Detection and risk factor analysis of avian colibacillosis associated with colistin-resistant Escherichia coli and Klebsiella pneumoniae

Abstract

Colibacillosis associated with colistin-resistant avian pathogenic Escherichia coli (E. coli) poses a threat to both food security and public health. The potential horizontal transmission of mobilized colistin-resistant (mcr) genes facilitates the co-emergence of Klebsiella pneumoniae. This study aimed to determine the prevalence, molecular detection, analyze the antibiogram and identify associated risk factors for colistin-resistant E. coli and Klebsiella pneumoniae isolated from broiler chicken in three districts of Punjab province, Pakistan. In total, 230 visceral organ samples were collected from 13 different chicken farms located in Sargodha, Jhang and Toba Tek Singh in Pakistan. Following isolation, the broth microdilution test was used to confirm phenotypic colistin resistance. Polymerase chain reaction was used to detect mcr-1 and mcr-2 genes associated with colistin resistance. Antimicrobial susceptibility test against 11 antibiotics was performed using the Kirby-Bauer disk diffusion method. Risk factors associated with colistin-resistant bacteria, including host attributes, farm management practices, environmental and agent characteristics, were analyzed. The prevalence of colistin-resistant E. coli and K. pneumoniae was 24.78% (95% CI, 19.6-30.7%) and 3.04% (95% CI, 1.5-6.1%), respectively. The prevalence of colistin-resistant E. coli varied between cities at 42, 23.61 and 5.55% for Jhang, Sargodha and Toba Tek Singh, respectively. The detection frequency of mcr-1 gene, 42.1% (24/57), was significantly (p < 0.01) higher than that of the mcr-2 gene, 14.03% (8/57). Phylogenetic analysis of lipid A phosphoethanolamine transferase sequences revealed greater similarity with mcr-1.5 variant. Isolates were found resistant to amoxicillin-clavulanic acid (84.21%), cefotaxime (70.17%), and trimethoprim-sulfamethoxazole (73.68%). The multivariate logistic regression predicted preceding viral infection of the respiratory tract as a significant association (OR = 4.808, p < 0.01), whereas daily removal/culling of dead/diseased chicken (OR = 0.308, p = 0.01) was a protective factor against the emergence of colistin-resistant strains. These findings indicate that the emergence of colistin-resistant strains deteriorate colibacillosis control efforts in poultry and serves as a possible reservoir for zoonotic infections.

Keywords: Escherichia coli; Klebsiella pneumoniae; avian colibacillosis; mcr; risk factors.

Figure 1

Description of various gross lesions found in necropsy examination of chicken infected with colibacillosis caused by colistin-resistant E. coli. (A) Multiple necrotic foci are shown on the surface of the heart and liver associated with pericarditis and perihepatitis. (B) Fibrous exudate diffusely deposited on the surface of the heart and liver. (C) Severe pericarditis marked by creamy fibrous discharge covering the heart surface.

Figure 2

PCR products were detected on a 1.2% agarose gel for mcr-1 and mcr-2 genes. Lane 1 and 13 (DNA ladder 100 bp, Solis BioDyne, Tartu, Estonia). Lanes 2 and 3 contain negative controls for mcr-1 and mcr-2, respectively. Lanes 4 and 5 contain positive controls for mcr-1 and mcr-2, respectively. Lanes 6, 7, and 10 contain positive samples for mcr-1 gene (320 bp). Lane 9 contains a positive sample for mcr-2 gene (576 bp). Negative samples (lanes 8, 11, and 12).

Figure 3

The phylogenetic tree was constructed for mcr gene product, lipid A phosphoethanolamine transferase, via MEGA (v 12.0.11) with the maximum likelihood method (bootstraps 500) and the WAG substitution model. Sequences obtained in the present study are marked with black squares. Node labels represent the bootstrap support values (percentage), and the scale bar indicates 0.50 amino acid substitutions per site.