Genomic and Functional Characterization of Poultry Escherichia coli From India Revealed Diverse Extended-Spectrum β-Lactamase-Producing Lineages With Shared Virulence Profiles

Abstract

Extended-spectrum β-lactamases (ESBLs) form the most important resistance determinants prevalent worldwide. Data on ESBL-producing Escherichia coli from poultry and livestock are scarce in India. We present data on the functional and genomic characterization of ESBL-producing E. coli obtained from poultry in India. The whole genome sequences of 28 ESBL-producing E. coli were analyzed comprising of 12 broiler chicken E. coli isolates, 11 free-range chicken E. coli isolates, and 5 human extraintestinal pathogenic E. coli. All of the 28 ESBL-producing E. coli isolates were tested for antibiotic susceptibilities, in vitro conjugation, and virulence-associated phenotypic characteristics. A total of 13 sequence types were identified from the poultry E. coli, which included globally successful sequence types such as ST117 (9%), ST131 (4.3%), and ST10 (4.3%). The most common ESBL gene detected in poultry E. coli genomes was bla _CTX-M-15 (17%). Also, FIB (73%) and FII (73%) were the most common plasmid replicons identified. Conjugation experiments demonstrated 54 (7/13), 30 (3/10), and 40% (2/5) of broiler, free-range, and human ExPEC E. coli to be able to transfer their ESBL genes, respectively. The in vitro virulence-associated phenotypic tests revealed the broiler, free-range, and human ExPEC isolates to be comparable in biofilm formation, resistance to serum bactericidal activity, adherence, and invasion capabilities. Our overall results showed prevalence of virulence phenotypes among the diverse ESBL-producing E. coli from poultry; while certain E. coli clones from broiler-poultry may indeed have the potential to cause infection in humans.

Keywords: ESBL; ExPEC; India; genomics; poultry Escherichia coli.

Figure 1

Antibiotic resistance pattern of 28 ESBL-producing E. coli isolates: tetracycline class includes tetracycline, oxytetracycline and chlortetracycline antibiotics. In addition, colistin antibiotic was also tested for which all isolates were found to be susceptible. Out of the 28 ESBL-producing E. coli, 22 (78.5%) were detected to be of MDR phenotype.

Figure 2

The core genome based consensus Maximum Likelihood phylogenetic tree of 25 E. coli genomes (12 broiler, 8 free-range and 5 human ExPEC genomes) with reference E. coli SE15 genome generated using Harvest. The output of Harvest was visualized using iTOL. The broiler and free-range E. coli isolates were present within same clusters indicating that they share similar genetic backgrounds. However, the human ExPEC isolates largely formed a distinct clade together with the genomes of broiler E. coli and no genome from free-range E. coli was represented within this clade (IV). Labeling at the tips include genome unique IDs (B01 to B12; Broiler E. coli, F01 to F08; Free-range E. coli, H01 to H05; Human ExPEC) followed by sequence types, acquired ESBL genes and the phylogroups.

Figure 3

Cluster heat map of 25 E. coli genomes (12 broiler, 8 free-range and 5 human ExPEC genomes) showing the presence and absence of 92 antibiotic resistance genes belonging to different categories (A) antibiotic efflux, (B) antibiotic inactivation, (C) target alteration, protection and replacement, (D) reduced permeability to antibiotics. Results of resistance clustering indicated that the three groups of E. coli isolates including broiler, free-range and human isolates represented clusters with mixed strains which shared resistance gene absence/ presence profile with each other. Gene names are represented on Y axis and the E. coli genomes are listed on the X axis (B01 to B12; Broiler E. coli, F01 to F08; Free-range E. coli, H01 to H05; Human ExPEC). Detail description of genomes can be found in Supplementary Table S1.

Figure 4

Cluster heat map of 25 E. coli genomes (12 broiler, 8 free-range and 5 human ExPEC genomes) showing the presence and absence of 143 virulence genes belonging to different categories (A) adherence, (B) toxins, (C) iron uptake, (D) auto-transporters, (E) secretion systems. The virulence based clustering indicated that the three groups of E. coli including broiler, free-range and human ExPEC shared virulence gene profiles with each other. Gene names are represented on Y axis and the E. coli genomes are listed on the X axis (B01 to B12; Broiler E. coli, F01 to F08; Free-range E. coli, H01 to H05; Human ExPEC). Detail description of genomes can be found in Supplementary Table S1.

Figure 5

In vitro functional characterization of 11 free-range chicken E. coli isolates, 12 broiler chicken E. coli isolates and 5 human ExPEC including the control E. coli DH5α. (A) Biofilm formation capabilities determined in M-63 minimal media using 96-well microtiter plate method. (B) Resistance to serum bactericidal activity against 50% human serum. (C) Adhesion and (D) invasion capabilities on T24 bladder epithelial cells. All 28 E. coli strains demonstrated significant correlation with the above tested virulence phenotypes compared to the negative control strain, E. coli DH5α. Experiments were performed twice (serum assay)/thrice (adhesion, invasion, and biofilm formation assays) in triplicates and statistical values were obtained using the nonparametric Mann-Whitney U test. 5.01. *p < 0.05; **p < 0.01; ***p < 0.001.