Characterization of the contribution to virulence of three large plasmids of avian pathogenic Escherichia coli chi7122 (O78:K80:H9)

Abstract

Despite the fact that the presence of multiple large plasmids is a defining feature of extraintestinal pathogenic Escherichia coli (ExPEC), such as avian pathogenic E. coli (APEC), and despite the fact that these bacteria pose a considerable threat to both human and animal health, characterization of these plasmids is still limited. In this study, after successfully curing APEC of its plasmids, we were able to investigate, for the first time, the contribution to virulence of three plasmids, pAPEC-1 (103 kb), pAPEC-2 (90 kb), and pAPEC-3 (60 kb), from APEC strain chi7122 individually as well as in all combinations in the wild-type background. Characterization of the different strains revealed unique features of APEC virulence. In vivo assays showed that curing the three plasmids resulted in severe attenuation of virulence. The presence of different plasmids and combinations of plasmids resulted in strains with different pathotypes and levels of virulence, reflecting the diversity of APEC strains associated with colibacillosis in chickens. Unexpectedly, our results associated the decrease in growth of some strains in some media with the virulence of APEC, and the mechanism was associated with some combinations of plasmids that included pAPEC-1. This study provided new insights into the roles of large plasmids in the virulence, growth, and evolution of APEC by showing for the first time that both the nature of plasmids and combinations of plasmids have an effect on these phenomena. It also provided a plausible explanation for some of the conflicting results related to the virulence of ExPEC strains. This study should help us understand the virulence of other ExPEC strains and design more efficient infection control strategies.

FIG. 1.

Plasmid and LPS profiles of different strains. (A) Plasmid profiles of strains in a 0.5% agarose gel stained with ethidium bromide. (B) LPS profiles of different strains in a silver-stained SDS-PAGE gel. Lane L, four plasmids (147 kb, 163 kb, 35.85 kb, and 6.9 kb) of strain 39R681 used as a ladder; lane 1, χ7122; lane 2, χ7368; lane 3, χ7394; lane 4, χ7392; lane 5, χ7367; lane 6, χ7561; lane 7, χ7562; lane 8, χ7274; lane 9, χ7145; lane 10, χ7167; lane 11, χ7193. Abbreviations: pAPEC-1,2,3, pAPEC-1, pAPEC-2, and pAPEC-3; pAPEC-1,2, pAPEC-1 and pAPEC-2; pAPEC-1,3, pAPEC-1 and pAPEC-3.

FIG. 2.

Iron uptake by different strains. (A) Iron uptake on CAS agar. (B) Diameters of orange haloes around colonies on CAS agar incubated for different times. Colony 1, χ7122; colony 2, χ7368; colony 3, χ7394; colony 4, χ7392; colony 5, χ7367; colony 6, χ7561; colony 7, χ7562; colony 8, χ7274; colony 9, χ6092; colony 10, χ7346; colony 11, χ7347; colony 12, χ7348. Abbreviations: pAPEC-1,2,3, pAPEC-1, pAPEC-2, and pAPEC-3; pAPEC-1,2, pAPEC-1 and pAPEC-2; pAPEC-1,3, pAPEC-1 and pAPEC-3; −, no plasmids.

FIG. 3.

Growth of bacteria in iron-restricted media. Bacteria were grown in LB medium containing 2,2′-dipyridyl at 37°C for 24 h. (A) Strains with wild-type background. (B) Strains with E. coli K-12 background. The data were obtained from at least 3 independent experiments in which each strain was tested in triplicate. Abbreviations: pAPEC-1,2,3, pAPEC-1, pAPEC-2, and pAPEC-3; pAPEC-1,2, pAPEC-1 and pAPEC-2; pAPEC-1,3, pAPEC-1 and pAPEC-3; −, no plasmids.

FIG. 4.

Genetic organization of the ABC ferrous iron feo (A) and enterobactin (B) regions of APEC χ7122. The black arrows represent known genes, the white arrows represent hypothetical protein genes, and the gray arrows represent insertion sequence genes.

FIG. 5.

Coomassie brilliant blue-stained SDS-PAGE profiles of outer membranes proteins (OMPs) of strains. (A and B) Bacteria with the wild-type background grown in the presence (A) or absence (B) of iron. Lane L, standard molecular weight markers (Bio-Rad); lane 1, χ7122; lane 2, χ7368; lane 3, χ7394; lane 4, χ7392; lane 5, χ7367; lane 6, χ7561; lane 7, χ7562; lane 8, χ7274. (C) Bacteria with the E. coli K-12 background grown in the absence of iron. Lane 1, χ 6092; lane 2, χ7346. The arrowheads indicate the IROMP bands. Abbreviations: pAPEC-1,2,3, pAPEC-1, pAPEC-2, and pAPEC-3; pAPEC-1,2, pAPEC-1 and pAPEC-2; pAPEC-1,3, pAPEC-1 and pAPEC-3.

FIG. 6.

Serum complement resistance of strains. The survival percentage was determined for each strain following incubation in 90% guinea pig (GP) or chicken (Ch) serum. (A) Bacteria with the wild-type background. (B) Bacteria with the E. coli K-12 background. The data were obtained from at least 3 independent experiments in which each strain was tested in triplicate. The error bars indicate the standard errors of the means. Significant differences are indicated by asterisks (*, P < 0.05 compared to the parent strain; **, P < 0.005 compared to the parent strain). Abbreviations: pAPEC-1,2,3, pAPEC-1, pAPEC-2, and pAPEC-3; pAPEC-1,2, pAPEC-1 and pAPEC-2; pAPEC-1,3, pAPEC-1 and pAPEC-3; −, no plasmids.

FIG. 7.

Pathogenicities of different strains in 1-day-old chicks. The survival percentages were evaluated for groups of chicks inoculated subcutaneously at 6 days after inoculation with either wild-type strain χ7122 or its plasmid-containing derivatives. Strains were classified as low-virulence (I), moderately virulent (II), and highly virulent (III) strains. Abbreviations: pAPEC-1,2,3, pAPEC-1, pAPEC-2, and pAPEC-3; pAPEC-1,2, pAPEC-1 and pAPEC-2; pAPEC-1,3, pAPEC-1 and pAPEC-3; −, no plasmids.

FIG. 8.

Growth curves for different strains in different media. The amounts of growth of wild-type strain χ7122 and its plasmid-containing derivatives in different media (LB medium, MM9 medium, RPMI-1640, and GFTS-2 medium) at different times were compared. The data were obtained from at least 3 independent experiments in which each strain was tested in triplicate. Statistically significant differences compared with the wild-type strain are indicated by an asterisk (P < 0.001).