Commensal bacteria contribute to the growth of multidrug-resistant Avibacterium paragallinarum in chickens

Abstract

Avibacterium paragallinarum-associated infectious coryza (IC) is an important threat in commercial poultry. Previous studies about the characteristics of A. paragallinarum are succeeded in revealing the course of IC disease, but whether and how resident microbes contribute to the infection remains unclear. To understand the role of commensal bacteria, we isolated 467 commensal bacteria, including 38 A. paragallinarum, from the respiratory tract of IC chicken. The predominant commensal isolates were Gram-positive bacteria belonging to Staphylococcus spp. [33.19%, 95% confidence interval (CI): 28.93-37.66%], Enterococcus spp. (16.49%, 95% CI: 13.23-20.17%), and Bacillus spp. (16.27%, 95% CI: 13.04-19.94%). These isolates were closely correlated with the survival of A. paragallinarum. We examined and found that commensal bacteria aggravate A. paragallinarum-associated infections because certain commensal species (28.57%, 95% CI: 15.72-44.58%) induced hemolysis and promoted the growth of A. paragallinarum in vitro. Notably, A. paragallinarum showed high resistance to routine antibiotics such as erythromycin (84.21%, 95% CI: 68.75-93.98%), tetracycline (73.68%, 95% CI: 56.90-86.60%) and carried diverse mobile resistance gene clusters. Overall, we found commensal bacteria especially Gram-positive bacteria facilitate the survival of multidrug-resistant A. paragallinarum to exacerbate infections, suggesting that novel strategies may diminish A. paragallinarum-associated infections by modulating the population dynamics of commensal bacteria.

Keywords: Avibacterium paragallinarum; antimicrobial resistance; commensal bacteria; satellitism; symbiosis.

FIGURE 1

Bacterial composition in upper respiratory tract from healthy chickens and IC chickens. In infection group, the bacterial genera with relative abundance of less than 2% were categorized into “Others” group, including Aerococcus spp. (0.21%), Carnobacterium spp. (0.21%), Enterobacter spp. (0.64%), Escherichia spp. (0.21%), Lysinibacillus spp. (1.71%), Paenibacillus spp. (0.21%), Streptococcus spp. (1.07%), Stenotrophomonas spp. (0.43%), Vibrio spp. (0.21%), and Kocuria spp. (1.71%).

FIGURE 2

Commensals facilitated the growth of A. paragallinarum. (A) Representative image of the satellitism that commensal bacterium was surrounded by A. paragallinarum. (B) The satellitism profiles of commensals from IC chicken. (C) The colocalization profiles of commensals with strong growth-promoting effect on A. paragallinarum. The full names of genera were replaced by the abbreviation in this figure, and the full name of genera was listed in Supplementary Table 1.

FIGURE 3

Isolation is positively correlated with co-colonization and satellitism at genus-level. The pink shaded areas represented the 95% confidence intervals. (A) Isolation is positively correlated with colocalization; (B) Colocalization is positively correlated with satellitism; (C) Isolation is positively correlated with satellitism.

FIGURE 4

Commensals exhibited extensive hemolytic activity. The selected commensal bacteria were both exhibiting strong or medium growth-promoting effects and colocalizing with A. paragallinarum. (A) Five representative species were selected to determine the change of hemolysis on blood agar at 12, 24, 48, and 72 h; (B) distribution of hemolytic activity among 16 species commensals in supernatant test at 48 h. Hemolysis rate greater than 10% is considered positive hemolysis in this assay.

FIGURE 5

Genetic environment of mobile virulence and antimicrobial resistance gene in A. paragallinarum. Different types of antimicrobial resistance gene clusters were shown in above figure, and numbers followed by genetic profile represented the total numbers of similar gene clusters in all A. paragallinarum. (A) Profiles of tetB cluster in A. paragallinarum. The major functional proteins in Tn10 transposon, including TetB (tetracycline efflux protein), TetR (TetR family transcriptional regulator), TetC (transposon Tn10 TetC protein), TetD (transposon Tn10 TetD protein), and two insertion sequence IS10L and IS10R. Tn10 transposon in Shigella flexneri (accession: AF162223) and tetB hybrid sequence in Glaesserella parasuis (accession: CP069308) served as reference sequences for the comparison of tetracycline resistance gene cluster in A. paragallinarum. The gray shading genes indicated that these determinants were dissimilarity among other sequences. (B) Diversity of mobile gene cluster in A. paragallinarum. Mobile gene clusters excluding tetB clusters were categorized into nine sequence types.