Molecular and Phenotypic Characteristics of Escherichia coli Isolates from Farmed Minks in Zhucheng, China

In this study, the prevalence, phenotypes, and clonal relationships of Escherichia coli (E. coli) strains isolated from minks were investigated. In July 2017, a total of 62 fresh faecal swab samples were randomly collected from one large-scale mink farm in Zhucheng, Shandong Province, China. In all the samples, 50 E. coli strains were isolated and then assigned to serotyping, antimicrobial susceptibility test, detection of antimicrobial resistance genes and the Class 1 integrons, and multilocus sequence typing (MLST). Four pathogenic serotypes were identified among all the isolates, while the most common serotype was enterohemorrhagic E. coli O104:H4 (6.0 %). Antimicrobial sensitivity testing revealed that most isolates were susceptible to cefoxitin (96.0 %) and amikacin (82.0 %), while most isolates were resistant to ampicillin (92.0 %) and tetracycline (90.0 %). An analysis of the nucleotide sequences revealed that 7 isolates (14.0%) carried 4 types of Class 1 integron cassette, including dfrA27+aadA2+qnrA (57.1%), dfrA17+aadA5 (14.3%), dfrA12+aadA2 (14.3%), and dfrA1+aadA1 (14.3%). PCR screening showed that 14 antibiotic resistance genes were presented in 50 isolates, while the most prevalent resistance gene was qnrS, which was detected in 60.0 % of isolates, followed by sul2 (40.0%) and oqxA (38.0%). MLST analysis showed that 32 sequence types (STs) were identified, while ST46 was the predominant genotype among all isolates. Clonal complex 3 (CC3) was dominant. Compared with 340 human E. coli STs reported in China, the ST10 clonal complex, known as the largest human clonal complex, was also found in the 50 mink E. coli isolates. Meanwhile, mink-derived strain ST206 formed a new clonal complex, CC206, which was different from human ST strains. Our results showed that farmed minks could be reservoirs of antimicrobial-resistant E. coli with Class 1 integron cassettes and resistance genes, which were likely to pose a threat to public health. Therefore, continuous inspections and monitoring of E. coli in minks are essential for detecting and controlling emerging E. coli with different serovars as well as antibiotic resistance.


Introduction
Since the advent of antibiotics, it had made great contributions to both human and animal health. Antibiotics that can kill or inhibit the growth of microorganisms have been used to treat a wide range of infectious diseases caused by bacteria, in livestock, poultry, wild, or fur animals (such as minks) and humans. Antibiotics play a very important role in dealing with pathogenic microorganism infections as well as in reducing morbidity and mortality [1,2].
According to the differences in pathogenicity, E. coli can be classified into commensal E. coli and pathogenic E. coli [3].
Antibiotics have long been considered as the first line of defense to prevent pathogenic E. coli infections. The treatment of pathogenic E. coli infections is becoming complicated because of the rapid emergence and dissemination of antibiotic-resistant strains, which may consequently 2 BioMed Research International result in an increasing number of clinical treatment failures in bacterial-mediated diseases and further threaten public health with the possibility of transmission to humans through aerosols, environmental contact or other methods [2,6]. The level of antibiotic resistance among pathogenic and commensal E. coli has steadily increased and has become a global health concern [7,8].
There are many resistance mechanisms underlying the emergence and prevalence of antimicrobial resistance. The acquisition of resistance genes through a mechanism involving mobile genetic elements, such as plasmids and transposons, is considered a major contributor to antimicrobial resistance [9,10]. A previous study also demonstrated that E. coli can carry resistance plasmids and can easily acquire resistance transfer genes [9,10]. The spread of antibiotic resistance among bacteria is mainly attributed to integrons. Integrons are DNA elements that are associated with the prevalence and horizontal transmission of antibiotic resistance [11][12][13]. Integrons can capture, express, and exchange gene cassettes and then convert them to functional genes [11,14].
E. coli may cause severe morbidity and mortality in fur animals such as minks. In recent years, several types of antibiotics have been used in Shandong Province to treat bacterial diseases, resulting in repercussions for bacterial antibiotic resistance [15,16]. To the best of our knowledge, in contrast to E. coli in humans, livestock, and poultry, there is still a lack of data about the antimicrobial resistance mechanisms of E. coli from minks in China. Therefore, in our study, 62 faecal samples were collected from mink farms in the Zhucheng area, and then E. coli strains were isolated for serotyping, antimicrobial susceptibility testing, detection of the Class 1 integron cassette, and resistance genes to provide information and guidance for the rational use of antibiotics in minks.

Materials and Methods
. . Sampling and Isolation. In July 2017, 62 faecal swabs were randomly collected from healthy minks in large-scale mink farms in the Zhucheng area in Shandong Province, China. Mink farms were chosen based on their scale with the requirement of the breeding stock being > 10000 heads. Ethical approval was not required for the study because the sampling process did not harm the minks. All the faecal samples were placed into an ice box and transferred to our laboratory within 6 h for further bacteriological analysis.
Isolation and identification of E. coli were performed according to the previously described method [17] with some slight modifications. Briefly, faecal swab samples were transferred to sterile culture tubes containing 4 mL of Luria-Bertani (LB) broth and mixed vigorously (220 r/min) at 37 ∘ C for 8 h. After enrichment, a loop of LB broth culture was streaked onto eosin-methylene blue medium (EMB) agar and incubated at 37 ∘ C for 24 h. Colonies showing a metallic sheen were considered presumptive E. coli isolates, and positive colonies were chosen for further biochemical identification using the API 20E system (Sysmex-bioMerieux, Tokyo, Japan).
. . Serotyping. To identify the E. coli serotype, all the positive strains were serotyped by E. coli diagnostic serum according to the manufacturer's instructions. The diagnostic serum kit consisted of EPEC diagnostic serum, EIEC diagnostic serum, ETEC diagnostic serum and EHEC (O104 and 0157) diagnostic serum, and E. coli H4 and H7 diagnostic serum (Tianrun Bio-Pharmaceutical, Ningbo, China). Briefly, the principle of the E. coli serotyping method was as follows: the diagnostic serum was directly mixed with a bacterial suspension on a slide, the specific agglutination reaction between antigens and the corresponding serum was observed, and then the serotype was determined.
E. coli (ATCC 25922) and Klebsiella pneumoniae (ATCC 700603) were used as the control strains. E. coli isolates that were resistant to more than three classes of antimicrobials were defined as multidrug resistance (MDR) isolates.
. . Statistical Analysis. We analysed the correlation between drug resistance phenotypes and drug resistance genes of 50 isolates. The statistical analyses were performed using SAS V8 software (SAS Inc., Raleigh, N Car, USA), employing the chi-square test. P values less than 0.05 were considered statistically significant.

. . Detection of the Class Integron Cassette and Resistance
Genes. E. coli isolates were characterized at the molecular level to detect their antimicrobial resistance mechanisms. Bacterial DNA was extracted using a TIANamp Bacterial DNA Kit (Tiangen, Beijing, China) according to the manufacturer's instructions. Gene cassette regions within the variable region of Class 1 integrons were detected via polymerase chain reaction (PCR). Primers were synthesized according to the references [19], and the sequences are shown in Table 1. The amplification consisted of an initial denaturation at 94 ∘ C for 5 min, 30 cycles of denaturation at 94 ∘ C for 60 s, annealing at 56 ∘ C for 55 s, and extension at 68 ∘ C for 6 min. A final extension for 10 min at 72 ∘ C was also applied. The PCR products were purified and then sequenced (Invitrogen, Beijing, China). The obtained DNA sequences were compared with those in GenBank using the Basic Local Alignment Search Tool (BLAST).
Twenty-four pairs of specific primers were designed according to known sequences and references in GenBank [20][21][22][23][24][25][26][27][28]. The primers used for PCR and the respective amplification lengths are shown in Table 1. The presence of  Primer  [29] were obtained from the NCBI repository and included in subsequent analyses for comparison. To analyse the distribution of STs in freerange mink farms, a minimum spanning tree was generated using BioNumerics software, version 7.6 (Applied Maths, Kortrijk, Belgium).
In order to determine the clonal and epidemiological relationships as well as the formation of clonal complexes (CCs), genetic similarity diagram analysis was performed by the program eBURSTv3 [30] (http://eburst.mlst.net/). Genetic similarity diagrams can demonstrate the genetic relationship among bacteria through their respective ST analysis. Then, the bacteria can be grouped into single locus variants (SLVs), double locus variants (DLVs), and triple locus variants (TLVs) or be present in isolation (singletons). If the STs exist and there is an allele difference among the ST types, the diagram also allows verification of the grouping of STs, representing the CC [31].
In order to examine the relatedness between strains and STs at the sequence-level resolution, concatenated sequence data of each distinct representative ST were imported into the MEGA 6.0 software package [http://www.megasoftware.net/]. Through complete deletion of alignment gaps, a total of 3423 positions were used in each concatenated sequence as a data set for phylogeny calculations. An evolutionary phylogeny was constructed in MEGA 6 using the maximum composite likelihood (MCL) to estimate evolutionary distances, and the topology was validated by bootstrapping (1000 replicates) [32][33][34]. To establish evolutionary relevance, E. coli strain 11 (ST11) was used as the tree root. The optimum tree generated was condensed where the bootstrap support for the clustering of taxa was <50 % of the replicates [35].
In order to analyse the strain resistance phenotype and strain resistance gene relatedness in the phylogenetic tree, a phylogenetic tree was introduced into the EvolView software package [http://www.evolgenius.info/evolview/#login].
Eighteen of the 31 ST genotypes originated from 10 clonal complexes, and the remaining 13 ST types did not have clonal complexes within the BURST algorithm. Ten different STs of the 50 E. coli strains formed three clonal complexes, CC10, CC46, and CC176. Among the three clonal complexes, CC10 was the largest, containing eight isolates, consisting of ST10, ST43, ST48, ST215, and ST744 in ST10 Cplx. The second largest clonal complex was CC46, which contained ten isolates, consisting of ST46 and ST1421 in ST10 Cplx and ST7110 without a clonal complex. The minimum clonal complex was CC176, consisting of ST5708 in ST10 Cplx and ST176 without a clonal complex (Figure 2).
As shown in the heatmap of the phylogenetic tree, most of the same ST strains were concentrated in the phylogenetic tree. The strains of the CC46 and CC176 clonal complexes were centrally distributed in the phylogenetic tree. The strains of the CC10 clonal complex were distributed in two regions in the phylogenetic tree.
There were significant differences between the drug resistance spectrum composition and drug resistance gene carriage status among most of the same ST strains or strains of the same clonal complex in the phylogenetic tree. There were no significant differences, or the differences were comparatively close between the drug resistance spectrum composition and drug resistance gene carriage status among the strains with close genetic relationships, such as 14-ST540 and 17-ST206.
There was no correlation between strains carrying different integrons or serotypes and their sequence type (Figure 4).

Discussion
In this study, 50 E. coli strains were recovered from minks in the Zhucheng region. The serotyping results showed that the most prevalent serovars were EHEC and ETEC, while there was no EPEC found in our study. It was worth mentioning that the O104:H4 EHEC strain was detected in our study. Some serotypes of EHEC carry Shiga toxins, especially E. coli O157, which commonly carries Shiga toxins (stx1 and stx2). Additionally, what makes this pathogen more important is that it is even capable of expressing stx1 and stx2 under the VBNC state according to the latest study [36]. In view of the relatively poor sanitation conditions in livestock breeding environments, the EHEC O104:H4 strain may be a potential pathogenic factor in mink farms. We should pay more attention to EHEC O104:H4 in the mink breeding industry and control measures should also be implemented.
In the antimicrobial susceptibility test, most strains were found to be susceptible to cefoxitin and amikacin, which was consistent with the results of a previous report focused on red

26-ST5143
Reference-ST11 Figure 4: Diversity profiles of the phylogenetic tree, drug resistance genes, antimicrobial resistance, and Class 1 integron structure. Note. Dendrogram of seven allele sequence types from 50 E. coli isolates from minks. ST11 is included as the reference. The three shaded parts in the figure represent three clonal complexes, CC10, CC46, and CC176 from top to bottom. The unit information is the number of isolated strains followed by the ST type and Class 1 integron structure IntA, dfrA27+aadA2+qnrA; IntB, dfrA17+aadA5; IntC, dfrA12+aadA2; or IntD, dfrA1+aadA1. (1) The 14 detected resistance genes were shown in the left matrix: a red square indicates that the strain carries the resistance gene, and a light blue square indicates that the resistance gene is not detected. (2) Antibiotics used in this experiment were shown in the right matrix: a blue square indicates that the strain is resistant to the antibiotic, and a light blue square indicates that the strain is not resistant.
foxes in Portugal [37] but was not consistent with a report on minks in Denmark, which may be due to differences in geography and rearing conditions [38]. Most isolates were resistant to ampicillin (46/50, 92.0 %) and tetracycline (45/50, 90.0 %), which was similar to a report on minks in Denmark [38,39], but the percentage of resistance to ampicillin and tetracycline was much higher than that of Pedersen's study (59.1 % and 75.5 %) [38]. Our study showed that 43 isolates (43/50, 86.0 %) were MDR, which was much higher than the MDR rate in minks in Denmark (60 %) [38].
Integrons are natural, highly efficient recombination and expression systems that can capture genes as part of genetic elements known as gene cassettes [40]. In recent studies, Class 1 integrons were investigated in animal, water, and human stool samples [41][42][43][44]; however, there have been few reports focusing on the prevalence of Class 1 integrons in strains of E. coli from minks. In this study, we investigated the occurrence and cassette region composition of Class 1 integrons among 50 E. coli isolates from minks. Among the 50 isolates recovered from minks, 7 isolates (7/50, 14.0 %) carried Class 1 integrons, which was similar to that (16.0 %) of a previous study in sheep [6] but was lower than that of rabbits and cattle [45,46]. These different results may be due to differences in species, environments and regions. Four transferable DHFR genes were detected in 50 isolates, dfrA , dfrA , dfrA , and dfrA ; these gene cassettes were encoded by the trimethoprim resistance gene. Three transferable AAD genes were detected in 50 isolates, aadA , aadA , and aadA ; these gene cassettes were encoded by the aminoglycoside resistance gene. In addition, qnrA was encoded by the quinolone resistance gene. Four kinds of gene cassette arrays, dfrA + aadA + qnrA, dfrA + aadA , dfrA + aadA , and dfrA + aadA , were identified in this study. These integrons were found not only in Salmonella [47,48] and E. coli [49,50] from chickens, ducks, and pigs, but also in Klebsiella pneumoniae [51] and Staphylococcus aureus [52] isolated from China, suggesting that gene cassette arrays can be transferred to different species of bacteria with the horizontal movement of integrons and then transmitted to different species. The horizontal transfer of integrons may BioMed Research International 9 have an important effect on the clinical use of antibiotics. Of note, Class 1 integrons are usually associated with MDR E. coli isolates, which is consistent with the results of this study.
A wide variety of resistance genes were found in nonpathogenic E. coli strains from minks. The inclusion of some resistance genes inside integrons may explain the spread of antibiotic resistance among minks in the Zhucheng area. Among the three types of -lactamase resistance genes detected in our study, 20.0 % of the E. coli isolates carried TEM and OXA , whereas 92.0 % of the isolates were resistant to ampicillin; this may be related to the expression level of TEM and OXA genes and requires further study. Our data showed that 40.0 % of the E. coli isolates carried sul , 32.0 % of the E. coli isolates carried sul , 24.0 % of the E. coli isolates carried sul , and .0 % of the isolates were resistant to sulfamethoxazole. The detection rates of -lactamase and sulfamethoxazole resistance genes in our study are similar to those in red foxes in northern Portugal [37]; however, there were some slight differences; for instance, both TEM and OXA were detected in our study, but only TEM was detected in the previous study [37]. For the sulfamethoxazole resistance genes, sul , sul , and sul , were all detected in our study, but only sul was detected in the red fox study [37]. These differences may be related to the differences in regions and species.
In our study, the quinolone genes (qnrS, qnrB, qnrD, and oqxA) were detected, alone or in combination in quinoloneresistant isolates, revealing that the frequent occurrence of quinolone genes may be associated with high antibiotic resistance rates to quinolone. Nevertheless, our results showed that there was no tetracycline resistance gene among mink E. coli isolates, which was not consistent with the resistance rate of tetracycline (90.0 %).
The MLST method provides a scalable typing system that reflects the population and evolutionary biology of bacteria and makes valid comparisons between results from different laboratories possible. MLST applies neutral or slowly accumulating genetic variations in housekeeping genes, which are not affected by the rapid evolution detected within genes encoding proteins that influence survival in a particular niche [53][54][55]. MLST analyses rely on the sequencing of seven housekeeping genes for each E. coli isolate. The sequences are compared using an online database (http://www.mlst.net) that attributes the allelic profile and performs the concatenation leading to the sequence type. MLST results are accurate and reproducible between laboratories and, over time, provide detailed information on the overall epidemiology of the organism [56,57]. STs are grouped in clonal complexes (CCs) of isolates that share five to seven alleles with another sequence type in the group [58]. Therefore, the clonal relationship of E. coli from minks was studied by MLST, and a phylogenetic tree was constructed based on the strain MLST data to understand the apparent partial characteristics of strains with different genetic relationships and the differences in drug resistance gene carriage.
In our study, 31 previously known STs were found, of which 18 STs (10,23,43,46,48,58,181,206,215,226,398,716,744,1421,1434,3075, 5708, and 7588) were clustered in 10 CCs (10, 23, 46, 155, 168, 206, 226, 398, 467, and 522) ( Figure 2), and the remaining 13 STs did not have CCs in the BURST algorithm. Ten previously known STs (10,43,48,215,744,1421,46,7110,176, and 5708) were found in 31 STs of 50 E. coli strains, which were clustered in three CCs (10, 46, and 176) (Figure 2). This finding differs from a previous study focused on E. coli of rabbits performed in Shandong Province [45]; these differences may indicate that, compared with other animals such as rabbits, E. coli strains from minks in Shandong Province have greater ST diversity and more CCs. The most frequently detected ST was ST46 (8/50, 16%) in the study. Compared with other reports, there were significant differences in STs and dominant STs detected from different regions and species. For example, ST302 was the dominant ST in rabbits in Shandong Province [45], ST710 was the dominant ST in non-O157 Shigatoxin-producing Escherichia coli (STEC) strains isolated from different resources in China [59], and ST101 was the dominant ST in edible animals in China [60]. This indicates that the genotypes of E. coli are widely distributed, and the dominant ST is obvious different among different regions. The reasons for this phenomenon may be related to the cloning and transmission of E. coli strains. Compared with 340 STs of human E. coli from China, we found that 17 STs of mink origin differed from the human STs by one allele and formed 9 clones (Figure 3). Four STs (10, 46, 361, and 2179) were the same as human STs (Figure 3). CC10 showed an interesting and unexpected result because the cluster contained 48 STs (8 from animals and 41 from humans). ST10 is a strong possible common ancestor candidate for CC10. ST48 from mink is a CC10 subgroup founder connecting five different STs (3127, 3932, 2739, 2434, and 4082) from human sources. These results show that some minkderived E. coli are closely related to human E. coli (in the Chinese population), and these STs might be related to some diseases (based on http://enterobase.warwick.ac.uk/species/ ecoli/search strains?Query=st search). To prevent threats to public health, therefore, continuous monitoring and detection of E. coli in minks are necessary.
By studying the results of the phylogenetic tree, we found that the strains of the same ST, such as ST10, ST46, and ST1434, were distributed in the same region on the phylogenetic tree and had the closest relationship with each other (Figure 4). Most strains of the same CC were concentrated in phylogenetic trees, but ST48 in CC10 was far from other strains in CC10. We speculated that the difference in the adK gene between ST48 and ST10 may be the cause of this distribution. Antibiotic resistance profiles and resistance gene profiles of ST genotypes with different genetic relationships were observed. We can conclude that the distribution of different serotypes and integron-carrying strains in phylogenetic trees has no obvious regularity. From the phylogenetic tree as a whole, there were clear differences in the composition of the drug resistance spectrum and the types of drug resistance genes carried by the closely related strains, but there were also some closely related strains with drug resistance spectrum composition and types of drug resistance gene carried that were very similar (17-ST206 and 14-ST540) (Figure 4). These strains have a high level of genetic similarity.

Conclusions
In summary, we conducted an epidemiological survey of E. coli on mink farms in Zhucheng. Our results showed that E. coli was highly prevalent in minks. The reported pathogenic serotypes and MDR E. coli strains contained abundant types of drug resistance genes and a certain number of gene cassettes. E. coli isolated from mink origin had similar genes, and some strains had high genetic similarity with E. coli strains isolated from humans in China. Therefore, the relevant breeding enterprises should manage and prevent these pathogenic bacteria to avoid harm to public health.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.