The current overuse of antibiotics in medicine and agriculture has led to the emergence of antimicrobial resistance, a serious threat to public health. However, current research has largely focused on clinical settings, with insufficient research examining the resistance characteristics of Escherichia coli from diseased animals, particularly the risk of animal-to-human transmission.
Recently, a research team from the Institute of Animal Husbandry and Veterinary Medicine of the Zhejiang Academy of Agricultural Sciences and the School of Life Sciences of the University of Science and Technology of China published a new study in the journal mBio. Using genomic technology, this study analyzed the antibiotic resistance of 114 strains of Escherichia coli from diseased pigs collected over a 12-year period in eastern China. The study identified, for the first time, enterotoxigenic E. coli (ETEC) strains carrying both the mcr-1 and mcr-3 genes.
Introduction
The inappropriate use of antibiotics in livestock farming and healthcare has led to the widespread spread of drug-resistant bacteria carrying "super-resistance genes" (such as blaNDM, mcr-1, and tet(X4)). This has weakened the efficacy of "last resort antibiotics" and resulted in a shortage of new drugs, creating a serious problem.
E. coli, as a major zoonotic pathogen, can both cause disease and spread drug-resistant genes across ecosystems (such as those carrying the high-risk genes blaNDM-5, mcr-1, and tet(X4/X5)). The combined effects of its high virulence and multidrug resistance exacerbate the risk. Whole-genome sequencing (WGS) is a key technology for identifying drug resistance and pathogenicity genes, providing a basis for the prevention and control of animal and human diseases.
Research Results
1. Prevalence of Escherichia coli in This Study
As shown in Figure 1A, this study analyzed 114 E. coli isolates from sick and dead pigs from 82 farms in 11 cities in Zhejiang Province between 2010 and 2021. These pigs suffered from diarrhea, splenomegaly, and hepatomegaly.
As shown in Figure 1B, genomic analysis revealed 39 sequence types (STs), of which ST88 (accounting for 15.8%) was the most common. The highest ST diversity was observed in Shaoxing and Hangzhou. Multiple STs coexisted across years and cities. Some ST types (such as ST88) were consistently prevalent over the nine-year period, while others (such as ST117 and ST48) appeared only during specific periods. Specimen identification was reliable, and small SNP differences between strains suggested possible cross-farm and time-varying transmission.
Figure 1. Epidemiological characteristics of 114 Escherichia coli isolates
Figure A: Sampling distribution and time in Zhejiang Province (blue markers)
Figure B: Association of strains with city, year, and high-risk drug-resistance genes. Line thickness corresponds to the number of strains.
Figure C: Minimum spanning tree constructed based on MLST. Node size represents the number of strains, and branch length reflects genetic variation.
Conclusion: E. coli isolates from infected pigs in Zhejiang exhibited high genetic diversity (39 ST types). However, subtle genetic differences between strains also suggest the risk of cross-farm transmission. The ST88 strain demonstrated sustained epidemic activity for up to nine years.
Figure 2. ANI of 114 E. coli Isolates
The names of E. coli strains isolated from 2010 to 2017 are marked in blue, and the names of E. coli strains isolated from 2018 to 2021 are marked in pink.
2. Antimicrobial Resistance Analysis of 114 E. coli Isolates
The 114 E. coli isolates from sick pigs generally exhibited severe multidrug resistance (99.12% were resistant to three or more drug classes (Figure 3B)). Resistance to ampicillin and combination preparations reached 100%, and resistance to ciprofloxacin and tetracycline exceeded 94% (Figure 3A). Notably, 21.05% of the strains were resistant to colistin. After 2018, the colistin resistance rate (15.71%) decreased significantly from the previous period (29.55%) (as shown in Figure 3C, 2018-2021). However, the florfenicol resistance rate increased to 94.29%, while resistance rates to other drugs remained relatively stable. This dynamic change may be related to adjustments in medication policies.
Figure 3. Antimicrobial resistance of 114 E. coli isolates
Figure A: Resistance rates of 114 isolates to 13 antibiotics; Figure B: Distribution of multidrug-resistant strains;
Figure C: Comparison of changes in resistance rates between 2010-2017 and 2018-2021, with chi-square tests used to analyze the significance of differences.
Conclusion: Severe multidrug resistance was prevalent in E. coli isolated from diseased pigs in Zhejiang Province, with colistin resistance being particularly prominent. However, after the ban in 2017, resistance rates decreased significantly, while florfenicol resistance increased. This dynamic change is closely related to adjustments in drug use policies.
3. Characterization of AMR-associated genomic features in E. coli isolates
The study found that the 114 E. coli isolates from diseased pigs carried an average of 4.9 plasmids, with the IncFIB plasmid being the most common (78.07% of the strains). All strains harbored at least two drug-resistance genes (ARGs), and 80.7% harbored more than 10 ARGs, with mdf(A), tet(A), floR, and sul2 being the most prevalent. Key findings revealed strong associations between specific plasmids and drug-resistance genes (e.g., IncHI2 type with aadA2b/sul3/tet(A), and IncI2 type with mcr-1), and experiments confirmed that IncI2 plasmids can effectively mediate the transfer of the mcr-1 gene between strains.
Figure 4. Prediction results of acquired resistance genes in 114 Escherichia coli isolates
Figure 5. Correlation coefficients between ARGs and plasmid replicons in 114 E. coli isolates
Conclusion: E. coli from diseased pigs commonly carry multiple plasmids and are enriched for resistance genes. IncI2 plasmids have been shown to be highly efficient vectors for the horizontal transfer of the key resistance gene mcr-1.
4. Identification of genomic features associated with virulence in E. coli isolates
The study found that all 114 E. coli isolates analyzed carried at least one virulence gene, with nearly 80% harboring at least 10. Among these, the terC gene was the most common (99.12%), followed by the traT gene (81.58%). Other key virulence genes, stb, sta1, stx2A, stx2B, and astA, occurred in 24.56% to 36.84% of isolates. The analysis revealed significant correlations between specific strain types (ST types) and certain virulence genes: ST501 was associated with stb and sta1, ST100 with astA and stb, and ST88 and ST4214 with stb, sta1, stx2A, and stx2B. Furthermore, significant co-occurrence relationships were observed between astA and stb, and between stb, sta1, stx2A, and stx2B. The study also identified 28 Shiga toxin-producing Escherichia coli (STEC), 43 enterotoxigenic Escherichia coli (ETEC), and one EPEC-producing Escherichia coli (EPEC).
Figure 6. Virulence Gene Prediction Results for 114 E. coli Isolates
Red grids indicate known high-risk E. coli virulence genes, while pink grids indicate common virulence genes.
Conclusion: E. coli isolated from diseased pigs commonly carry multiple virulence genes. Specific strain types (such as ST501/ST100) are significantly associated with key toxin genes, and pathogenic subtypes such as STEC, ETEC, and EPEC were successfully identified.
5. Genomic Characterization of Plasmids Carrying mcr-1 and mcr-3
The study revealed that two E. coli strains carry both the mcr-1 and mcr-3 genes. These genes are located on different plasmids: mcr-1 is located on an IncI2 plasmid, which has a simple structure and high transmission potential, while mcr-3 is located on an IncHI2 plasmid, which is complex and diverse and carries multiple drug resistance genes.
Figure 7. Comparative analysis of plasmids carrying mcr and tet(X4) with previously reported plasmids using BLAST tools.
Conclusion: The mcr-1 and mcr-3 genes in E. coli were found to be located on structurally simple (IncI2) and complex (IncHI2) plasmids, respectively, suggesting different mechanisms of resistance gene transmission.
6. Genomic Characterization of tet(X4)-Carrying Plasmids
The study found that two strains of tigecycline-resistant E. coli carried two structurally distinct tet(X4) plasmids: the small plasmid p626A1-38K-tetX4 (IncX1 type, simple structure, containing tet(X4) and floR) and the large plasmid p802A1-191K-tetX4 (complex type, containing tet(X4) and five other resistance genes). Both are similar to the backbone of known epidemic plasmids, and there are mobile regions around tet(X4) containing multiple resistance genes and insertion sequences, suggesting a high-level transmission risk.
Figure 8. Analysis of the Genetic Environment of the mcr Gene
A: Genetic environment of the *mcr-3* gene in plasmids p204A1-223K-mcr3 and p602A1-220K-mcr3;
B: Genetic environment of the *mcr-1* gene in plasmids p204A1-63K-mcr1 and p602A1-65K-mcr1.
Conclusion: This study found that the two structurally distinct tet(X4) plasmids carried by tigecycline-resistant E. coli both possess backbone features similar to those of prevalent plasmids and contain mobile regions surrounding the genes, indicating a high risk of transmission.
Conclusions
This study found widespread multidrug resistance (including to last-line drugs) in Escherichia coli isolated from diseased pigs in eastern China. Key resistance genes, mcr-1/mcr-3/tet(X4), were transmitted via a complex plasmid (IncI2/IncHI2 type). This confirms that plasmids are the core vectors of drug resistance transmission and urges immediate intervention to prevent the spread of drug-resistant bacteria from animals to humans and the environment.
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