Antibiotic-Resistant Escherichia coli Strains Isolated from Captive Giant Pandas: A Reservoir of Antibiotic Resistance Genes and Virulence-Associated Genes
by
, , , , , , , ,
Siping Fan
1,†,
Shaoqi Jiang
1,2,†,
Lijun Luo
1,†,
Ziyao Zhou
1,†,
Liqin Wang
3,†,
Xiangming Huang
4,
Haifeng Liu
1,
Shaqiu Zhang
1,
Yan Luo
1,
Zhihua Ren
1,
Xiaoping Ma
1,
Suizhong Cao
1,
Liuhong Shen
1,
Ya Wang
1,
Liping Gou
1,
Yi Geng
1,
Guangneng Peng
1,
Yanqiu Zhu
1,
Wei Li
1,
Yalin Zhong
1,
Xianpeng Shi
1,
Ziqi Zhu
1,
Keyun Shi
5,* and
Zhijun Zhong
1,*add
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1
Key Laboratory of Animal Disease and Human Health of Sichuan, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2
Leshan Vocational and Technical College, Leshan 614000, China
3
The Chengdu Zoo, Institute of Wild Animals, Chengdu 610081, China
4
Chengdu Research Base of Giant Panda Breeding, Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Chengdu 610081, China
5
Jiangsu Yixing People’s Hospital, Yixing 214200, China
*
Authors to whom correspondence should be addressed.
†
These authors have contributed equally to this work.
Vet. Sci. 2022, 9(12), 705; https://doi.org/10.3390/vetsci9120705
Submission received: 26 October 2022
/
Revised: 14 December 2022
/
Accepted: 15 December 2022
/
Published: 18 December 2022
(This article belongs to the Special Issue Extraintestinal Pathogenic Escherichia coli)
Abstract
:Simple Summary
Escherichia coli (E. coli) carrying antibiotic resistance genes (ARGs) and virulence-associated genes (VAGs), which are considered as emerging environmental pollutants, undoubtedly causes adverse health effects on humans and animals. With the implementation of the Wild Release Plan of captive giant pandas, antibiotic-resistant E. coli carrying ARGs/VAGs in giant pandas’ feces that are discharged into the environment may become pollutants for nature. To evaluate the potential threat of E. coli strains from captive giant pandas, we analyzed the distribution and the association of ARGs and VAGs in antibiotic-resistant E. coli. Our results revealed a high prevalence of ARGs and a significant positive association among ARGs and VAGs, suggesting that the continuous monitoring of the impact of the spread of antibiotic-resistant E. coli (carried various ARGs and VAGs) is needed in future studies.
Abstract
Recent studies showed that Escherichia coli (E. coli) strains isolated from captive giant pandas have serious resistance to antibiotics and carry various antibiotic resistance genes (ARGs). ARGs or virulence-associated genes (VAGs) carried by antibiotic-resistant E. coli are considered as a potential health threat to giant pandas, humans, other animals and the environment. In this study, we screened ARGs and VAGs in 84 antibiotic-resistant E. coli strains isolated from clinically healthy captive giant pandas, identified the association between ARGs and VAGs and analyzed the phylogenetic clustering of E. coli isolates. Our results showed that the most prevalent ARG in E. coli strains isolated from giant pandas is blaTEM (100.00%, 84/84), while the most prevalent VAG is fimC (91.67%, 77/84). There was a significant positive association among 30 pairs of ARGs, of which the strongest was observed for sul1/tetC (OR, 133.33). A significant positive association was demonstrated among 14 pairs of VAGs, and the strongest was observed for fyuA/iroN (OR, 294.40). A positive association was also observed among 45 pairs of ARGs and VAGs, of which the strongest was sul1/eaeA (OR, 23.06). The association of ARGs and mobile gene elements (MGEs) was further analyzed, and the strongest was found for flor and intI1 (OR, 79.86). The result of phylogenetic clustering showed that the most prevalent group was group B2 (67.86%, 57/84), followed by group A (16.67%, 14/84), group D (9.52%, 8/84) and group B1 (5.95%, 5/84). This study implied that antibiotic-resistant E. coli isolated from captive giant pandas is a reservoir of ARGs and VAGs, and significant associations exist among ARGs, VAGs and MGEs. Monitoring ARGs, VAGs and MGEs carried by E. coli from giant pandas is beneficial for controlling the development of antimicrobial resistance.
1. Introduction
Escherichia coli (E. coli) is one of the most important intestinal floras of humans and animals [1,2]. The carrying of antibiotic resistance genes (ARGs) and virulence-associated genes (VAGs) among E. coli undoubtedly causes adverse health effects on humans and animals [3,4]. Antibiotic-resistant E. coli is also considered as an emerging potential environmental pollutant [5,6,7]. ARGs and VAGs carried by E. coli are widely studied due to the risk of transmission to the environment [8,9,10,11]. The giant panda (Ailuropoda melanoleuca) is one of the endangered animals in the world; the number of giant pandas (especially captive giant pandas) has increased with the implementation of protective projects [12]. However, giant pandas are still threatened by bacterial diseases, especially the diseases caused by intestinal pathogens (such as Escherichia coli, Klebsiella spp., Campylobacter jejuni, Pseudomonas aeruginosa, Yersinia enterocolitica and Clostridium welchii) [13]. Overusing antibiotics for captive animals promotes the resistance of microorganisms [14,15,16]. E. coli is one of the most common microorganisms and frequently occurs in nature and the gastrointestinal tract of animals. E. coli is usually considered as an indicator organism for antimicrobial resistance (AMR) [7,17]. Research indicated that E. coli strains from giant pandas are highly resistant to antibiotics such as tetracyclines, sulfonamides and β-lactams [12,18,19]. Wang et al. showed that streptomycin was the most prevalent resistance phenotype and strB was the most prevalent ARG in E. coli strains from wild giant pandas, depicting an association between phenotype resistance and ARGs [19]. Our previous study proved a significant association between AMR and mobile genetic elements (MGEs) in the E. coli of captive giant pandas, while the associations of ARGs with AMR and MGEs are still unknown [12].
Previous researchers showed that an association existed between ARGs and VAGs in E. coli isolated from animals (yaks, weaned piglets and waterfowls) [20,21,22]. The uropathogenic E. coli (UPEC) isolated from patients with urinary tract infections (UTIs) showed antibiotic resistance related to particular VAGs [23]. However, the association between ARGs and VAGs in E. coli isolated from giant pandas is unknown.
With the implementation of the Wild Release Plan of captive giant pandas [24,25], AMR E. coli carrying ARGs/VAGs in giant pandas’ feces that are discharged into the environment may increase the risk of infections in animals and humans. To better understanding the characteristics of antibiotic-resistant E. coli, we detected ARGs and VAGs in 84 strains isolated from clinically heathy captive giant pandas and analyzed the associations of ARGs with AMR, VAGs and MGEs in this study.
2. Materials and Methods
2.1. Bacterial Strains
Eighty-four E. coli strains were isolated and identified from the fecal samples of 84 clinically healthy captive giant pandas and preserved in the Key Laboratory of Animal Disease and Human Health of Sichuan (Chengdu, China) [12].
2.2. Antibiotic Sensitivity and Resistance
Antibiotics for the antimicrobial susceptibility test were selected based on the information provided by breeders at Chengdu Giant Panda Breeding & Research Base (CGPBRB) and other reports about antimicrobial susceptibility in wildlife. Antibiotic susceptibility results (22 antibiotics in six categories) of all the E. coli strains have been reported in our previous publication [12]. The antibiotic susceptibility results were interpreted according to the CLSI criteria (Clinical and Laboratory Standards Institute, 2022).
2.3. DNA Extraction and Screening for ARGs and VAGs
Total genomic DNA was extracted from isolates using the TIANamp Bacteria DNA kit (Tiangen Biotech, Beijing, China), according to the manufacturer’s instructions. DNA samples were stored at −20 °C for subsequent PCR detection. We further amplified 16sRNA (16 S rRNA Primer: 5′-GAGTTTGATCCTGGCTCAG-3′; 5′-AGAAAGGAGGTGATCCAGCC-3′) [26] to confirm the quality of the extracted DNA before screening for related genes.
A total of 22 ARGs (six categories), 3 phylogenetic grouping associated genes (chuA, yjaA and TspE4.C2) and 17 VAGs (five categories) were screened by PCR. Phylogenetic groups (A, B1, B2 and D) were assigned as previously described by Clermont et al. [27]. Primers were synthesized by Huada Gene Technology Co., Ltd. (Shenzhen, China). The primer sequences and the amplification conditions are shown in Tables S1 and S2, as previously described [10,27,28,29,30,31]. 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) at 120 volts for 50 min, stained with GoldViewTM (Sangon Biotech, Shanghai, China) and photographed under ultraviolet light using a 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). The sequences were then analyzed online using BLAST (http://blast.ncbi.nlm.nih.gov, accessed on 24 September 2022).
2.4. Association Analysis between ARGs and AMR/VAGs/MGEs
A χ2 test was used to compare the association between ARGs and AMR/VAGs/MGEs. Eleven MGEs have been identified, as reported in our previous study [12]. p-values < 0.05 were considered to be statistically significant. Odds ratios (OR) and 95% confidence intervals were also determined for statistically significant associations. An OR < 1 and an OR > 1 represent negative and positive associations, respectively. Data analysis was performed with SPSS Statistics (version 26.0).
3. Results
3.1. Distribution of ARGs, VAGs and Phylogenetic Grouping
The patterns of the ARGs and VAGs carried by the 84 antibiotic-resistant E. coli strains isolated from captive giant pandas are shown in Table S3. A total of 14 ARGs were detected from 22 ARGs in six categories (amide alcohols, aminoglycosides, β-lactams, sulfonamide, tetracyclines and quinolones). As shown in Figure 1A, the detection rate of the β-lactam resistance gene blaTEM (100.00%, 84/84) was the highest, followed by the tetracycline resistance gene tetA (86.90%, 73/84) and the sulfonamide resistance gene sul3 (61.90%, 52/84). The detection rates for the other ARGs were lower than 50%: the amide alcohol resistance genes cmlA (48.81%, 41/84) and flor (47.62%, 40/84), the β-lactam resistance genes blaCTX-M (42.86%, 36/84) and blaSHV (10.71%, 9/84), the sulfonamide resistance genes sul1 (39.29%, 33/84) and sul2 (9.52%, 8/84), the quinolone resistance genes qnrS (36.90%, 31/84) and oqxAB (13.10%, 11/84), the tetracycline resistance genes tetC (29.76%, 25/84) and tetG (2.38%, 2/84) and the aminoglycosides resistance gene aacC2 (1.19%, 1/84). The remaining eight ARGs (aacC4, aadA1, aphA3, tetB, tetD, tetE, cat1 and qnrA) were not detected in any strains. A total of 9 VAGs out of 17 in four categories (adhesion-related genes, antiserum survival factor, invasion- and toxin-related genes and iron transport-related genes) were detected in 84 strains. As shown in Table 1 and Figure 1B, the highest detection rate was fimC (91.67%, 77/84), followed by ompT (63.10%, 53/84) and sitA (55.95%, 47/84). The detection rates of the other VAGs were iroN (44.05%, 37/84), fyuA (39.29%, 33/84), irp2 (38.10%, 32/84), eaeA (21.43%, 18/84), cvaC (4.76%, 4/84) and papA (2.38%, 2/84). The remaining eight VAGs (tsh, hlyA, hlyF, iss, astA, estA, estB and elt) were not detected in any strains. We further analyzed the distribution of VAGs related to diarrheagenic Escherichia coli (DEC) and extraintestinal pathogenic Escherichia coli (ExPEC). For the four VAGs (eaeA, elt, estA and estB) related to DEC, only eaeA (21.43%, 18/84) was detected in 18 strains. The detection rates of VAGs related to ExPEC (papA, fimC, fyuA, iroN, irp2, sitA, cvaC and ompT) ranged from 2.38% (papA, 2/84) to 91.67% (fimC, 77/84).
The phylogenetic grouping results showed that group B2 (67.86%, 57/84) was the most prevalent, followed by group A (16.67%, 14/84), group D (9.52%, 8/84) and group B1 (5.95%, 5/84) (Table S3). We further analyzed the number of VAGs in different phylogenetic groups. The average number of VAGs per isolate was 0.86 in group A and 2 in groups B1 and D, and the highest average number of VAGs per isolate was 4.65 in group B2.
3.2. Associations between ARGs and AMR
The distribution of resistance phenotypes and ARGs is shown in Table 2. For tetracyclines antibiotics, the resistance rates of tetracycline and doxycycline ranged from 48.81% (41/84) to 61.90% (52/84), and the detection rates of the tetracyclines resistance gene tet (including tetA, tetC and tetG) ranged from 2.38% (2/84) to 86.90% (73/84). For amide alcohols antibiotics, the detection rate of flor was 47.62% (40/84), and that of cmlA was 48.81% (41/84). The detection rate of amide alcohols antibiotics resistance genes was 21.43% for both CHL and FFC. Only one aminoglycoside-resistant gene, aacC2 (1.19%, 1/84), was detected in this study, and the proportions of strains with aminoglycoside-resistant phenotypes ranged from 3.57% (3/84) to 10.71% (9/84). We detected a high prevalence of the β-lactam antibiotic gene blaTEM (100%, 84/84), but only three β-lactams (AML, AMP, AMC) were found to have resistance rates higher than 46.43% (39/84). Only two of the four quinolone resistance genes were detected, and all the detection rates were below 36.90% (31/84). The resistance rates of the quinolones antibiotic ranged from 7.14% (6/84) to 10.71% (9/84). The detection rates of the three sulfonamide resistance genes ranged from 9.52% (8/84) to 61.90% (52/84), while the resistance rate of SXT was only 17.86% (15/84). The associations between ARGs and AMR were also analyzed (Table S4). A total of 88 pairs of significant associations (p < 0.05) were observed between ARGs and AMR, of which 86 pairs were positively associated. The strongest was observed for CEZ and qnrS (OR, 24.86; 95% CI, 2.98–205.71), followed by AMP and cmlA (OR, 22.43; 95% CI, 4.80–104.83). Negative associations were only observed between AMC and blaCTX-M/sul2.
3.3. Associations among 13 ARGs and among 9 VAGs
Table 3 summarizes the associations among 13 ARGs (except for blaTEM due to no results being available) detected in 84 E. coli strains from captive giant pandas. The results showed that a total of 30 pairs of ARGs were positively associated (OR > 1). Only one negative association was observed between sul3 and oqxAB (OR, 0.18; 95% CI, 0.05–0.76). The strongest positive association was detected between sul1 and tetC (OR, 133.33; 95% CI, 15.96–1113.64). Statistically significant associations (p < 0.05) were observed among nine VAGs (Table 4). A total of 14 pairs’ positive associations were detected among VAGs, while no negative associations were detected. The positive association between fyuA and iroN was observed as the strongest (OR, 294.40; 95% CI, 32.82–2640.87). No significant association was observed between fimC/papA/cvaC and other VAGs.
3.4. Association of ARGs with VAGs or MGEs
The association between ARGs and VAGs detected in E. coli strains isolated from captive giant pandas is shown in Table 5. The results showed that 45 pairs of ARGs were positively associated with VAGs. Only one negative association was observed for tetC and sitA (OR, 0.25; 95% CI, 0.09–0.64). The strongest positive association between ARGs and VAGs was observed in sul1 and eaeA (OR, 23.06; 95% CI, 4.80–110.85). There was no significant association between blaSHV/tetG/oqxAB/aacC2 and VAGs. Associations between ARGs and MGEs were further analyzed (Table S5). A total of 46 pairs showed significant positive associations (p < 0.05). The strongest association was observed between flor and intI1 (OR, 79.86; 95% CI, 9.90–643.30), followed by sul2 and IS26 (OR, 75.00; 95% CI, 6.73–835.97) and cmlA and intI1 (OR, 72.80; 95% CI, 9.07–584.18).
4. Discussion
Recently, antibiotic resistance E. coli as well as antibiotic resistance genes (ARGs) and virulence-associated genes (VAGs) carried by E. coli strains have been extensively studied as environmental pollutants, which may cause adverse effects to ecology, human and animal health [21,32,33,34]. To evaluate the potential threat of E. coli strains from captive giant pandas, the distribution and association of ARGs and VAGs in antibiotic-resistant E. coli were analyzed. Our results showed that many ARGs (especially β-lactam resistance genes, 100.00% for blaTEM) and VAGs (especially adhesion-related virulence genes, 91.67% for fimC) were detected from the 84 E. coli strains. Fourteen ARGs were detected, and the top five ARGs ranked by detection rates were blaTEM (100.00%), tetA (86.90%), sul3 (61.90%), cmlA (48.81%) and flor (47.62%). Another study from the Dujiangyan Giant Panda Conservation and Research Center and the Wolong Nature Reserve showed that the top five ARGs detected were sul1 (45%), blaCTX-M (44%), ant(3′)-Ia (38%), tetA (37%) and qnrB (35%) [35]. Those data showed a high prevalence of ARGs (especially for β-lactam, tetracyclines and sulfonamides resistance genes) in E. coli strains from giant pandas, and the carrying rate of the ARGs in the E. coli isolated in our study is higher than that in strains from Dujiangyan and Wolong. Notably, all of the 84 strains were positive for blaTEM in our study. E. coli strains containing blaTEM were reported in other wild animals (such as owls, foxes, wild rabbits, genets, etc.) [36]. Another study showed that 5 out of 13 ARGs were detected in E. coli isolated from wild giant pandas, but blaTEM was not detected [13]. The different detection rates of ARGs (especially for blaTEM) in captive and wild giant pandas may result from their different living environments [37]. The results from non-human primates have also shown that the carrying rates of ARGs in E. coli are affected by living environments [10,38]. As far as we know, this is the first report of a high prevalence of blaTEM detected in E. coli strains from captive giant pandas. Some studies showed that blaCTX-M and sul1 are prevalent in E. coli from captive giant pandas and wild birds [35,39]. The most prevalent ARGs in E. coli isolated from captive giant pandas in Dujiangyan and Wolong were blaCTX-M (44%) and sul1 (45%), which is consistent with our results (blaCTX-M was 42.86% and sul1 was 39.29%) [35]. E. coli carrying various ARGs was considered as an environmental pollutant, since the spread of antibiotic-resistant bacteria may pose a potential threat to human health [40,41,42]. Referring to the previous report on wild gulls, the antibiotic-resistant E. coli strains from giant pandas may serve as a pool of ARGs [43]. Moreover, VAGs related to diarrheagenic Escherichia coli (DEC) or extraintestinal pathogenic Escherichia coli (ExPEC) were screened [44,45,46]. In the 84 E. coli strains, 9 out of 17 VAGs were detected, of which the top 5 resistance genes were fimC (91.67%), ompT (63.10%), sitA (55.95%), iroN (44.05%) and fyuA (39.29%). None of the isolates were positive regarding tsh, hlyA, hlyF, iss, astA, estA, estB and elt. Only eaeA (which is a marker of enteropathogenic E. coli, EPEC) was detected in 18 strains. Most strains (91.67%, 77/84) carried at least one VAG related to ExPEC, which raises a health concern. Similarly, ExPEC-associated VAGs were observed in a higher proportion than those associated with DEC in E. coli from waterfowl and yaks [20,21]. VAGs related to a high-pathogenicity island (fyuA and irp2) were observed in 33 strains in our study. The detection rate of the VAGs in our study (ranging from 2.38% to 91.67%) is higher than the result of Wang et.al (all detection rates below 25%) [19]. Notably, the most prevalent VAG in our study is fimC, which belongs to adhesion-related virulence genes. Deng et al. showed that an E. coli strain from a urine sample of a giant panda carried the largest number (42.45%, 239/563) of adhesion- and invasion-related virulence genes [47]. Our present study showed E. coli strains from clinically healthy captive giant pandas carrying a large number of VAGs, which was also observed in healthy chickens and pigs [48,49]. As the phylogenetic grouping is closely related to virulence genes (group A and group B1 carry fewer virulence genes, while group B2 and group D carry more virulence genes [31]), we further analyzed the phylogenetic grouping and the number of virulence genes carried by each E. coli strain from giant pandas. Our results showed that the B2 group is the most prevalent group (67.86%, 57/84) which carries the highest number of virulence genes. Another study on E. coli strains of wild giant pandas showed that the B2 group was the most prevalent (38%, 31/82) [13], which is consistent with our study. In summary, our results showed that the antibiotic-resistant E. coli derived from giant pandas is a natural reservoir of ARGs and VAGs. In our previous study, we detected a high prevalence of MGEs in E. coli strains isolated from giant pandas [12]. Another study also proved that the release of wild animals may spread ARGs through MGEs [50]. With the release program of captive giant pandas being implemented in China [24,25], E. coli strains carrying various ARGs and VAGs may be harmful when spreading to the surrounding environment, breeders and tourists, posing a higher risk to public health.
In general, the antibiotic resistance phenotype is determined by the carrying and expression of ARGs [51,52,53]. ARGs carried by E. coli strains are closely related to the antibiotic resistance phenotype [54]. Thus, we further analyzed the relations between antibiotic resistance phenotypes and ARGs carried by E. coli from captive giant pandas. As shown in Table 2, for amide alcohols, aminoglycosides and quinolones, the antibiotic resistance phenotypes were all matched with the detection rates of ARGs. However, for tetracyclines, β-lactams and sulfonamides, the detection rates of related ARGs fluctuated (such as the resistance rate of sulfonamides being 17.86%, while the detection rate of sul ranged from 9.52% to 61.90%). The detection rates of ARGs are not strictly correlated with the corresponding antibiotic resistance phenotypes [10]. This might be due to the fact that the expression of ARGs is just one of the mechanisms that mediate bacterial resistance or due to the abnormal expression or low expression of ARGs that cannot cause the drug resistance [51,55,56]. Previous studies have shown an association between ARGs and VAGs carried by E. coli in wild waterfowls and weaned piglets [21,22]. We further analyzed the association among 13 ARGs and 9 VAGs, respectively. We observed 30 pairs of positive associations and 1 negative association among 13 ARGs, of which the strongest positive association (p < 0.05, OR > 1) was observed for sul1 and tetC. Similarly, positive associations among different ARGs were observed in another study on wild birds [21]. For the VAGs, 14 pairs of VAGs showed positive associations, and none of the VAGs pairs were negatively associated, which is consistent with the results of E. coli strains from Qinghai Plateau yaks [20]. The strongest positive association (p < 0.05, OR > 1) was observed for fyuA and iroN within VAGs. Associations among ARGs and VAGs in E. coli from captive giant pandas were further analyzed. Our results showed that positive associations were more common than negative ones (45 pairs were positive and only 1 was negative). The strongest positive association was observed for sul1 and eaeA between ARGs and VAGs. Interestingly, a study on waterfowls showed that negative associations were more common [21]. According to the available information, this is the first report about the association among ARGs and VAGs carried by E. coli from captive giant pandas. Previous studies have shown that specific microorganisms (such as endophytes or pathogenic E. coli) are enriched for ARGs [57,58,59], and significant associations exist between VAGs and AMR [60]. However, the direction of associations (positive or negative) among ARGs and VAGs is unknown. We speculate that carrying VAGs increases the probability of strains carrying ARGs, and vice versa. Considering that MGEs mediate the horizontal transmission of ARGs and that associations exist among MGEs and ARGs [61,62,63], we further analyzed the association between ARGs and MGEs. Only significant positive associations (p < 0.05, OR > 1) were observed between ARGs and MGEs. Positive associations for ARGs and MGEs were also reported in E. coli strains from wastewaters and activated sludge reactors [64,65]. Our present results showed that E. coli strains from captive giant pandas are a reservoir of ARGs, VAGs and MGEs; ARGs are significantly associated (p < 0.05) with VAGs and MGEs. As captive giant pandas are released into the wild, various ARGs, VAGs and MGEs carried by E. coli strains from captive giant pandas may have an impact on the environment.
5. Conclusions
The present study showed that antibiotic-resistant E. coli from captive giant pandas is a pool of ARGs, VAGs and MGEs, and significant associations exist among them. Positive associations are more prevalent than negative ones among ARGs and VAGs, and only positive associations were observed between ARGs and MGEs in E. coli from captive giant pandas. Antibiotic-resistant E. coli strains that carry various ARGs, VAGs and MGEs are a potential threat not only to the health of giant pandas but also to the environment. We strongly recommend implementing surveillance programs for the use of antibiotics in giant pandas and monitoring the impact of antibiotic-resistant E. coli (carrying various ARGs, VAGs and MGEs) on humans, other animals and the environment in future studies.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/vetsci9120705/s1, Table S1: PCR primer and conditions for the antibiotic resistance genes used in this study; Table S2: PCR primer and conditions for the virulence-associated genes used in this study; Table S3: Antibiotic resistance genes and virulence-associated genes pattern for Escherichia coli isolates from captive giant pandas; Table S4: Odds ratios (OR) of antibiotic resistance genes (ARGs) associated with antimicrobial resistance (AMR) in Escherichia coli isolates from captive giant pandas (n = 84); Table S5: Odds ratios (OR) of antibiotic resistance genes (ARGs) associated with mobile genetic elements (MGEs) in Escherichia coli isolates from captive giant pandas (n = 84).
Author Contributions
Conceptualization, Z.Z. (Zhijun Zhong), K.S., X.H. and G.P.; methodology, Z.Z. (Zhijun Zhong), K.S., X.H. and G.P.; software, Z.Z. (Zhijun Zhong), K.S., X.H. and G.P.; validation, Y.G., Y.Z. (Yanqiu Zhu), W.L. and Y.Z. (Yalin Zhong); formal analysis, Y.G., Y.Z. (Yanqiu Zhu), W.L. and Y.Z. (Yalin Zhong); investigation, S.Z., Y.L., Z.Z. (Ziyao Zhou), Z.R., X.S., Y.W., L.G. and X.M.; resources, Z.Z. (Zhijun Zhong); data curation, S.F., S.J., Z.Z. (Ziyao Zhou), H.L. and Z.Z. (Zhijun Zhong); writing—original draft preparation, S.F., S.J., Z.Z. (Ziqi Zhu), H.L. and Z.Z. (Zhijun Zhong); writing—review and editing, S.F., L.W., L.L. and Z.Z. (Zhijun Zhong); visualization, S.Z., Y.L., Z.Z. (Ziyao Zhou), Z.R., X.S., Y.W., L.G. and X.M.; supervision, S.C., L.S. and Z.Z. (Zhijun Zhong); project administration, Z.Z. (Zhijun Zhong). All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the National Key Research and Development Program of China (No. 2018YFD0500900, 2016YFD0501009) and the Chengdu Giant Panda Breeding Research Foundation (No. CPF2017-05, CPF2015-4).
Institutional Review Board Statement
The study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University under permit number DYY-2018203039. Prior to the collection of fecal specimens from captive giant pandas, permission was obtained from the Chengdu Giant Panda Breeding & Research Base in Sichuan, China.
Informed Consent Statement
Informed Consent Statement was obtained from the Chengdu Giant Panda Breeding & Research Base in Sichuan, China.
Data Availability Statement
The data presented in this study are available in the article/supplementary material; further inquiries can be directed to the corresponding authors.
Acknowledgments
We thank Junai Gan for the English language revision.
Conflicts of Interest
The authors declare no conflict of interest.
References
- Vila, J.; Sáez-López, E.; Johnson, J.R.; Römling, U.; Dobrindt, U.; Cantón, R.; Giske, C.G.; Naas, T.; Carattoli, A.; Martínez-Medina, M.; et al. Escherichia coli: An old friend with new tidings. FEMS Microbiol. Rev. 2016, 40, 437–463. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Foster-Nyarko, E.; Pallen, M.J. The microbial ecology of Escherichia coli in the vertebrate gut. FEMS Microbiol. Rev. 2022, 46, fuac008. [Google Scholar] [CrossRef] [PubMed]
- Arbab, S.; Ullah, H.; Wang, W.; Zhang, J. Antimicrobial drug resistance against Escherichia coli and its harmful effect on animal health. Vet. Med. Sci. 2022, 8, 1780–1786. [Google Scholar] [CrossRef]
- Pakbin, B.; Bruck, W.M.; Rossen, J.W.A. Virulence Factors of Enteric Pathogenic Escherichia coli: A Review. Int. J. Mol. Sci. 2021, 22, 9922. [Google Scholar] [CrossRef]
- Anand, U.; Reddy, B.; Singh, V.K.; Singh, A.K.; Kesari, K.K.; Tripathi, P.; Kumar, P.; Tripathi, V.; Simal-Gandara, J. Potential Environmental and Human Health Risks Caused by Antibiotic-Resistant Bacteria (ARB), Antibiotic Resistance Genes (ARGs) and Emerging Contaminants (ECs) from Municipal Solid Waste (MSW) Landfill. Antibiotics 2021, 10, 374. [Google Scholar] [CrossRef]
- Song, J.; Jongmans-Hochschulz, E.; Mauder, N.; Imirzalioglu, C.; Wichels, A.; Gerdts, G. The Travelling Particles: Investigating microplastics as possible transport vectors for multidrug resistant E. coli in the Weser estuary (Germany). Sci. Total Environ. 2020, 720, 137603. [Google Scholar] [CrossRef] [PubMed]
- Zhang, S.; Abbas, M.; Rehman, M.U.; Huang, Y.; Zhou, R.; Gong, S.; Yang, H.; Chen, S.; Wang, M.; Cheng, A. Dissemination of antibiotic resistance genes (ARGs) via integrons in Escherichia coli: A risk to human health. Environ. Pollut. 2020, 266, 115260. [Google Scholar] [CrossRef] [PubMed]
- Feng, G.; Huang, H.; Chen, Y. Effects of emerging pollutants on the occurrence and transfer of antibiotic resistance genes: A review. J. Hazard. Mater. 2021, 420, 126602. [Google Scholar] [CrossRef]
- Li, L.G.; Huang, Q.; Yin, X.; Zhang, T. Source tracking of antibiotic resistance genes in the environment—Challenges, progress, and prospects. Water Res. 2020, 185, 116127. [Google Scholar] [CrossRef]
- Zhu, Z.; Jiang, S.; Qi, M.; Liu, H.; Zhang, S.; Liu, H.; Zhou, Z.; Wang, L.; Wang, C.; Luo, Y.; et al. Prevalence and characterization of antibiotic resistance genes and integrons in Escherichia coli isolates from captive non-human primates of 13 zoos in China. Sci. Total Environ. 2021, 798, 149268. [Google Scholar] [CrossRef]
- Larsson, D.G.J.; Flach, C.F. Antibiotic resistance in the environment. Nat. Rev. Microbiol. 2022, 20, 257–269. [Google Scholar] [CrossRef] [PubMed]
- Zhu, Z.; Pan, S.; Wei, B.; Liu, H.; Zhou, Z.; Huang, X.; Luo, Y.; Zhou, L.; Zhang, S.; Ma, X.; et al. High prevalence of multi-drug resistances and diversity of mobile genetic elements in Escherichia coli isolates from captive giant pandas. Ecotoxicol. Environ. Saf. 2020, 198, 110681. [Google Scholar] [CrossRef] [PubMed]
- Chen, D.; Zou, W.; Xie, S.; Kong, L.; Chen, Y.; Zhang, X.; Li, J.; Wang, H.; Cheng, G.; Qin, Y.; et al. Serotype and Antimicrobial Resistance of Escherichia coli Isolated from Feces of Wild Giant Pandas (Ailuropoda melanoleuca) in Sichuan Province, China. J. Wildl. Dis. 2018, 54, 691–699. [Google Scholar] [CrossRef]
- Dolejska, M.; Papagiannitsis, C.C. Plasmid-mediated resistance is going wild. Plasmid 2018, 99, 99–111. [Google Scholar] [CrossRef] [PubMed]
- Guyomard-Rabenirina, S.; Reynaud, Y.; Pot, M.; Albina, E.; Couvin, D.; Ducat, C.; Gruel, G.; Ferdinand, S.; Legreneur, P.; Le Hello, S.; et al. Antimicrobial Resistance in Wildlife in Guadeloupe (French West Indies): Distribution of a Single bla (CTX-M-1)/IncI1/ST3 Plasmid Among Humans and Wild Animals. Front. Microbiol. 2020, 11, 1524. [Google Scholar] [CrossRef]
- Bungau, S.; Tit, D.M.; Behl, T.; Aleya, L.; Zaha, D.C. Aspects of excessive antibiotic consumption and environmental influences correlated with the occurrence of resistance to antimicrobial agents. Curr. Opin. Environ. Sci. Health 2020, 19, 100224. [Google Scholar] [CrossRef]
- Chandler, J.C.; Aljasir, S.F.; Hamidi, A.; Sylejmani, D.; Gerow, K.G.; Bisha, B. Short communication: A countrywide survey of antimicrobial-resistant indicator bacteria in Kosovo’s dairy farms. J. Dairy Sci. 2018, 101, 6982–6989. [Google Scholar] [CrossRef] [Green Version]
- Guo, L.; Long, M.; Huang, Y.; Wu, G.; Deng, W.; Yang, X.; Li, B.; Meng, Y.; Cheng, L.; Fan, L.; et al. Antimicrobial and disinfectant resistance of Escherichia coli isolated from giant pandas. J. Appl. Microbiol. 2015, 119, 55–64. [Google Scholar] [CrossRef]
- Wang, X.; Yan, Q.; Xia, X.; Zhang, Y.; Li, D.; Wang, C.; Chen, S.; Hou, R. Serotypes, virulence factors, and antimicrobial susceptibilities of vaginal and fecal isolates of Escherichia coli from giant pandas. Appl. Environ. Microbiol. 2013, 79, 5146–5150. [Google Scholar] [CrossRef] [Green Version]
- Rehman, M.U.; Zhang, H.; Iqbal, M.K.; Mehmood, K.; Huang, S.; Nabi, F.; Luo, H.; Lan, Y.; Li, J. Antibiotic resistance, serogroups, virulence genes, and phylogenetic groups of Escherichia coli isolated from yaks with diarrhea in Qinghai Plateau, China. Gut Pathog. 2017, 9, 24. [Google Scholar] [CrossRef]
- Zhang, S.; Chen, S.; Rehman, M.U.; Yang, H.; Yang, Z.; Wang, M.; Jia, R.; Chen, S.; Liu, M.; Zhu, D.; et al. Distribution and association of antimicrobial resistance and virulence traits in Escherichia coli isolates from healthy waterfowls in Hainan, China. Ecotoxicol. Environ. Saf. 2021, 220, 112317. [Google Scholar] [CrossRef] [PubMed]
- Do, K.H.; Seo, K.W.; Byun, J.W.; Lee, W.K. Relationship between virulence factors and antimicrobial resistance genes of pathogenic Escherichia coli from diarrheic weaned piglets. Res. Vet. Sci. 2022, 150, 137–143. [Google Scholar] [CrossRef]
- Malekzadegan, Y.; Khashei, R.; Sedigh Ebrahim-Saraie, H.; Jahanabadi, Z. Distribution of virulence genes and their association with antimicrobial resistance among uropathogenic Escherichia coli isolates from Iranian patients. BMC Infect. Dis. 2018, 18, 572. [Google Scholar] [CrossRef] [Green Version]
- Dai, Q.L.; Li, J.W.; Yang, Y.; Li, M.; Zhang, K.; He, L.Y.; Zhang, J.; Tang, B.; Liu, H.P.; Li, Y.X.; et al. Genetic Diversity and Prediction Analysis of Small Isolated Giant Panda Populations After Release of Individuals. Evol. Bioinform. Online 2020, 16, 1176934320939945. [Google Scholar] [CrossRef] [PubMed]
- Kang, D.; Li, J. Role of nature reserves in giant panda protection. Environ. Sci. Pollut. Res. Int. 2018, 25, 4474–4478. [Google Scholar] [CrossRef]
- Seghal Kiran, G.; Anto Thomas, T.; Selvin, J.; Sabarathnam, B.; Lipton, A.P. Optimization and characterization of a new lipopeptide biosurfactant produced by marine Brevibacterium aureum MSA13 in solid state culture. Bioresour. Technol. 2010, 101, 2389–2396. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Vale, A.P.; Shubin, L.; Cummins, J.; Leonard, F.C.; Barry, G. Detection of bla (OXA-1), bla (TEM-1), and Virulence Factors in E. coli Isolated From Seals. Front. Vet. Sci. 2021, 8, 583759. [Google Scholar] [CrossRef] [PubMed]
- Paixão, A.C.; Ferreira, A.C.; Fontes, M.; Themudo, P.; Albuquerque, T.; Soares, M.C.; Fevereiro, M.; Martins, L.; de Sá, M.I.C. Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates. Poult. Sci. 2016, 95, 1646–1652. [Google Scholar] [CrossRef]
- Fujimoto, Y.; Inoue, H.; Kanda, T.; Ijiri, M.; Uemura, R. Virulence-Associated Gene Profiles of Escherichia coli Isolated from Chickens with Colibacillosis in Japan and Their Correlation with Pathogenicity in Chicken Embryos. Avian Dis. 2021, 65, 401–405. [Google Scholar] [CrossRef]
- Algammal, A.M.; Hetta, H.F.; Batiha, G.E.; Hozzein, W.N.; El Kazzaz, W.M.; Hashem, H.R.; Tawfik, A.M.; El-Tarabili, R.M. Virulence-determinants and antibiotic-resistance genes of MDR-E. coli isolated from secondary infections following FMD-outbreak in cattle. Sci. Rep. 2020, 10, 19779. [Google Scholar] [CrossRef] [PubMed]
- Clermont, O.; Bonacorsi, S.; Bingen, E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 2000, 66, 4555–4558. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Abia, A.L.; Schaefer, L.; Ubomba-Jaswa, E.; Le Roux, W. Abundance of Pathogenic Escherichia coli Virulence-Associated Genes in Well and Borehole Water Used for Domestic Purposes in a Peri-Urban Community of South Africa. Int. J. Environ. Res. Public Health 2017, 14, 320. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Badura, A.; Luxner, J.; Feierl, G.; Reinthaler, F.F.; Zarfel, G.; Galler, H.; Pregartner, G.; Riedl, R.; Grisold, A.J. Prevalence, antibiotic resistance patterns and molecular characterization of Escherichia coli from Austrian sandpits. Environ. Pollut. 2014, 194, 24–30. [Google Scholar] [CrossRef]
- Di Cesare, A.; Pinnell, L.J.; Brambilla, D.; Elli, G.; Sabatino, R.; Sathicq, M.B.; Corno, G.; O'Donnell, C.; Turner, J.W. Bioplastic accumulates antibiotic and metal resistance genes in coastal marine sediments. Environ. Pollut. 2021, 291, 118161. [Google Scholar] [CrossRef] [PubMed]
- Zou, W.; Li, C.; Yang, X.; Wang, Y.; Cheng, G.; Zeng, J.; Zhang, X.; Chen, Y.; Cai, R.; Huang, Q.; et al. Frequency of antimicrobial resistance and integron gene cassettes in Escherichia coli isolated from giant pandas (Ailuropoda melanoleuca) in China. Microb Pathog. 2018, 116, 173–179. [Google Scholar] [CrossRef] [PubMed]
- Costa, D.; Poeta, P.; Sáenz, Y.; Vinué, L.; Coelho, A.C.; Matos, M.; Rojo-Bezares, B.; Rodrigues, J.; Torres, C. Mechanisms of antibiotic resistance in Escherichia coli isolates recovered from wild animals. Microb Drug Resist. 2008, 14, 71–77. [Google Scholar] [CrossRef]
- Swift, B.M.C.; Bennett, M.; Waller, K.; Dodd, C.; Murray, A.; Gomes, R.L.; Humphreys, B.; Hobman, J.L.; Jones, M.A.; Whitlock, S.E.; et al. Anthropogenic environmental drivers of antimicrobial resistance in wildlife. Sci. Total Environ. 2019, 649, 12–20. [Google Scholar] [CrossRef]
- Sacristán, I.; Esperón, F.; Acuña, F.; Aguilar, E.; García, S.; López, M.J.; Cevidanes, A.; Neves, E.; Cabello, J.; Hidalgo-Hermoso, E.; et al. Antibiotic resistance genes as landscape anthropization indicators: Using a wild felid as sentinel in Chile. Sci. Total Environ. 2020, 703, 134900. [Google Scholar] [CrossRef]
- Yuan, Y.; Liang, B.; Jiang, B.W.; Zhu, L.W.; Wang, T.C.; Li, Y.G.; Liu, J.; Guo, X.J.; Ji, X.; Sun, Y. Migratory wild birds carrying multidrug-resistant Escherichia coli as potential transmitters of antimicrobial resistance in China. PLoS ONE 2021, 16, e0261444. [Google Scholar] [CrossRef]
- Chen, H.; Bai, X.; Jing, L.; Chen, R.; Teng, Y. Characterization of antibiotic resistance genes in the sediments of an urban river revealed by comparative metagenomics analysis. Sci. Total Environ. 2019, 653, 1513–1521. [Google Scholar] [CrossRef]
- Huijbers, P.M.; Graat, E.A.; Haenen, A.P.; van Santen, M.G.; van Essen-Zandbergen, A.; Mevius, D.J.; van Duijkeren, E.; van Hoek, A.H. Extended-spectrum and AmpC β-lactamase-producing Escherichia coli in broilers and people living and/or working on broiler farms: Prevalence, risk factors and molecular characteristics. J. Antimicrob Chemother. 2014, 69, 2669–2675. [Google Scholar] [CrossRef]
- Nnadozie, C.F.; Odume, O.N. Freshwater environments as reservoirs of antibiotic resistant bacteria and their role in the dissemination of antibiotic resistance genes. Environ. Pollut. 2019, 254, 113067. [Google Scholar] [CrossRef] [PubMed]
- Atterby, C.; Börjesson, S.; Ny, S.; Järhult, J.D.; Byfors, S.; Bonnedahl, J. ESBL-producing Escherichia coli in Swedish gulls-A case of environmental pollution from humans? PLoS ONE 2017, 12, e0190380. [Google Scholar] [CrossRef] [PubMed]
- Bujňáková, D.; Karahutová, L.; Kmeť, V. Escherichia coli Specific Virulence-Gene Markers Analysis for Quality Control of Ovine Cheese in Slovakia. Microorganisms 2021, 9, 1808. [Google Scholar] [CrossRef] [PubMed]
- Rybak, B.; Krawczyk, B.; Furmanek-Blaszk, B.; Wysocka, M.; Fordon, M.; Ziolkowski, P.; Meissner, W.; Stepniewska, K.; Sikorska, K. Antibiotic resistance, virulence, and phylogenetic analysis of Escherichia coli strains isolated from free-living birds in human habitats. PLoS ONE 2022, 17, e0262236. [Google Scholar] [CrossRef] [PubMed]
- Yu, Y.; Hu, B.; Fan, H.; Zhang, H.; Lian, S.; Li, H.; Li, S.; Yan, X.; Wang, S.; Bai, X. Molecular Epidemiology of Extraintestinal Pathogenic Escherichia coli Causing Hemorrhagic Pneumonia in Mink in Northern China. Front. Cell Infect. Microbiol. 2021, 11, 781068. [Google Scholar] [CrossRef] [PubMed]
- Deng, W.; Li, C.; Zhao, S.; Li, R.; He, Y.; Wu, D.; Yang, S.; Huang, Y.; Zhang, H.; Zou, L. Whole genome sequencing reveals the distribution of resistance and virulence genes of pathogenic Escherichia coli CCHTP from giant panda (in Chinese with English abstract). Hereditas 2019, 41, 1138–1147. [Google Scholar] [CrossRef]
- Messaili, C.; Messai, Y.; Bakour, R. Virulence gene profiles, antimicrobial resistance and phylogenetic groups of fecal Escherichia coli strains isolated from broiler chickens in Algeria. Vet. Ital. 2019, 55, 35–46. [Google Scholar] [CrossRef]
- Hernández-Fillor, R.E.; Brilhante, M.; Marrero-Moreno, C.M.; Baez, M.; Espinosa, I.; Perreten, V. Characterization of Third-Generation Cephalosporin-Resistant Escherichia coli Isolated from Pigs in Cuba Using Next-Generation Sequencing. Microb. Drug Resist. 2021, 27, 1003–1010. [Google Scholar] [CrossRef] [PubMed]
- Power, M.L.; Emery, S.; Gillings, M.R. Into the wild: Dissemination of antibiotic resistance determinants via a species recovery program. PLoS ONE 2013, 8, e63017. [Google Scholar] [CrossRef] [PubMed]
- James, J.; Yarnall, B.; Koranteng, A.; Gibson, J.; Rahman, T.; Doyle, D.A. Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance. Microb. Cell Fact 2021, 20, 13. [Google Scholar] [CrossRef] [PubMed]
- Ma, T.; McAllister, T.A.; Guan, L.L. A review of the resistome within the digestive tract of livestock. J. Anim. Sci. Biotechnol. 2021, 12, 121. [Google Scholar] [CrossRef] [PubMed]
- Van Camp, P.J.; Haslam, D.B.; Porollo, A. Bioinformatics Approaches to the Understanding of Molecular Mechanisms in Antimicrobial Resistance. Int. J. Mol. Sci. 2020, 21, 1363. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Suarez, S.A.; Martiny, A.C. Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli. Microbiol. Spectr. 2021, 9, e0028921. [Google Scholar] [CrossRef]
- Munita, J.M.; Arias, C.A. Mechanisms of Antibiotic Resistance. Microbiol. Spectr. 2016, 4. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Srinivasan, V.; Gillespie, B.E.; Lewis, M.J.; Nguyen, L.T.; Headrick, S.I.; Schukken, Y.H.; Oliver, S.P. Phenotypic and genotypic antimicrobial resistance patterns of Escherichia coli isolated from dairy cows with mastitis. Vet. Microbiol. 2007, 124, 319–328. [Google Scholar] [CrossRef] [PubMed]
- Shabana, I.I.; Al-Enazi, A.T. Investigation of plasmid-mediated resistance in E. coli isolated from healthy and diarrheic sheep and goats. Saudi J. Biol. Sci. 2020, 27, 788–796. [Google Scholar] [CrossRef] [PubMed]
- Wang, F.H.; Qiao, M.; Chen, Z.; Su, J.Q.; Zhu, Y.G. Antibiotic resistance genes in manure-amended soil and vegetables at harvest. J. Hazard. Mater. 2015, 299, 215–221. [Google Scholar] [CrossRef]
- Cerqueira, F.; Matamoros, V.; Bayona, J.; Elsinga, G.; Hornstra, L.M.; Piña, B. Distribution of antibiotic resistance genes in soils and crops. A field study in legume plants (Vicia faba L.) grown under different watering regimes. Environ. Res. 2019, 170, 16–25. [Google Scholar] [CrossRef] [Green Version]
- Zhao, F.; Yang, H.; Bi, D.; Khaledi, A.; Qiao, M. A systematic review and meta-analysis of antibiotic resistance patterns, and the correlation between biofilm formation with virulence factors in uropathogenic E. coli isolated from urinary tract infections. Microb Pathog. 2020, 144, 104196. [Google Scholar] [CrossRef]
- Babakhani, S.; Oloomi, M. Transposons: The agents of antibiotic resistance in bacteria. J. Basic Microbiol. 2018, 58, 905–917. [Google Scholar] [CrossRef] [PubMed]
- Stevenson, C.; Hall, J.P.; Harrison, E.; Wood, A.; Brockhurst, M.A. Gene mobility promotes the spread of resistance in bacterial populations. ISME J. 2017, 11, 1930–1932. [Google Scholar] [CrossRef] [Green Version]
- Zhang, R.; Gu, J.; Wang, X.; Li, Y. Antibiotic resistance gene transfer during anaerobic digestion with added copper: Important roles of mobile genetic elements. Sci. Total Environ. 2020, 743, 140759. [Google Scholar] [CrossRef] [PubMed]
- Kayali, O.; Icgen, B. intI1 Type Mobile Genetic Elements Co-selected Antibiotic-Resistant Genes in Untreated Hospital Wastewaters. Bull. Environ. Contam. Toxicol. 2021, 106, 399–405. [Google Scholar] [CrossRef] [PubMed]
- Zhao, R.; Feng, J.; Huang, J.; Li, X.; Li, B. Reponses of microbial community and antibiotic resistance genes to the selection pressures of ampicillin, cephalexin and chloramphenicol in activated sludge reactors. Sci. Total Environ. 2021, 755, 142632. [Google Scholar] [CrossRef]
Figure 1.
Heat map demonstrating the distribution of antibiotic resistance genes (ARGs) and virulence-associated genes (VAGs) in Escherichia coli (E. coli) isolated from captive giant pandas in Sichuan, China. The color scale and the corresponding value indicate the number of E. coli isolates carrying corresponding abscissa and ordinate genes. The prevalence of ARGs (A), VAGs (B), ARGs and VAGs (C) in E. coli isolates. blaTEM, tetA and fimC were the highly prevalent genes among all tested ARGs and VAGs.
Figure 1.
Heat map demonstrating the distribution of antibiotic resistance genes (ARGs) and virulence-associated genes (VAGs) in Escherichia coli (E. coli) isolated from captive giant pandas in Sichuan, China. The color scale and the corresponding value indicate the number of E. coli isolates carrying corresponding abscissa and ordinate genes. The prevalence of ARGs (A), VAGs (B), ARGs and VAGs (C) in E. coli isolates. blaTEM, tetA and fimC were the highly prevalent genes among all tested ARGs and VAGs.
Table 1.
Distribution of virulence-associated genes detected in E. coli isolated from captive giant pandas (n = 84).
Table 1.
Distribution of virulence-associated genes detected in E. coli isolated from captive giant pandas (n = 84).
| Category of Virulence | Genes | No. of Positive Isolates | Percentage of Positive Isolates (%) |
|---|---|---|---|
| Adhesion-related genes | tsh | 0 | 0.00 |
| eaeA | 18 | 21.43 | |
| papA | 2 | 2.38 | |
| fimC | 77 | 91.67 | |
| Hemolysin-related genes | hlyA | 0 | 0.00 |
| hlyF | 0 | 0.00 | |
| Invasion- and toxin-related genes | elt | 0 | 0.00 |
| estA | 0 | 0.00 | |
| estB | 0 | 0.00 | |
| astA | 0 | 0.00 | |
| Iron transport-related genes | fyuA | 33 | 39.29 |
| iroN | 37 | 44.05 | |
| irp2 | 32 | 38.10 | |
| sitA | 47 | 55.95 | |
| Antiserum survival factor | cvaC | 4 | 4.76 |
| ompT | 53 | 63.10 | |
| iss | 0 | 0.00 |
Table 2.
Distribution of antibiotic resistance phenotypes and antibiotic resistance genes detected in E. coli strains isolated from captive giant pandas (n = 84).
Table 2.
Distribution of antibiotic resistance phenotypes and antibiotic resistance genes detected in E. coli strains isolated from captive giant pandas (n = 84).
| Category of Antibiotic | Antibiotic | Results of the Antibiotic Sensitivity Test | Antibiotic Resistance Genes | No. of Positive Isolates (%) | ||
|---|---|---|---|---|---|---|
| No. of Resistant Ones (%) | No. of Intermediate Ones (%) | No. of Sensitive Ones (%) | ||||
| Tetracyclines | DOX | 52 (61.90) | 20 (23.81) | 12 (14.29) | tetA | 73 (86.90) |
| tetB | - | |||||
| tetC | 25 (29.76) | |||||
| TET | 41 (48.81) | 34 (40.48) | 9 (10.71) | tetD | - | |
| tetE | - | |||||
| tetG | 2 (2.38) | |||||
| Amide alcohols | CHL | 18 (21.43) | 10 (11.90) | 56 (66.67) | flor | 40 (47.62) |
| FFC | 18 (21.43) | 3 (3.57) | 63 (75.00) | cmlA | 41 (48.81) | |
| Aminoglycosides | AMK | 3 (3.57) | 4 (4.76) | 78 (92.86) | aacC2 aacC4 aadA1 aphA3 | 1 (1.19) - - - |
| KAN | 6 (7.14) | 4 (4.76) | 74 (88.10) | |||
| STR | 9 (10.71) | 4 (4.76) | 71 (84.52) | |||
| NEO | 3 (3.57) | 6 (7.14) | 75 (89.29) | |||
| GM | 5 (5.95) | 0 (0.00) | 79 (94.05) | |||
| β-lactams | AML | 39 (46.43) | 35 (41.67) | 10 (11.90) | blaCTX-M blaTEM blaSHV | 36 (42.86) 84 (100.00) 9 (10.71) |
| AMP | 58 (69.05) | 21 (25.00) | 5 (5.95) | |||
| SAM | 1(1.19) | 6 (7.14) | 77 (91.67) | |||
| AMC | 68 (80.95) | 7 (8.33) | 9 (10.71) | |||
| AZM | 6 (7.14) | 7 (7.14) | 73(86.90) | |||
| CRO | 6 (7.14) | 3 (3.57) | 75 (89.29) | |||
| CTX | 8 (9.52) | 9 (10.71) | 67 (79.76) | |||
| CEZ | 7 (8.33) | 4 (4.76) | 73 (86.90) | |||
| Quinolones | CIP | 6 (7.14) | 2 (2.38) | 76 (90.48) | qnrA | - |
| NA | 9 (10.71) | 1 (1.19) | 74 (88.10) | qnrS | 31 (36.90) | |
| NOR | 7 (8.33) | 0 (0.00) | 77 (91.67) | cat1 | - | |
| OFX | 6 (7.14) | 1 (1.19) | 77 (91.67) | oqxAB | 11 (13.10) | |
| Sulfonamides | SXT | 15 (17.86) | 0 (0.00) | 69 (82.14) | sul1 | 33 (39.29) |
| sul2 | 8 (9.52) | |||||
| sul3 | 52 (61.90) | |||||
Note: DOX, Doxycycline; TET, Tetracycline; CHL, Chloramphenicol; FFC, Florfenicol; AMK, Amikacin; KAN, Kanamycin; STR, Streptomycin; NEO, Neomycin; GM, Gentamicin; AML, Amoxicillin; AMP, Ampicillin; SAM, Ampicillin/Sulbactam; AMC, Amoxicillin/Clavulanic Acid; AZM, Aztreonam; CRO, Ceftriaxone; CTX, Cefotaxime; CEZ, Cefazolin; CIP, Ciprofloxacin; NA, Nalidixic Acid; NOR, Norfloxacin; OFX, Ofloxacin; SXT, Trimethoprim-Sulfamethoxazole. - indicates a negative detection.
Table 3.
Odds ratios (OR) of antibiotic resistance genes in E. coli isolates from captive giant pandas (n = 84).
Table 3.
Odds ratios (OR) of antibiotic resistance genes in E. coli isolates from captive giant pandas (n = 84).
| Gene (No.) | OR (95% Confidence Interval) | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| blaCTX-M (36) | blaSHV (9) | tetA (73) | tetC (25) | tetG (2) | sul3 (52) | sul1 (33) | sul2 (8) | qnrS (31) | flor (40) | oqxAB (11) | cmlA (41) | aacC2 (1) | |
| blaCTX-M (36) | NS | - | - | 15.40 (4.55–52.12) | - | 4.50 (1.66–12.25) | 45.57 (12.24–169.72) | 11.35 (1.33–96.99) | 6.81 (2.534–18.27) | 5.00 (1.96–12.74) | - | 5.72 (2.21–14.81) | - |
| blaSHV (9) | - | NS | - | - | - | - | - | 7.00 (1.34–36.69) | - | - | 14.38 (3.03–68.21) | - | - |
| tetA (73) | - | - | NS | - | - | 23.18 (2.80–192.27) | 7.81 (0.95–64.18) | - | NA | 11.47 (1.40–94.28) | - | NA | - |
| tetC (25) | 15.40 (4.55–52.12) | - | - | NS | - | 11.90 (2.57–55.07) | 133.33 (15.96–1113.64) | 22.56 (2.60–195.77) | 40.74 (10.05–165.14) | 28.41 (6.03–133.98) | - | 15.44 (4.11–58.03) | - |
| tetG (2) | - | - | - | - | NS | - | - | - | - | - | - | - | - |
| sul3 (52) | 4.50 (1.66–12.25) | - | 7.81 (0.95–64.18) | 11.90 (2.57–55.07) | - | NS | 8.83 (2.71–28.78) | - | 42.273 (5.36–333.64) | 40.714 (8.58–193.17) | 0.18 (0.05–0.76) | 103.33 (12.74–838.13) | - |
| sul1 (33) | 45.57 (12.24–169.72) | - | 7.81 (0.95–64.18) | 133.33 (15.96–1113.64) | - | 8.83 (2.71–28.78) | NS | 13.46 (1.57–115.36) | 12.36 (4.29–35.63) | 13.15 (4.44–38.96) | - | 8.91 (3.19–24.95) | - |
| sul2 (8) | 11.35 (1.33–96.99) | 7.00 (1.34–36.69) | - | 22.56 (2.60–195.77) | - | - | 13.46 (1.57–115.36) | NS | 15.17 (1.77–130.25) | NA | - | 8.65 (1.01–73.75) | - |
| qnrS (31) | 6.81 (2.534–18.27) | - | NA | 40.74 (10.05–165.14) | - | 42.273 (5.36–333.64) | 12.36 (4.29–35.63) | 15.17 (1.77–130.25) | NS | 55.36 (11.41–168.57) | - | NA | - |
| flor (40) | 5.00 (1.96–12.74) | - | 11.47 (1.40–94.28) | 28.41 (6.03–133.98) | - | 40.714 (8.58–193.17) | 13.15 (4.44–38.96) | NA | 55.36 (11.41–168.57) | NS | - | 70.20 (17.47–282.02) | - |
| oqxAB (11) | - | 14.38 (3.03–68.21) | - | - | - | 0.18 (0.05–0.76) | - | - | - | - | NS | - | - |
| cmlA (41) | 5.72 (2.21–14.81) | - | NA | 15.44 (4.11–58.03) | - | 103.33 (12.74–838.13) | 8.91 (3.19–24.95) | 8.65 (1.01–73.75) | NA | 70.20 (17.47–282.02) | - | NS | - |
| aacC2 (1) | - | - | - | - | - | - | - | - | - | - | - | - | NS |
Note: Only antibiotic resistance genes with a significant association (p < 0.05) are shown. OR, odds ratio for significant associations between antibiotic resistance genes (95% confidence interval in parentheses); NA, no results available or they could not be calculated; - indicates no significant associations (p ≥ 0.05); NS, no statistics were determined for the same gene.
Table 4.
Odds ratios (OR) of virulence-associated genes in E. coli isolates from captive giant pandas (n = 84).
Table 4.
Odds ratios (OR) of virulence-associated genes in E. coli isolates from captive giant pandas (n = 84).
| Gene (No.) | OR (95% Confidence Interval) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| fimC (77) | papA (2) | fyuA (33) | irp2 (32) | eaeA (18) | iroN (37) | sitA (47) | ompT (53) | cvaC (4) | |
| fimC (77) | NS | - | - | - | - | NA | - | - | - |
| papA (2) | - | NS | - | - | - | - | - | - | - |
| fyuA (33) | - | - | NS | NA | 4.29 (1.42–12.98) | 294.40 (32.82–2640.87) | 76.80 (9.60–614.43) | 20.43 (4.41–94.69) | NA |
| irp2 (32) | - | - | NA | NS | 4.60 (1.51–13.98) | 237.67 (27.26–2072.08) | 69.75 (8.74–556.40) | 18.91 (4.09–87.55) | NA |
| eaeA (18) | - | - | 4.29 (1.4215–12.98) | 4.60 (1.51–13.98) | NS | 4.55 (1.45–14.33) | 20.40 (2.56–162.34) | 14.17 (1.78–112.74) | - |
| iroN (37) | NA | - | 294.40 (32.82–2640.87) | 237.67 (27.26–2072.08) | 4.55 (1.45–14.33) | NS | 29.64 (7.74–113.47) | 16.70 (4.48–62.30) | - |
| sitA (47) | - | - | 76.80 (9.60–614.43) | 69.75 (8.74–556.40) | 20.40 (2.56–162.34) | 29.6 (7.743–113.47) | NS | 19.86 (6.19–63.64) | - |
| ompT (53) | - | - | 20.43 (4.41–94.69) | 18.91 (4.09–87.55) | 14.17 (1.78–112.74) | 16.70 (4.48–62.30) | 19.86 (6.19–63.64) | NS | - |
| cvaC (4) | - | - | NA | NA | - | - | - | - | NS |
Note: Only virulence-associated genes with a significant association (p < 0.05) are shown. OR, odds ratio for significant associations between virulence-associated genes (95% confidence interval in parentheses); the meaning of NA/NS/- is the same as that in Table 3.
Table 5.
Odds ratios (OR) of antibiotic resistance genes associated with virulence-associated genes in E. coli isolates from captive giant pandas (n = 84).
Table 5.
Odds ratios (OR) of antibiotic resistance genes associated with virulence-associated genes in E. coli isolates from captive giant pandas (n = 84).
| Gene (No.) | OR (95% Confidence Interval) | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| blaCTX-M (36) | tetA (73) | tetC (25) | sul3 (52) | sul1 (33) | sul2 (8) | qnrS (31) | flor (40) | cmlA (41) | |
| fyuA (33) | 8.67 (3.18–23.62) | - | 14.15 (4.45–45.03) | 6.30 (2.10–18.91) | 12.44 (4.35–35.61) | 13.46 (1.57–115.36) | 7.18 (2.67–19.32) | 13.15 (4.44–38.96) | 6.84 (2.54–18.43) |
| irp2 (32) | 10.00 (3.58–27.95) | - | 15.57 (4.88–50.33) | 5.83 (1.94–17.49) | 14.33 (4.89–42.01) | 14.28 (1.67–122.50) | 8.02 (2.94–21.88) | 11.76 (4.00–34.59) | 6.18 (2.30–16.59) |
| eaeA (18) | 7.00 (2.06–23.79) | - | 11.70 (3.50–39.09) | 3.92 (1.04–14.84) | 23.06 (4.80–110.85) | - | 10.09 (2.92–34.88) | 8.20 (2.16–31.18) | - |
| iroN (37) | 6.82 (2.60–17.89) | - | 14.11 (4.19–47.48) | 4.87 (1.79–13.27) | 13.51 (4.64–39.20) | 10.73 (1.26–91.70) | 8.01 (2.92–21.99) | 9.07 (3.35–24.59) | 8.14 (3.04–21.81) |
| sitA (47) | 6.91 (2.51–18.98) | 7.23 (1.46–35.93) | 0.25 (0.09–0.64) | 6.66 (2.04–21.82) | 3.43 (1.37–8.62) | - | 7.92 (2.63–23.90) | 7.73 (2.86–20.90) | 5.23 (2.04–13.44) |
| cvaC (4) | 4.14 (1.52–11.27) | 5.80 (1.41–23.88) | 6.62 (1.79–24.55) | 6.21 (2.34–16.51) | 12.17 (3.29–45.06) | - | 10.45 (2.83–38.63) | 14.29 (4.31–47.37) | 8.10 (2.82–23.31) |
Note: Only antibiotic resistance genes vs. virulence-associated genes with a significant association (p < 0.05) are shown. OR, odds ratio for significant associations between antibiotic resistance genes and virulence-associated genes (95% confidence interval in parentheses); - indicates no significant associations (p ≥ 0.05).
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Fan, S.; Jiang, S.; Luo, L.; Zhou, Z.; Wang, L.; Huang, X.; Liu, H.; Zhang, S.; Luo, Y.; Ren, Z.; et al. Antibiotic-Resistant Escherichia coli Strains Isolated from Captive Giant Pandas: A Reservoir of Antibiotic Resistance Genes and Virulence-Associated Genes. Vet. Sci. 2022, 9, 705. https://doi.org/10.3390/vetsci9120705
AMA Style
Fan S, Jiang S, Luo L, Zhou Z, Wang L, Huang X, Liu H, Zhang S, Luo Y, Ren Z, et al. Antibiotic-Resistant Escherichia coli Strains Isolated from Captive Giant Pandas: A Reservoir of Antibiotic Resistance Genes and Virulence-Associated Genes. Veterinary Sciences. 2022; 9(12):705. https://doi.org/10.3390/vetsci9120705
Chicago/Turabian StyleFan, Siping, Shaoqi Jiang, Lijun Luo, Ziyao Zhou, Liqin Wang, Xiangming Huang, Haifeng Liu, Shaqiu Zhang, Yan Luo, Zhihua Ren, and et al. 2022. "Antibiotic-Resistant Escherichia coli Strains Isolated from Captive Giant Pandas: A Reservoir of Antibiotic Resistance Genes and Virulence-Associated Genes" Veterinary Sciences 9, no. 12: 705. https://doi.org/10.3390/vetsci9120705
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