Virulence characteristics and antibiotic resistance analysis of multi-drug resistant Enterotoxigenic Escherichia coli in healthy pigs from Zhejiang Province, China

Abstract

Background: Enterotoxigenic Escherichia coli (ETEC) is a predominant diarrheal pathogen worldwide, employing adhesins and secreted enterotoxins to cause disease in mammalian hosts. The report about antimicrobial resistance (AMR) in ETEC have increased, detrimentally affecting both livestock industry and food safety. Despite this, there is a paucity of studies on the AMR genes, virulence factors and the correlation of them in ETEC.

Results: Six ETEC-positive samples collected from healthy pigs across three regions yielded 16 distinct ETEC isolates. Among these, 13 isolates (81.3%) exhibited multidrug resistance (MDR), while all 16 isolates demonstrated tolerance to working concentrations of sodium hypochlorite. 15 isolates harbored both heat-labile enterotoxin genes eltAB and heat-stable enterotoxin gene stb. One isolate uniquely carried eltAB, heat-stable enterotoxin genes sta, stb, and Shiga toxin gene stx2e. Galleria mellonella infection assays confirmed the virulence of representative strains in our study. Whole-genome sequencing of one isolate per sample (n = 6), integrated with NCBI porcine ETEC data, revealed broad distribution of sequence types (STs). A previously unreported ST7070 strain was identified as an enterotoxin gene carrier (eltAB + stb). The results of correlation analysis revealed that co-transfer in aminoglycoside resistance genes aph(3'')-Ib, aph(6')-Id, sulfonamide resistance gene sulIII and co-localized cluster of arr-3 (rifampicin resistance), aac(6')-Ib-cr (aminoglycoside resistance), catB (phenicol resistance), and bla_OXA (β-lactam resistance). Significant co-transfer observed between disinfectant resistance gene sugE(p) and extended-spectrum β-lactamase gene bla_CMY.

Conclusions: ETEC strains isolated from healthy animals exhibited both pathogenicity and MDR phenotypes. Besides, they harbored diverse AMR genes and disinfectant resistance determinants. Integrated analysis with NCBI-deposited ETEC genomic data further revealed co-transfer events of AMR and disinfectant resistance genes among ETEC populations.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12917-025-05044-9.

Keywords: Antimicrobial resistance; Bioinformatic analysis; ETEC; Pigs.

Fig. 1

The results of antimicrobial susceptibility test of 16 ETEC (The shade of color represents the extent. The MIC value of each strain was divided by the intermediate breakpoint for the corresponding antimicrobial agents and then normalized. More negative values indicate greater sensitivity, whereas more positive values indicate greater resistance)

Fig. 2

Phylogenetic analysis of porcine-derived ETEC strains from NCBI exhibiting genomic homology with isolates in our study (n = 49)

Fig. 3

Virulence potential of ETEC using a G. mellonella infection model

Fig. 4

Genetic environment of the pathogenicity island of eltAB in ETEC

Fig. 5

Distribution of enterotoxin genes in swine ETEC from different districts

Fig. 6

Relationships of colonization factors and enterotoxin groups

Fig. 7

Correlational analysis of ETEC genotypes among virulence-based subgroups in porcine isolates (NCBI database and our research) *, p < 0.05 **, p < 0.01

Fig. 8

Correlational analysis of ETEC genotypes in porcine isolates (NCBI database and our research) *, p < 0.05 **, p < 0.01

Fig. 9

Correlational analysis of ETEC genotypes in porcine isolates which carried eltAB + stb (NCBI database and our research) *, p < 0.05 **, p < 0.01