Antimicrobial Resistance and Prevalence of Extended Spectrum β-Lactamase-Producing Escherichia coli from Dogs and Cats in Northeastern China from 2012 to 2021

(1) Background: there has been a growing concern about pet-spread bacterial zoonosis in recent years. This study aimed to investigate the trend in drug-resistance of canine Escherichia coli isolates in northeast China between 2012–2021 and the differences in drug-resistance of E. coli of different origins in 2021. (2) Methods: E. coli were isolated from feces or anal swab samples from dogs and cats, and their antibiotic susceptibility profiles and phylogenetic grouping were identified. PCR was applied on the extended spectrum β-lactamase (ESBL) E. coli for antibiotic resistance genes. (3) Results: five hundred and fifty-four E. coli isolates were detected in 869 samples (63.75%). The multidrug resistance (MDR) rates of E. coli in pet dogs showed a decreasing trend, but working dogs showed the opposite trend. Resistance genes blaCTX-M and blaCTX-M+TEM were dominant among the ESBL producers (n = 219). The consistency between the resistance phenotypes and genes was high except for fluoroquinolone-resistant ESBL E. coli. All ESBL E. coli-carrying blaNDM were isolated from working dogs, and one of the strains carried mcr-1 and blaNDM-4. Phylogroup B2 was the dominant group in pet cats, and more than half of the isolates from companion cats were ESBL E. coli. (4) Conclusions: the measures taken to reduce resistance in China were beginning to bear fruit. Companion cats may be more susceptible to colonization by ESBL E. coli. The problem of resistant bacteria in working dogs and pet cats warrants concern.


Introduction
As the first choice for the treatment of bacterial infection, antibiotics play an important role in the clinical treatment of humans and animals [1] and can also promote the growth of livestock and poultry. Before China's Ministry of Agriculture and Rural Development issued a ban on the addition of antibiotics to feed, nearly half of the antibiotics used every year were mixed in animal feed to improve the growth of livestock and poultry. Antimicrobial resistance has become one of the most challenging problems in public health due to the overuse of antibiotics [2].
Most Escherichia coli are considered important components of the intestinal flora, but some E. coli carrying virulence genes can act as conditional pathogens to cause intestinal or extraintestinal infections, such as diarrhea, urinary tract infections, sepsis and meningitis, and a proportion of extraintestinal pathogenic E. coli can inhabit the intestine as normal flora and cause extraintestinal infections [3,4]. E. coli is one of the most widespread organisms in the intestinal flora and is chronically exposed to antibiotic selection pressure; therefore,

Drug Resistance Phenotypes
In total, 86.46% of E. coli isolates exhibited resistance to at least one antibiotic. E. coli exhibited diverse antimicrobial resistance phenotypes, with 230 different resistance patterns detected (Table S1). The MDR rates of E. coli isolates from pet dogs decreased from 76.92% from 2012-2013 to 62.42% in 2021 (p < 0.05), but working dogs showed the opposite trend (an increase from 41.18 to 72.86%, p < 0.001). There was no significant difference in the proportions of MDR E. coli between healthy companion animals and those with diarrhea [59.18% (29/49) vs. 60.71% (17/28), respectively, p > 0.05]. In pet dogs, the proportion of E. coli with multiple antibiotic resistance (MAR) index ≥ 0.2 gradually declined between 2012-2021, but working dogs showed the opposite trend. E. coli in pet cats showed more serious drug resistance than that in pet dogs in 2021 ( Figure 1).
The resistance rates to different antibiotics and drug resistance patterns of isolates from different sources between 2012-2021 are shown in Tables S2 and S3 and Figure S1. There was a general trend of decreasing resistance to various antibiotics in pet dog E. coli isolates between 2012-2021, while the opposite was true for working dog E. coli ( Figure 2). Working dog isolates in 2021 showed higher resistance to β-lactamase inhibitors and aminoglycosides than isolates from pet dogs. Notably, almost all isolates in this study were sensitive to colistin and carbapenems, and only 1.99% (n = 11) were resistant to colistin and 1.99% (n = 11) to carbapenem antibiotics. No carbapenem resistance phenotype was found in isolates from pet cats. The resistance rates to different antibiotics and drug resistance patterns of isolates from different sources between 2012-2021 are shown in Tables S2 and S3 and Figure S1. There was a general trend of decreasing resistance to various antibiotics in pet dog E. coli isolates between 2012-2021, while the opposite was true for working dog E. coli ( Figure  2). Working dog isolates in 2021 showed higher resistance to β-lactamase inhibitors and aminoglycosides than isolates from pet dogs. Notably, almost all isolates in this study were sensitive to colistin and carbapenems, and only 1.99% (n = 11) were resistant to colistin and 1.99% (n = 11) to carbapenem antibiotics. No carbapenem resistance phenotype was found in isolates from pet cats. F Figure 2. Trends of resistance to various antibiotics of 554 E. coli isolates from different origins between 2012-2021 (the depth of color of the rectangle represents the degree of resistance of the isolates to the antibiotic, and the darker the color, the stronger the drug resistance). AMK, amikacin; GEN, gentamicin; MEN, meropenem; CAZ, ceftazidime; AMP, ampicillin; CST, colistin; SXT, trimethoprim-sulfamethoxazole; CHL, chloramphenicol; TET, tetracycline. Notes: the antibiotics listed in Figure 2 only include the antibiotics tested against all E. coli.  The resistance rates to different antibiotics and drug resistance patterns of isolates from different sources between 2012-2021 are shown in Tables S2 and S3 and Figure S1. There was a general trend of decreasing resistance to various antibiotics in pet dog E. coli isolates between 2012-2021, while the opposite was true for working dog E. coli ( Figure  2). Working dog isolates in 2021 showed higher resistance to β-lactamase inhibitors and aminoglycosides than isolates from pet dogs. Notably, almost all isolates in this study were sensitive to colistin and carbapenems, and only 1.99% (n = 11) were resistant to colistin and 1.99% (n = 11) to carbapenem antibiotics. No carbapenem resistance phenotype was found in isolates from pet cats. Trends of resistance to various antibiotics of 554 E. coli isolates from different origins between 2012-2021 (the depth of color of the rectangle represents the degree of resistance of the isolates to the antibiotic, and the darker the color, the stronger the drug resistance). AMK, amikacin; GEN, gentamicin; MEN, meropenem; CAZ, ceftazidime; AMP, ampicillin; CST, colistin; SXT, trimethoprim-sulfamethoxazole; CHL, chloramphenicol; TET, tetracycline. Notes: the antibiotics listed in Figure 2 only include the antibiotics tested against all E. coli.
All ESBL-producing E. coli showed resistance to cefazolin, ampicillin, and piperacillin. The resistance rates of ESBL producers to fluoroquinolone antibiotics continued to decline from 2012 to 2021 (Table S4). It is noteworthy that 2.74% (n = 6) of ESBL-E. coli in 2021 were resistant to 11-12 antimicrobial categories (Table S5).

Phylogenetic Groups
The analysis of phylogroups showed that phylogroups A (37.77%) and B1 (30.16%) were the dominant groups among E. coli isolates in 2021, followed by phylogroups B2 (16.03%), F (6.79%), E (4.89%), C (2.99%), D (1.09%) and E clades (0.27%), respectively (Table S7). The isolates from different origins exhibited different structural characteristics. Phylogroups A and B1 were the dominant groups of canine E. coli; however, phylogroup B2 was the dominant group in E. coli from companion cats, and no isolates were assigned to phylogroup D. Phylogroups A and B1 were the dominant phylogroups of canine ESBL E. coli, except for those isolated in 2015, and phylogroups B1 and B2 were dominant phylogroups in feline ESBL E. coli (Table S8).

Discussion
Drug-resistant bacteria can be transmitted through close contact between pets and humans (such as petting, licking, or physical injuries) or through the home environment (such as contamination of food and furniture), posing a risk to children, the elderly, and pregnant or immunocompromised individuals [11]. Some high-income countries have investigated the antimicrobial resistance of E. coli carried by companion animals, such as Antibiotics 2022, 11, 1506 6 of 13 the United Kingdom [12], the United States [13], Spain [14], Canada [15], Australia [16], Japan [17], and Thailand [18]. The prevalence of drug-resistant E. coli (86.64%) in this study was much higher than in most developed and even developing countries. Previous studies have shown that the occurrence of MDR E. coli in companion animals varies considerably between provinces in China, from 24 to 86% [19][20][21][22][23]. E. coli exhibited a variety of antimicrobial resistance patterns in our study.
Since China began to include antibiotics in the management of veterinary prescription drugs in 2013, relevant authorities have released a number of policies to curb the increase in antimicrobial resistance in animal-derived bacteria. In 2015, China issued a decision on the discontinuation of the use of four veterinary drugs (namely, lomefloxacin, pefloxacin, ofloxacin, and norfloxacin) in food animals [24]; in 2016, China banned the use of colistin sulfate to promote animals' growth and, in 2018, China advocated avoiding the use of 3rd/4th generation cephalosporins and fluoroquinolone antibiotics in veterinary antimicrobial selection. In 2019, China began a complete ban on the addition of growth-promoting additives in feed. In our study, the resistance of E. coli in pet dogs to fluoroquinolones and 3rd/4th generation cephalosporins continued to decline from 2012 to 2021, the resistance to colistin decreased between 2015-2021, and the resistance of E. coli to various types of antibiotics in pet dogs was largely on a downward trend between 2015-2021. Although China does not allow carbapenems to be used in animals [25], a few E. coli with resistance to these antibiotics and related resistance genes were still detected in this experiment, which may have been caused by the transmission of drug resistance genes from humans to pets. Additionally, there have been occasional reports of companion animals carrying relevant resistance genes in recent years in China and elsewhere [25,26], so the government still needs to control these types of antibiotics strictly, because the spread of resistance to these antibiotics will cause a global health crisis [27].
It is reported that bacterial resistance is positively correlated with antibiotic use [11]. Antimicrobial resistance was generally consistent between our isolates and clinical isolates from the Chinese antimicrobial surveillance network (CHINET; https://www.chinets.com/, accessed on 21 April 2022). The possible causes are as follows: (1) The inability of companion animals to communicate the specificity and severity of their symptomatology may prompt pet owners to take precautionary and preventative measures to protect their pets, such as the use of antibiotics [28]; (2) Resistance genes may spread within the household; (3) The development of veterinary drugs is limited, and it is difficult to meet the needs of various pet diseases, so some human clinical antibiotics are used in animals; (4) Both self-medication with antibiotics and inappropriate antibiotic prescribing are prevalent in China. Self-administered antibiotics are mainly from over-the-counter purchases and leftover antibiotics from previous prescriptions [29].
As an important component of AMR E. coli, the prevalence of ESBL E. coli in companion animals varies among continents, ranging from 0.63% (Oceania) to 16.56% (Africa) in companion dogs and from 0% (Oceania) to 16.56% (Asia) in cats [30]. In this study, 41.30% (171/414) of E. coli isolated from companion animals were ESBL E. coli, which was significantly higher than the global prevalence of ESBL E. coli [30]. The rational use of antibiotics to reduce resistance remains a huge challenge in China. It has been reported that the prevalence of ESBL producers is higher in diarrheic animals than in healthy animals [31,32]. However, the prevalence of ESBL producers in healthy and diarrheic companion animals showed no significant difference in this study (p > 0.05), but the frequent and uncontrolled defecation of diarrheic pets increases the likelihood of spread of the ESBL-producing E. coli to the owner [33].
In 2021, ESBL E. coli was isolated from working dogs for the first time (34.29%). Resistance rates to most antibiotics were much lower in working dogs than in companion dogs between 2012-2013, a phenomenon that began to change in 2021. The possible reasons are as follows: (1) The use of antibiotics. Some antibiotics have been widely used in working dogs after 2020, such as penicillin and gentamicin; (2) Working dogs may acquire more drugresistance genes from the environment during training, such as when picking up target objects from the ground. Notably, for the first time, an E. coli resistant to all 13 categories of antibiotics tested (except aztreonam) was isolated from working dog samples, and it carried both mcr-1 and bla NDM-4 . E. coli carrying both genes has been previously isolated only from broiler feces in China [34] and from human patients in north Lebanon [35]. Study has shown that the fitness cost of bla NDM-4 carriage is not lower and that its presence might indicate rare occurrence and transmission among strains of different origins [34], but relevant genes were still detected in working dogs. The other two strains of E. coli carrying bla NDM-5 were also isolated from working dogs, and the fitness cost of bla NDM-5 is low [34], which undoubtedly increases the difficulty of prevention and treatment of drug-resistant bacterial infections and poses a potential threat to working dogs and related practitioners. It is necessary to take action to solve the problem of the spread of drug-resistant E. coli in working dogs.
Many researchers have reported that ESBL E. coli had a higher prevalence in dogs than in cats [36,37], but the trend in this study was the opposite. This phenomenon may be caused by the following: (1) A higher usage rate of the 3rd generation cephalosporins in cats than in dogs, because cats have poor adherence to oral medication and are less likely to ingest medication in food [38]; (2) The transmission frequency of drug resistance genes between cats and owners is higher than for dogs; a study in Mexico showed that participants spent more time stroking, brushing, and hugging their cats than their dogs [39]. According to the survey data of the white paper on China's pet industry in 2021, the number of cats surpassed that of dogs in 2021, becoming the most preferred pet; therefore, it is necessary to strengthen the monitoring of drug resistance in the intestinal flora of cats.
Most of the early ESBLs described were of the TEM or SHV type, but the CTX-M type gradually became the most prevalent ESBL type throughout the world after the emergence of the CTX-M type [40], and bla CTX-M was the dominant bla gene in this study. ESBL E. coli resistant to amikacin in this study were resistant to gentamicin, so amikacin can be used as an indicator of aminoglycoside antibiotic resistance. Compared with aac (6 )-Ib, aac (3)-II had a higher detection rate in gentamicin-resistant strains, which is consistent with the prevalence of aminoglycoside-modifying enzymes in Chinese human clinical E. coli isolates [41]. The resistance of ESBL E. coli from dogs and cats to amikacin in this study is mainly caused by rmtB, which is consistent with the prevalence of 16S rRNA methylases in Asia [42]. In this study, armA were isolated from ESBL E. coli in pet cats for the first time in China, and there is only one report of E. coli carrying armA isolated from companion dogs in the world [43]. Some strains carrying sulR genes are sensitive to trimethoprim-sulfamethoxazole, which may be related to dfr, which endows bacteria with trimethoprim resistance [44]. The sul2 genes are on large multi-resistance plasmids, indicating that it is unrealistic to eliminate the resistance genes widely distributed on mobilegenetic elements by reducing the use of antibiotics in the medium and short term [45]. The detection rates of PMQR (plasmid-mediated quinolone resistance) genes (qnr, aac (6 )-Ib-cr) in fluoroquinolone resistant strains were low. It is reported that high fluoroquinolone resistance is mainly obtained through the accumulation of PMQR genes and quinolone resistance-determining regions (QRDR) mutations [46]. PMQR genes do not confer a high level of resistance to fluoroquinolones but, rather, confer reduced susceptibility to these antimicrobial agents [44], so the prevalence of qnrS in sensitive and intermediate strains is much higher than that in resistant isolates. The high detection rates of tet (A) and tet (B) indicate that the main mechanism of tetracycline resistance in E. coli in dogs and cats in northeast China is active efflux, which is in line with the epidemic trend of tetracycline resistance genes in E. coli of animal origin. Resistance genes cmlA and floR are often found on plasmids and can be transferred horizontally between different species of bacteria. The high detection rate of cmlA in the chloramphenicol strains of intermediate sensitivity may be caused by the non-expression or low-level expression of the cmlA gene in vivo [47]. Most of the drug-resistant genes detected in this study exist in plasmids. The resistance rates to sulfonamides, fluoroquinolones and gentamicin were significantly higher in bla CTX-M positive strains than in bla CTX-M negative strains because these drug-resistant genes are on the same plasmid or can be co-transferred with bla CTX-M [22,48,49]. Once these genes are co-transferred with bla CTX-M , they will pose a huge risk to human health.
All phylogroups were found among ESBL producers in this study, which was only previously reported in the United States and Europe [30]. It has been reported that phylogenetic group B2 was more common in diarrheic animals than in healthy animals [50], but there was no significant difference in phylogroup between diarrheic and healthy companion animals in this study (p > 0.05). A large number of canine E. coli isolated in 2021 belonged to phylogroups A and B1, which was consistent with the prevalence trend of E. coli phylogroups in domestic animals [51]. However, a predominance of phylogroup B2 (42.25%), followed by phylogroup B1 (35.21%) was observed in pet cats, consistent with the dominant phylogroup of E. coli isolated from the urine of cats in the United States [13]. B2 and D are the most common phylogenetic groups of urinary tract infection isolates in dogs and cats, and phylogroup B2 is highly associated with urinary tract infections in humans [52], so isolates in cats are even more worrying. Integrons were less frequent in B2 than non-B2 isolates [53], and there were more feline ESBL E. coli belonging to phylogroup B1 than phylogroup B2, suggesting a trade-off between resistance and virulence [49].

Sample Collection
With the approval of the Laboratory Animal Welfare and Ethics Committee of the Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, professionals collected 869 feces or anal swabs, placed them in a refrigerated box, and sent them with ice packs to the laboratory for testing within 24 h. The animals sampled included pet dogs (n = 544, collected from Animal Hospitals in Changchun, Jilin Province), pet cats (n = 119, collected from animal hospitals in Changchun, Jilin Province), and working dogs (n = 206, collected from Harbin, Heilongjiang Province). In this study, samples were collected three times. Samples from working dogs and pet dogs were collected between 2012-2013, samples from pet dogs were collected in 2015, and samples from working dogs, pet dogs, and cats were collected in 2021.

Isolation and Identification of E. coli
Each sample was placed in a 4 mL centrifuge tube, to which 2 mL physiological saline was added. After vortexing and mixing, 20 µL fecal suspension were placed on MacConkey agar plates (Qingdao Hope Bio-Technology Co., Ltd., Qingdao, China) and cultured at 37 • C for 16-18 h. A red single colony was selected and purified until no miscellaneous bacteria were found. Specific E. coli 16S rDNA primers [54] were used to identify the isolates. The biochemical indices of the PCR positive strains were further determined using the BD Phoenix-100 ™ automatic microbiological identification/drug susceptibility system (Becton, Dickinson and Company, Franklin Lakes, New York, NJ, USA). The E. coli strains were stored at −80 • C for further analysis (one strain per sample).
The ESBL producers from 2012-2013 were re-tested for their antimicrobial resistance phenotype using the BD Phoenix-100 ™ automatic microbiological identification/drug susceptibility system, as described above. The MAR index was determined according to the method described by Krumperman [55].

Phylogenetic Grouping
DNA templates were made by the boiling method [56]. All ESBL producers between 2012-2021 and E. coli isolates obtained in 2021 were amplified with chuA, yjaA, TspE4C2, and arpA, according to the multiplex PCR method described by Clermont et al. [57]. The phylogroups (A, B1, B2, C, D, E, and F) were identified according to the different amplified fragment sizes. Sterile distilled water was the negative control, and the positive control was the strain template mixture preserved in this trial.

Statistical Analysis
We tested for statistical significance using the chi-square test of independence in SPSS 20.0 software (IBM, Armonk, New York, USA). Pearson's chi-squared test, Yate's correction for continuity, and Fisher's exact test were used in statistical significance when no/one/two or more cell counts in a 2 × 2 table were less than 5, respectively. A p-value < 0.05 was regarded as the criterion for statistical significance.

Conclusions
With the restrictions on the use of antibiotics in China, the antimicrobial resistance of E. coli in companion dogs is on the decline. Companion cats may be potential hosts for E. coli, causing extraintestinal disease in humans. Compared with pet dogs, the problem of drug-resistant bacteria carried by pet cats has become prominent; in particular, the high prevalence of ESBL E. coli, which belong to the B2 group in fecal samples from pet cats deserves further study. Our data helped to fill the gap in the knowledge of intestinal E. coli resistance in working dogs. All ESBL E. coli carrying bla NDM were isolated from working dogs, and one of them carried mcr-1 and bla NDM-4 . The problem of drug-resistant bacteria in working dogs is often neglected and needs to be paid attention. As animals in close contact with humans, the public health risk associated with dogs and cats carrying MDR zoonotic pathogens requires continued concern.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.