version). Detailed responses are as follows.

Reviewer #1:

General:

Comment 1：As the study serves the purpose characterising E. coli strains in order to monitor its prevalence and distribution, I strongly advise the authors to work with a writing coach or copyeditor to improve the language and readability.

Response 1：Thanks for your comments. We have carefully checked the English expression and made extensive English revisions under the scrutiny of native English speakers with appropriate research background.

Comment 2：The authors need to rerun the phylogenetic grouping and/or MLST, as it is highly unlikable that ST10 belongs to phylogroup B2. Please recheck!

Response 2：Thanks for your comments. We reran the phylogenetic grouping and MLST to check our results carefully, and our results were correct. Actually, this phenomenon was also observed in E. coli isolates from chickens in China (1) and the ExPEC isolates from humans in Turkey (2). Previous studies showed that ST10 strains identified in E. coli from pigs in China (3) and humans in Spain (4) were mostly belonged to A or D group. These results indicates that the relationship between ST and phylogroups may vary in different E. coli isolated from different hosts. We have also added the corresponding discussion content for this phenomenon in our revised manuscript (Tracked Version) (Line 593-598).

Commet 3：The authors should reexamine the references cited in the discussion to determine whether the comparisons thus drawn are indeed based on comparable study conditions.

Response 3：Thanks for your comments. We have carefully checked the references to ensure that the comparisons drawn are indeed based on comparable study conditions, and we have modified the references in our revised manuscript.

Comment 4: The authors should reconsider, why they are drawing associations between VAGs and ARGs and explain the importance of these results.

Response 4：Thanks for your comments. we have added content in discussion for possible reasons of the associations between VAGs and ARGs and explanation of their importance in “discussion” section in our revised manuscript (Tracked Version) (Line 521-543): The possible reasons for drawing associations between VAGs and ARGs may be related to the co-location on the same mobile genetic elements, while negative associations indicated gene incompatibilities (5). Recent studies also showed that many ARGs were inserted into conjugative plasmids carrying VAGs (6), which also may provide conditions for drawing positive association between ARGs and VAGs.

In our study, the strongest positive association was observed between ompT and sul2, which is usually detected in plasmids (7,8), indicating the positive association between ompT and sul2 may be related to the plasmid. The positive association between ARGs and VAGs implied the high risk of plasmid-mediated co-transmission of ARGs and VAGs in E. coli, which could accelerate the spread of VAGs and ARGs within E. coli populations and enhance the emergence possibility of new pathogens with increased virulence and resistance potential.

Negative associations observed in our present study may also be related to mobile genetic elements. For example, the VAG cvaC usually be found in virulent IncF plasmids (9), while blaCTX-M is usually found in integron (10), the different location on mobile genetic elements may be related to the negative associations between blaCTX-M and cvaC in our present study. While the specific mechanism for associations observed in our present study required more comprehensive studies to validated.

Comment 5: Please add limitations of the study.

Response 5：Thanks for your comments. We have added “Limitations” in our revised manuscript (Line 612-616): In our present study, the number of samples included was limited and the sampling site was only at our Veterinary Teaching Hospital. Future investigations should include more samples and expand sampling areas. Moreover, the use of High-throughput sequencing or Whole Genome Sequencing could provide more comprehensive information on MDR E. coli strains from diarrheal dogs.

Specifics:

Line 52-53: The authors should clarify the statement. Reference 11 was unable to find link between the examined resistance and virulence genes.

Response: Thanks for your comments. We have deleted the reference in our revised manuscript.

Line 58: The authors should revise the use of the term „virulent groups “ (B2 and D), as Clermont et al (Reference 19) refer to virulent extra-intestinal strains belonging to groups B2 and D. Please check throughout the manuscript.

Response：Thanks for your comments, we have checked the whole manuscript and changed the “virulent groups” into “virulent extraintestinal-related groups” in our revised manuscript (Tracked Version) (Line 331, 554, 558, 569, and Line 945 in legends of Fig 4).

Line 58, 216, 224, 304, 342, 360: Italics in the scientific name

Response：Thanks for your comments, we have modified the italics in our revised manuscript (Tracked Version) (Line 66, 253, 428, line 923 in legends of Fig 2, line 943 in legends of Fig 4).

Line 61-63: The authors should clarify the sentence to avoid confusion.

Response：Thanks for your comments. We have modified the sentence in our revised manuscript (Tracked Version) (Line 68-75): Recent studies have identified a high population diversity of STs which related to zoonotic or pathotype in E. coli isolates from pet dogs, such as ST354, ST393, and ST457 E. coli were observed in companion animals from Australia. And the high-risk ExPEC clones associated with humans and multidrug resistance, including sequence type (ST) 38, ST131, ST224, ST167, ST354, ST410, ST617 and ST648, have been identified in cats and dogs in Thailand which suggested there may be clonal dissemination between pets and humans.

Line 70-71: Bulletin of the Ministry of Agriculture and Rural People’s Republic of China, 2020). (27). Please check punctuation.

Response：Thanks for your comments, we deleted the extra punctuation in our revised manuscript (Tracked Version) (Line 80-81).

Line 76: Please change diarrheal in diarrheal disease or diarrhea.

Response：Thanks for your comments, we have changed diarrheal into diarrhea in our revised manuscript (Tracked Version) (Line 96).

Line 99: Please change 16 S rRNA into 16S rRNA and Primer:5’-G to Primer: 5’-G

Response：Thanks for your comments, we have change 16 S rRNA into 16S rRNA and Primer:5’-G to Primer: 5’-G in our revised manuscript (Line 116-117).

Line 114, 173, 332, 347, 349: Please use the correct abbreviation for SXT.

Response：Thanks for your comments, we have checked the whole manuscript and modified the abbreviation for SXT in our revised manuscript (Tracked Version) (Line 142, 207, 382, and 409).

Line 116: Since there are no breakpoints for tobramycin, ciprofloxacin, cefuroxime,

cefotaxime, cefepime, cefoxitin, aztreonam in CLSI Vet06 2023, the authors should clarify how they obtained the breakpoints.

Response：Thanks for your comments. The references insert here is wrong. The correct referenced standard for our present study was the CLSI 2023 (Performance standards for antimicrobial susceptibility testing, M100-Ed33), and the Interpretive Categories and Zone Diameter Breakpoints for the antimicrobial agents which we tested are showed in Table S6. We have revised this reference in our revised manuscript.

Response：Thanks for your comments. Firstly, the protocol of Clermont was still widespread used in recent studies of E. coli isolates from diarrheal dogs (11,12). Secondly, phylogenetic protocol from Clermont has shown that virulent extraintestinal strains mainly belong to groups B2 and D, whereas strains of groups A and B1 are usually devoid of extraintestinal virulence factors, indicating a link between phylogroups and extraintestinal virulence (13,14). Our present study focuses on the antibiotic resistance characteristics of MDR E. coli from diarrheal dogs and preliminary analysis of the phylogroups and MLST for MDR E. coli. Our results showed the most common ST was ST10 and ST10 strains mostly belonged to B2 groups. As the reviewer suggests that the new protocol from 2013 can get more comprehensive phylogroups, we will consider using the new protocol in our future research.

Line 177-178: Please change Results s howed to Results showed

Response：Thanks for your comments, the “showed” has been modified to “revealing “in our revised manuscript (Tracked Version) (Line 212).

Line 179-180: Please change were o bserved to were observed

Response：Thanks for your comments, we have changed o bserved into observed in our revised manuscript (Tracked Version) (Line 213).

Figure 1.B: Please change the image in favor of clarity and quality. For example, summarize combinations that occur only once and enlarge the remaining that occur multiple times.

Response：Thanks for your comments. We have added red boxes in the figure to highlight the prevalent resistant-phenotypes patterns (occur three times) in our revised Figure 1. And the other combinations (without red box) occur only once or twice. The corresponding explanation for the red boxes has been added to the figure legends of Fig 1.B (Line 919-921).

Line 186: Please change 0 R to 0R according to figure 1.A. Please revise the language in the following sentences

Table 1: Please change“Antibiotics”in the top heading to “Antibiotic”. Please insert a space between number of isolates and percentage in the first column. Please change 1rd/2th to 1st/2nd. As the interpretation of zone diameter or MIC changes with revised breakpoints every few years, a table with the results of the zone diameters should be added as a supplementary figure.

Response：Thanks for your comments. We have change 0 R to 0R according to figure 1.A (Line 914). For Table 1, we have changed“Antibiotics”in the top heading to “Antibiotic”, inserted a space between number of isolates and percentage in the first column, and change 1rd/2th to 1st/2nd (Line 228). Moreover, the zone diameter breakpoints for the antimicrobial susceptibility testing in our present study are showed in Table S6.

Line 201, 212, 206, 207, 373: Please check gene names.

Response：Thanks for your comments, we have modified the gene names in our revised manuscript (Tracked Version) (Line 235, 248, 239, 239, 443).

Line 218-220: The authors should clarify the sentence to avoid confusion.

Response：Thanks for your comments, we have modified the sentence to avoid confusion in our revised manuscript (Tracked Version) (Line 255-257): The average number of VAGs carried by per DEC-related strain (8.36) was significantly higher than that of DEC-unrelated VAGs strains (5.56) (P < 0.01).

Response：Thanks for your comments, we have removed the % from the percentage of resistance to beta-lactams in our revised manuscript (Tracked Version) (Table 2). For the phenomenon of strains showing resistance to doxycycline and at the same time susceptible to tetracycline, we checked the detailed results of AMR, there are four strains resistant to doxycycline and at the same time susceptible to tetracycline. Moreover, the similar phenomenon was observed in our previous study for E. coli from captive non-human primates (15) and E. coli from captive giant pandas (16). While the reason for this phenomenon is unclear and needs to be investigated in further experiments.

Response：Thanks for your comments, we have changed it in our revised manuscript (Line 247): The other 2 pairs (blaCTX-M/tsh, blaCTX-M/cvaC) were negative association.

Line 284: Please check sentence with keeping in mind that no strains belonged to phylogroup A.

Response：Thanks for your comments, we have rewritten in our revised manuscript (Tracked Version) (Line 325).

Line 286-288: Please explain the marking representing significance in Figure 1.B.

Response：Thanks for your comments, we have added the sentence of “Marking * represents significant difference, *, P < 0.05; **, P < 0.01; ***, P < 0.001” in revised legends of Figure 4.B (Line 948-949).

Figure 4.C and 4.D: Please clarify the meaning of the different coloured outlines of the circles.

Response：Thanks for your comments. 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. We have added the meaning of the different colored outlines of the circles in our revised Figure legends (Line 954-957).

Figure 4.E: Please clarify on which data the tree is based.

Response：Thanks for your comments. The Heatmap was performed by “pheatmap” in R-studio based on the distribution of phylogenetic groups and MLST for MDR strains in our study. The distribution of phylogenetic groups and MLST was transformed to “0” or “1”. The partial data of the transformation results are shown in the table below. For example, data for CD01 in the table below means the strain belonged to B2 group and ST10, CD02 in the table below means the strain belonged to D group and ST58.

NAME B1 B2 D ST10 ST58 ST361 ST1276

CD01 0 1 0 1 0 0 0

CD02 0 0 1 0 1 0 0

CD03 0 1 0 0 0 1 0

CD04 0 1 0 1 0 0 0

CD05 0 0 1 0 0 0 1

CD06 0 1 0 0 0 0 0

……

Line 368-369: You stated you found blaTEM-1, blaTEM-135, and blaTEM-176. Neither of these is a ESBL-encoding gene. Please rephrase the sentence accordingly.

Response：Thanks for your comments. We have checked the whole manuscript and changed the ESBL-encoding gene to β-lactam resistance genes in order to avoid confusion (Line 439).

Line379-380: Please add in Material and Methods, how you located the blaCTX-M-14 on the plasmid and present the data in results.

Response：Thanks for your comments. In our present study, we did not investigate the location of blaCTX-M-14. This sentence was proposed based on the references which provided to indicate the potential transmission risk of blaCTX-M-14 strains. And the references have been added at the end of the sentence (Line 449-450).

Response：Thanks for your comments. In our present study, the high prevalence of blaCTX-M-14-positive and blaCTX-M-55-positive strains was observed, which are considered as high-risk strains for transmission between different hosts, thus, “paying more attention” means that more studies should focus on the capability of clonal transmission between different hosts for these strains in the future. We have added the corresponding content in our revised manuscript (Tracked Version) (Line 458-460).

Moreover, the other uncommon variants of blaCTX-M were not the focus of our discussion, so we have deleted the “diversity and evolution” in our revised manuscript.

Line 399-400: The authors are repeating the sentence from 391-392. Please amend.

Response：Thanks for your comments. We have deleted the repeating sentence in our revised manuscript.

Line 405-406: The authors speak in very general terms. The manuscript would improve by the use of examples.

Response：Thanks for your comments. We have modified the sentence in our revised manuscript (Tracked Version) (Line 480-485): The similar phenomenon was also found in E. coli from captive non-human primates and waterfowl in China which indicated the different expression of ARGs, such as the abnormal expression of ARGs and the expression of ARGs has not reached that level which can activate the antibiotic resistance, may be a possible reason the incomplete consistency between AMR and ARGs.

Line 412: Please cross-reference the percentages presented with the sources.

Response：Thanks for your comments. We have checked the percentages in reference and changed the sentence to “the ranging from 0.6% to 81.6% in Shaanxi and 2.53% to 87.34% in Shandong, respectively” in our revised manuscript (Tracked Version) (Line 501).

Line 412-414: As in the cited study a different set of virulence genes was tested, a comparison with the results of the study appears difficult. Please revise or delete.

Response：Thanks for your comments. We have deleted the sentence in our revised manuscript.

Response：Thanks for your comments. In our present study, we separated the VAGs into categories of ExPEC-related VAGs (17–20) and DEC-related VAGs (21–23) according to previous studies. And 75.68% (56/74) of the strains carried at least one DEC-related VAG, which also carried at least one ExPEC-related VAG. We have added the corresponding data of strains combining ExPEC-related and DEC-related VAGs simultaneously in the “Results” section of our revised manuscript (Tracked Version) (Line 254-255).

Line: 428-429: The authors repeat the results already given in discussion. Please delete.

Response：Thanks for your comments. We have deleted the repeating sentence in our revised manuscript.

Response 4：Thanks for your comments. We have added corresponding content in discussion for the possible reasons for the other two positive pairs and negative association pairs in our revise manuscript (Tracked Version) (Line 531-543): Similarly, the location of aacC, which usually was detected in the variable region of integron (24), may be related to the other two positive associations (aacC2/ompT, aacC4/astA) observed in our present study.

For the negative associations, the gene incompatibilities may be one of possible reasons according to previous researches (5,25). For example, the VAG cvaC usually be found in virulent IncF plasmids (9), while blaCTX-M is usually found in integron (10), the different location on mobile genetic elements may be related to the reason for negative association between blaCTX-M and cvaC observed in our present study. While the specific mechanism for the associations observed in our present study required more comprehensive studies to validate.

Line 442-443: Please clarify the sentence to avoid the reader’s confusion.

Response：Thanks for your comments. We have modified the sentence in our revised manuscript (Tracked Version) (Line 556-559): our present results showed a high prevalence of strains which belonged to virulent extraintestinal-related groups (B2 and D) were detected among MDR E. coli from pet dogs.

Line 448-449: Please clarify the sentence to avoid the reader’s confusion.

Response：Thanks for your comments. We have modified the sentence to “These phenomena indicated that the antimicrobial resistance may increase the possibility for carrying more virulent factor (for example, the number of VAGs carried) in E. coli strains” in our revised manuscript (Tracked Version) (Line 570-572). Actually, the similar phenomenon was found in previous study, which indicated that the probability of identifying virulence factor, such as STb, VT2, AIDA, was higher in strains carrying dhfrXIII, dhfrI, and tetB, respectively (5).

Line 452: According to the reference, ST131 has been identified the most common sequence type in ExPEC. Please rephrase.

Response：Thanks for your comments. We have modified the sentence to “which has been identified as the most common ST in ExPEC, covering all geographic regions” in our revised manuscript (Tracked Version) (Line 583).

Line 455: The reader might wonder as ST10 is the most prevalent sequence type in the study, if there is anything known about its distribution?

Response：Thanks for your comments. Actually, ST10 also was widely detected in E. coli from other animals in China, such as pigs (3,26) and chickens (1), indicating that the ST10 may be the prevalent ST in E. coli strains from animals in China. And we have also added the corresponding content in our revised manuscript (Tracked Version) (Line 591-593).

Line 462-463: Please specify.

Response：Thanks for your comments. We have changed the sentence to “Seventy-four MDR strains were observed among 135 E. coli isolates from pet dogs, of which 12 ARGs and 14 VAGs were detected, implying that the E. coli isolates can be considered as a pool of ARGs and VAGs” in our revised manuscript (Tracked Version) (Line 601-604).

Response to Reviewer 2 Comments

Response to Reviewer:

Thank you very much for your time and thoughtful comments, many of which have been incorporated into the revised manuscript (Tracked Version). Detailed responses are as follows.

Reviewer #2:

(1) Abstract: So far I know, any abstract has four essential parts with few additional or optional parts, such as objective(s), methodology, findings, and concluding remarks as essential parts and background, limitations, future indication etc could be present as additional portion(s). In this abstract, the objectives and methodology were not properly written, even absent. The abstract should be re-written properly, concisely and with proper structures.

Response 1：Thanks for your comments. As the reviewer suggested, we have modified the “Abstract” in our revised manuscript (Tracked Version) (Line 15-41).

(2) Introduction: In my opinion, research gaps, justification and novelty of the present research were not mentioned or presented properly.

Response 2：Thanks for your comments. Recently, increasing studies have focused on antimicrobial resistant E. coli from companion animals, indicating that the companion animals are reservoirs of MDR strains with a high risk of transmission between dogs and humans (27). However, the research on MDR strains from companion animals in China is limited, especially in Sichuan Province, which is considered to be a major province of pets keeping in western China (28). In our present study, we not only analyze the antibiotic resistance of E. coli isolated from pet dogs, but also pay more attention to the ARGs, VAGs and population structure (ST and phylogenetic groups) for MDR strains to evaluate the potential threat. Moreover, more and more studies have observed the association which existed in between ARGs and VAGs in E. coli from animals, such as pandas (29) and waterfowl (19), but studies for the association between ARGs and VAGs of E. coli from pet dogs was limited, we further analyzed the association between ARGs and VAGs in MDR strains. Above all, our research helps to better understand the characteristics of MDR strains from pet dogs and fills the research gaps of MDR E. coli from pet dogs in Sichuan Province. And we have modified the “Introduction” section in our revised manuscript (Tracked Version) (Line 46-90).

(3) Methodology:

(A) Sample size could be larger, and could be determined using formula. However, the less number of samples could be explained properly in the 2.1 section.

Response 3A: Thanks for your comments. Actually, we collected about 250 fecal samples in the initial phase of the present study, while some of the samples did not meet the sampling standards for “Samples were excluded if the pet dogs were prescribed antimicrobial therapy or veterinary admission within the previous 3 months” in our present study. The sampling standards were also shown in 2.1 section (Line 83-84). Thus, only 185 fecal samples were ultimately included. We have added the explanation for limited number of samples in the “Limitations” section in our revised manuscript (Tracked Version) (Line 612-616).

(B) Line 77: why this findings unnecessarily came here?

Response 3B: Thanks for your comments. We have deleted this finding in 2.1 section and added it to “Results” section in our revised manuscript (Tracked Version) (Line 199-200).

Line 80-81: Could you please provide the permission number?

Response: Thanks for your comments. The permission number is DYY-2020303164, and we have added it in our revised manuscript (Tracked Version) (Line 100).

Response 3C: Thanks for your comments. We have simplified the section 2.2 in our revised manuscript (Tracked Version) (Line 111-127): The isolation and identification of E. coli was performed as previous studies described. Fecal samples were enriched in Luria-Bertani (LB) broth at 37 °C, 120 r/min in a shaking incubator for 24 h. All isolates were identified presumptively by using phenotypic methods, including Gram staining, MacConkey agar growth and Eosin Methylene Blue agar growth. We further used 16S rDNA sequences (Primer: 5’-GAGTTTGATCCTGGCTCAG-3’; 5’-AGAAAGGAGGTGATCCAGCC-3’) for final identification of E. coli. The confirmed isolates were stored in Luria-Bertani (LB) broth containing 50% glycerol at −20 °C for further analysis.

(D) Line 113-115: these information could be added in discussion.

Response 3D: Thanks for your comments. Actually, the information is the criteria for our selection of antimicrobial agents for susceptibility testing in our present study, it is more scientific to place the information in “2.3” section, and we have modified the sentence to“For the 16 antimicrobial agents, the CN and TOB in aminoglycosides, DOX in tetracyclines, KZ, CTX, AMP, AMC in β-lactams and CIP in quinolones were used in pet animals at the location of our present study. The other antimicrobial agents (TET, C, CXM, FEP, ATM, IPM, FOX and SXT) have been found with E. coli resistance in dogs according to previous studies” in our revised manuscript (Tracked Version) (Line 139-143).

(E) Line 124-125: please rephrase the sentence.

Response 3E: Thanks for your comments. We have rephrased the sentence to “Primers of 23 ARGs (including 5 blaCTX-M alleles for group1, 2, 8, 9 and 25) in 6 categories and 25 VAGs in 5 categories were synthesized by Huada Gene Technology Co. Ltd (Shenzhen, China), and the PCR primer and conditions for the ARGs and VAGs which been chosen in our present study were showed in S1 Table and S2 Table, respectively” in our revised ma

Attachment

Submitted filename: Response to Reviewers.docx

pone.0298053.s008.docx^{(67.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0298053.r003

Decision Letter 1

Md Tanvir Rahman

17 Jan 2024

Characteristics of MDR E. coli strains isolated from Pet Dogs with clinic diarrhea: A Pool of Antibiotic Resistance Genes and Virulence-Associated Genes

PONE-D-23-30998R1

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Additional Editor Comments (optional):

Thanks for addressing all the comments of the reviewers.

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0298053.r004

Acceptance letter

Md Tanvir Rahman

5 Feb 2024

PONE-D-23-30998R1

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. PCR primer and conditions for antibiotic resistance genes (ARGs) used in this study.

(XLSX)

pone.0298053.s001.xlsx^{(12.4KB, xlsx)}

S2 Table. PCR primer and conditions for virulence-associated genes (VAGs) used in this study.

(XLSX)

pone.0298053.s002.xlsx^{(12.2KB, xlsx)}

S3 Table. PCR primer and conditions of 7 housekeeping genes for MLST and 3 related genes for phylogroups used in this study.

(XLSX)

pone.0298053.s003.xlsx^{(10.4KB, xlsx)}

S4 Table. Distribution of sequence types (STs) in MDR E. coli strains from pet dogs (n = 74).

(XLSX)

pone.0298053.s004.xlsx^{(10.2KB, xlsx)}

S5 Table. Details of STs, phylogroups, AMR, ARGs and VAGs pattern for MDR E. coli isolated from pet dogs.

(XLSX)

pone.0298053.s005.xlsx^{(16.7KB, xlsx)}

S6 Table. The performance Standards for Antimicrobial Susceptibility testing in the present study.

(XLSX)

pone.0298053.s006.xlsx^{(10.3KB, xlsx)}

S7 Table. Clinical history of the pet dogs which successfully isolated E. coli strains in our present study.

(XLSX)

pone.0298053.s007.xlsx^{(16.2KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0298053.s008.docx^{(67.8KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

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

Yu Yuan

Yan Hu

Xiaoli Zhang

Wenhao Zhong

Shulei Pan

Liqin Wang

Ziyao Zhou

Haifeng Liu

Shaqiu Zhang

Guangneng Peng

Ya Wang

Qigui Yan

Yan Luo

Keyun Shi

Zhijun Zhong

Roles

Abstract

1. Introduction

2. Materials and methods

2.1 Sample collection

2.2 Strains isolation

2.3 Screening of MDR strains by antimicrobial susceptibility test

2.4 DNA extraction and detection of ARGs, VAGs

2.5 Phylogenetic grouping and MLST

2.6 Association analysis between ARGs and Antimicrobial resistance phenotypes (AMRs) or VAGs

3. Results

3.1 AMRs of 135 isolates and Screening of MDR strains

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

Table 1. Antimicrobial resistance (AMR) detected in E. coli strains isolated from pet dogs (n = 135).

3.2 Distribution of ARGs and VAGs in MDR E. coli strains

Table 2. Details of antimicrobial resistance (AMR) and antibiotic resistance genes (ARGs) detected in MDR E. coli strains isolated from pet dogs (n = 74).

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

Table 3. Detection rate of virulence-associated genes (VAGs) in MDR E. coli isolated from pet dogs (n = 74).

3.3 Associations between ARGs and AMRs or VAGs in MDR E. coli strains

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

3.4 Phylogenetic Grouping and MLST of 74 MDR E. coli isolates

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

4. Discussion

5. Conclusions and limitations

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Md Tanvir Rahman

Roles

Author response to Decision Letter 0

Decision Letter 1

Md Tanvir Rahman

Roles

Acceptance letter

Md Tanvir Rahman

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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