Migratory wild birds carrying multidrug-resistant Escherichia coli as potential transmitters of antimicrobial resistance in China

Migratory birds play an important role in the spread of multidrug-resistant (MDR) bacteria. To investigate the prevalence of MDR Escherichia coli in migratory birds in China and potential relationships with the environment, a total of 1387 samples (fecal samples, cloacal swabs, or throat swabs) were collected from migratory birds from three different river basins in China. The collected samples were processed and subjected to bacteriological examinations. Antimicrobial susceptibility testing of the recovered isolates was performed using the E-test for the detection of minimum inhibitory concentrations (MICs). Some antibiotic resistance genes were detected and the PCR products were confirmed by sequencing. In total, 478 (34.7%) E. coli isolates were recovered. The results showed that the drug-resistant E. coli isolates were highly resistant to β-lactams (43.7%) and tetracycline (22.6%), and 73 (15.3%) were MDR, including eight that were extended spectrum β-lactamase-positive. The retrieved strains harbored the blaCTX-M, blaTEM-1, tet(A), tet(B), tet(M), sul1, sul2, sul3, cmlA, floR, and intI1 genes with a prevalence of 5.9%, 36.4%, 80.5%, 11.9%, 6.8%, 6.8%, 47.5%, 12.7%, 50.8%, 37.3%, and 61.0%, respectively. The drug resistance rate of the isolates from southern China was higher than those from northern China. The E. coli samples collected for migratory birds in the Pearl River Basin had the highest proportion (46.7%) MDR isolates. Furthermore, MDR bacteria carried by migratory birds were closely related to the antibiotic content in the basin, which confirms that MDR bacteria carried by migratory birds are likely acquired from the environment. This study also confirmed that migratory birds are potential transmitters of MDR bacteria, demonstrating the need to reduce the use and emission of antibiotics and further in-depth studies on the mechanisms underlying drug resistance of bacteria.


Sample collection
All migratory bird samples were collected from May 2017 to June 2019 from six provinces in China (Fig 1, obtained from the USGS National Map Viewer). The procedures for handling and sampling of migratory birds were approved by the State Forestry Administration and the Laboratory Animal Welfare and Ethics Committee of the Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences (AMMS- 11-2020-11), and conducted in accordance with the Guidelines for the Care and Use of Animals in Research. No anesthesia, euthanasia, or animal sacrifice was conducted in this study. The sampling provinces located in Northeast China, Northwest China, Southern China and other regions were all within the range of the East Asian-Australasian Flyway. Feces, cloacal swabs, and throat swabs were collected under the supervision of the Wild Animal Sources and Diseases Inspection Station,

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Antibiotic resistance of E. coli in wild birds National Forestry and Grassland Bureau of China. All precautions were made to avoid any potential harm to the birds. The swabs were stored in physiological saline containing 20% glycerol at -80˚C for a short period and transported to our laboratory on dry ice.

Isolation and identification of E. coli
The samples (n = 1387) were resuspended in physiological saline, plated on MacConkey ager (BD Biosciences, San Jose, CA, USA), and incubated overnight at 37˚C. One suspected E. coli colony was selected from each plate and re-cultured on McConkey agar for subsequent analysis. The identification and antimicrobial susceptibility of presumptive E. coli isolates were determined using the NMIC/ID 4 panel of the BD Phoenix™ Automated Identification and Susceptibility Testing System (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) [22]. Bacterial strains were preliminarily classified as extended spectrum beta-lactamase (ESBL)-producers by the ESBL screen flow application of the same system. The isolates found to be resistant to at least three different classes of antimicrobial agents were classified as MDR bacteria [23].

Detection of minimum inhibitory concentration (MIC)
The MIC of E. coli isolates with the drug resistant phenotype was tested on Mueller-Hinton agar (bioMérieux, Marcy-l'Étoile, France) plates using commercially available E-test strips (Liofilchem SRL, Roseto degli Abruzzi, Italy) containing different types of antibiotics. Antimicrobial susceptibility testing include aminoglycosides (amikacin and gentamicin), β-lactams (cefazolin, cefotaxime, cefepime, aztreonam, ampicillin, piperacillin, amoxicillin/clavulanic and ampicillin/sulbactam), sulfonamides (trimethoprim and sulfamethoxazole), quinolones (ciprofloxacin and levofloxacin), and tetracycline (chloramphenicol). The disk diffusion method was conducted in accordance with the 2019 Clinical and Laboratory Standards Institute guidelines. E. coli ATCC 25922 was used as a control strain.

Detection of ARGs and integrons
All isolates obtained from the examined samples were subjected to genotyping using polymerase chain reaction (PCR). The template DNA consisted of boiled lysates prepared from the isolates. The primer sequences, sizes of the amplified fragments, PCR conditions, and references are described in Table 1. For PCR amplification, each 25-μl reaction contained 1 μL of the DNA template, 12.5 μL of 2×Taq DNA Master Mix (CWBio, Beijing, China), 0.5 μL of each primer at a concentration of 10 μM, and 10.5 μL of ddH 2 O. PCR reactions were performed to detect the ESBL genes bla CTX-M , bla CTX-M genotype groups 1, 2, 9, and bla TEM , the tetracycline resistance genes tet(A), tet(B), tet(C), tet(D), tet(M), and tet(W), the sulfonamide resistance genes sul1, sul2, sul3, and sulA, the chloramphenicol resistance genes cat1, cmlA, and floR, the colistin resistance gene mcr-1, and the integrase genes intI1 (for class 1 integrons), intI2 (for class 2 integrons), and intI3 (for class 3 integrons). Then, the PCR products were separated by electrophoresis with a 1% agarose gel and visualized under ultraviolet light. The positive amplicons of the ARGs in most MDR strains were sequenced (Comate Bioscience Co., Ltd., Changchun, China) and the sequences were analyzed for homology using the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST/).

Statistical analyses
Statistically significant differences of isolation rate and percentage of MDR E. coli isolates among the different surveilled regions were assessed using one-way analysis of variance. All

E. coli isolation
Following overnight incubation at 37˚C, suspected E. coli colonies appearing with peach or reddish coloration, smooth, and wet on McConkey agar were selected for identification. The morphologic tests showed that the selected colonies were all composed of Gram-negative rodshaped bacteria. Biochemical testing was conducted using the NMIC/ID 4 panel of the BD Phoenix™ Automated Identification and Susceptibility Testing System to obtain a more definitive identification of E. coli. Of the 1387 fecal, cloacal, and throat samples from migratory birds in six provinces in China, 478 (34.7%) E. coli isolates were obtained (

ARGs and integrons
The β-lactam resistance genes bla CTX-M and bla TEM-1 , the tetracycline resistance genes tet(A), tet(B), and tet(M), the sulfonamide resistance genes sul1, sul2, and sul3, the chloramphenicol resistance genes cmlA and floR, and the integrase gene intI1 were identified in most of MDR isolates. Class 1 integrons were present in 72 (61.0%) of the 118 E. coli isolates, most of which were found in MDR E. coli (65/73). Homology analysis of the sequences showed that the PCR results were not false positives (S1 Table). The detection results of drug resistance phenotypes, ARGs, and integrase genes of all MDR strains are shown in Table 3.

Discussion
E. coli is an important pathogen that causes severe infections in humans and animals, and acts as a donor and as a recipient of AGRs involving other bacteria. The main mechanisms of AMR among E. coli strains include (a) inactivation of antibiotics by producing inactivating enzymes or hydrolases; (b) changes to antibiotic target sites; (c) changes to bacterial membrane permeability; and (d) resistance associated with drug efflux pumps. E. coli has a great capacity to accumulate ARGs, mostly through horizontal gene transfer. Some mobile genetic elements seem to play a major role in the dissemination of ARGs. In general, antimicrobial resistance in E. coli is considered a major challenges in both humans and animals and must be considered as an urgent public health concern [34].
Many studies have shown that migratory birds transport antibiotic-resistant bacteria over long distances [3,35]. The East-Asian Australasian flyway is considered to be used by the most species of migratory birds [19]. Migratory birds can acquire and transmit MDR bacteria along the long migratory journey from Siberia to Australia [36]. A study conducted in Russia detected high levels of resistance to critically important antimicrobials, such as extended-spectrum cephalosporins, fluoroquinolones, colistin, and carbapenems, in wild birds [37]. The sampling sites in this study were located along this migratory route and were divided into two geographic locations by the Aihui-Tengchong Line. Almost half (43.8%) of the land southeast of the Aihui Tengchong line is inhabited by 94.1% of the population in China. The Aihui Tengchong line has also become the dividing line of urbanization level of China to some extent. In this study, the distribution of MDR E. coli was greater southeast of the Aihui-Tengchong Line than northwest. The significant difference in the drug resistance rate among the E. coli isolates collected from northern and southern China might be related to the impact of various human activities.
The significant difference in the drug resistance rate among E. coli isolates between swimming and wading birds is likely related to the difference in environments and feeding habits of migratory birds. Various birds previously identified as carriers of ESBL-producing E. coli are considerably mobile and often cross continents [38,39]. Among the eight ESBL-positive isolates, seven were from the Pearl River basin, and all were collected from wading birds. Wading birds mainly feed underwater or on underwater sediments, such as sludge, which may be related to the presence of drug resistance genes. To date, relatively few studies have investigated MDR bacteria carried by wading birds. Thus, follow-up analysis based on these results is warranted.
Overall, the prevalence of MDR E. coli was higher in the eastern and southern sampling sites than in the northwest, which was also consistent with the antibiotic emission density in China [34]. The level of drug resistance in a certain area is closely related to specific regional factors, such as local economic and agricultural development. Samples collected from the Pearl River basin had the highest level of drug resistance possibly because of the high discharge of antibiotics and industrial sewage in the region [40,41]. Notably, the high levels of antibiotics in most of the sampling sites in the Pearl River basin were due to closer proximity to human habitats or by birds feeding on human garbage [37]. Previous studies have shown that the concentrations of quinolones, macrolides, and β-lactams are much higher in the sediments of the Pearl River basin as compared to those of the Yellow River and Yangtze River basins [42,43].
Strains isolated from the Yellow River basin were most commonly resistant to tetracycline, followed by β-lactams. These results are basically consistent with those of previous studies on the content of antibiotics in drinking water in the Yellow River basin and coastal cities [42,44]. Tetracyclines were the first major category of broad-spectrum antibiotics used in humans and animals globally [45]. In general, E. coli of animal origin are often resistant to older antimicrobial agents, including tetracyclines and sulfonamides. The active efflux gene tetA and ribosomal protection gene tetM detected in this study can be transferred between bacteria through plasmids and transposons, resulting in extensive drug resistance [46]. Among all the sampling sites in this study, no drug-resistant E. coli was isolated from the Dali Lake samples, which was likely due to the distances of the sampling sites from human habitats, as these areas had lower concentrations of antibiotics in the environment and, thus, little impact on migratory birds.
The samples from the Yellow River basin not only contained more tet(A) genes, but also a certain amount of intI1. Integrons can rapidly obtain and disseminate various genes encoding resistance to antibiotics [47,48] and are classified as class 1, 2, or 3 based on the integrase gene (intI). Class 1 integrons are the most common and, thus, were monitored in this study. Interestingly, intI1 was detected in 88.7% (63/71) of MDR E. coli in the present study, which seems to support the idea that the occurrence of multidrug resistance among microbes is associated with mobile genetic elements [49].
The wetland area of Poyang Lake is among the top 10 ecological conservation areas in China and also the largest bird reserve and habitat for migratory birds in the world [50]. The isolation rate of drug-resistant bacteria from samples collected from birds around Poyang Lake was low (14.7%), which may reflect the low antibiotic emission in this area. A previous study reported that the concentrations of antibiotics around Poyang Lake are relatively moderate to below average as compared to other lakes in China [51]. Although the prevalence of drug-resistant bacteria around Poyang Lake area was low, considering the high mobility of migratory birds and the important geographical location of Poyang Lake, the levels of antibiotics in this area should be closely monitored. The dominant genes in the Poyang Lake samples were the tetracycline resistance gene tet(A), ESBL gene bla TEM-1 , and sulfonamide resistance gene sul2, which is generally consistent with the findings of previous studies [52]. Sulfonamide, tetracycline, and quinolone resistance genes are the most frequently detected ARGs in lakes and rivers and, therefore, have been suggested as possible indicators of environment pollution of antibiotics [53]. In addition, tetracyclines and sulfonamides (i.e., sulfadiazine, sulfamethoxazole, sulfamethazine, and sulfachlorpyridazine) are considered as priorities for control of antibiotics [54]. However, although migratory birds in different areas were sampled, this study did not take into consideration the timing in the same environment.

Conclusion
The result of this study confirmed the relationships of migratory birds with the environment and the spread of bacterial drug resistance. Migratory wild birds carrying MDR E. coli might be act as potential transmitters of antimicrobial resistance in China. Whether the drug-resistant bacteria carried by these migratory birds can colonize the host for long periods and spread with migration remains to be further studied. The results also demonstrated regional differences in MDR E. coli carried by migratory birds in China and the drug resistance rate was closely related to the population density and antibiotic emission density of different drainage areas. Although migratory birds, as carriers of drug-resistant bacteria, have a limited influence on the environment, the long-term impact should not be ignored. Recent works have shown that even treated waste can impact the acquisition of ARGs by avian wildlife [36]. Therefore, it is not only necessary to pay attention to the important role of migratory birds in the transmission of drug-resistant bacteria, but also to reduce the use of antibiotics in order to fundamentally reduce the transmission of ARGs.
Supporting information S1