APEC Pathogenesis

We have found that large plasmids (extrachromosomal ´accessory´ DNA present in some bacteria) are a defining trait of the APEC pathotype, occurring in ~80% of APEC and harboring many of the genes thought to be linked to APEC´s abilities to cause disease and resist therapy. Such plasmids are often transmissible to recipient bacteria, and acquisition of these plasmids results in an enhancement of the recipient´s virulence and resistance (Rodriguez-Siek et al 2005a; Johnson et al 2005, Johnson et al 2006 a, b,c; Skyberg et al 2006, Skyberg et al 2008; Johnson et al 2010). For these reasons alone, study of these plasmids seems justifiable, but other reasons exist to study these plasmids. For instance, E. coli, containing large R (resistance) plasmids, have been found in rivers, irrigation waters, and sediments polluted by agricultural and municipal run-offs, where they may enter the water and food supply (Cernat 2002 a,b; Roe 2003). Once there, they may become involved in human disease or serve as reservoirs of virulence and resistance genes for human pathogens. Too, analysis of bacterial chromosomes has revealed a more prominent role of plasmids in bacterial evolution than previously recognized (Smets and Barclay 2005). Despite this new appreciation for the role of plasmids in bacterial evolution, plasmid genomics continues to lag behind that of chromosomal genomics. Given the importance of plasmids to animal and human disease, environmental processes, antimicrobial resistance, and microbial evolution, this paucity of useable plasmid genome data is a serious oversight. Therefore, in order to clarify the role of APEC-like plasmids in disease, in the evolution of APEC and other pathogens, and as reservoirs of virulence and resistance genes for human disease, we are working to increase the numbers of plasmid genomes in public databases and enhance the tools for their analysis. To date, we have sequenced a number of APEC plasmids (Johnson et al 2005, 2006 a, b, c; 2010), and several have been characterized for their contributions to disease (e.g., Skyberg et al 2006, 2008; Johnson et al 2010). We are exploiting their presence to track APEC in the environment (Johnson et al 2009), assess bacterial changes over time (unpublished data), and improve APEC diagnostics and control (Lynne et al 2006; Johnson et al 2008). Also, by studying APEC isolated over the past 40 years for certain plasmid genes, we have demonstrated that APEC have become better able to cause disease and resist therapy over time (unpublished data).

We seek to enhance food security through improved understanding of APEC pathogenesis with the ultimate goal of improving control of avian colibacillosis. Colibacillosis is an economically devastating disease for the world´s poultry industries due to mortality, morbidity, condemnations at processing, and decreased production. Recent completion of the APEC and chicken genomes and generation of APEC and chicken DNA microarrays and other expression technologies has provided an unprecedented opportunity to understand the mechanisms of APEC pathogenesis, the relationship between APEC and its host during infection, and the traits that underlie effective host resistance to APEC infection. Such "systems approaches" will provide a degree of insight into the APEC: chicken interaction that has not been previously achievable. As a result of such studies, it is anticipated that designers of future colibacillosis control strategies can target both host and pathogen, which should result in improved control of this important disease.

To date, some of the things we know about APEC virulence is that these organisms possess various virulence traits enabling their extraintestinal lifestyle. APEC have acquired genes by horizontal gene transfer that encode virulence traits and distinguish APEC from avian fecal E. coli commensals (Rodriguez-Siek et al 2005a). These genes are often clustered into chromosomal or plasmid pathogenicity islands (Barnes et al 2008). Portions of plasmid-located pathogenicity islands occur widely among APEC isolated from different parts of the world (e.g., Maurer et al 1998; Janssen et al 2001; Altekruse et al 2002; Delicato et al 2002; Delicato et al 2003; Ewers et al 2004; Yang et al 2004; McPeake et al 2005; Rodriguez Siek et al 2005a, b; Vandkerchove et al 2005; Zhao et al 2005), various avian host species (e.g., Altekruse et al 2002; McPeake et al 2005; Rodriguez Siek et al 2005 a, b), and different syndromes (e.g., De Brito et al 2003; Rodriguez Siek et al 2005a) and are considered a defining trait of the APEC pathotype (Rodriguez Siek et al 2005a; Johnson et al 2008). Other virulence factors have been described in APEC, including adhesins (e.g., Naveh et al 1984; Yerushalmi et al 1990; Arne et al 2000, Babai et al 2000; Gophna et al 2001a; La Ragione et al 2002; Lymberopoulos et al 2006), toxins (e.g., Parreira et al 1998; Salvadori et al 2001a,b; Parreira et al 2002; La Ragione et al 2002, Rodriguez Siek et al 2005 a,b; Rosario et al 2004), and hemolysins (e.g., Nagai et al 1998; Reingold et al 1999; Altekruse et al 2002; Morales et al 2004; Wyborn et al 2004; Rodriguez Siek et al 2005 a,b; McPeake et al 2005; Johnson et al 2006a,b). Also, the ability of APEC to obtain iron is well documented and likely due to several iron acquisition mechanisms (e.g., Dho and Lafont 1984; Lafont et al 1987; Wittig et al 1988; Gophna et al 2001b; Dozois et al 2003; Sabri et al 2006). In addition, ability to resist serum is a common trait among APEC, regardless of the syndrome or host of origin (Nolan et al 2003). Though a recent transcriptome analysis of an APEC following growth in serum failed to confirm the role of certain traits expected to contribute to serum resistance, several other virulence genes; genes involved in adaptive metabolism, protein transport, biosynthesis pathways, stress resistance, virulence regulation; and genes of unknown function, including those found in a plasmid pathogenicity island were identified as contributing to survival in serum (Li et al 2010). In addition, APEC´s ability to resist phagocytes is another important virulence determinant (e.g., Kottom et al 1997; Mellata et al 2003).

Extraintestinal pathogenic Escherichia coli (ExPEC), including uropathogenic E. coli, neonatal meningitis E. coli, and septicemia-causing E. coli, are responsible for significant morbidity and mortality in human beings, resulting in hundreds of thousands of deaths and millions of days of lost productivity annually (Russo and Johnson 2003). Every year in the US alone, it is estimated that ExPECcaused diseases result in over $4 billion in direct healthcare costs (Russo and Johnson 2003). Thus, no matter how we measure the costs of ExPEC-caused diseases, they are unacceptably high. While it is generally agreed that the source of ExPEC causing human disease is the victim´s own colonic flora, it is not known how ExPEC come to inhabit the colon. Similarities in APEC and ExPEC in the diseases they cause, content of virulence traits, and genomic sequences have led to the hypothesis that ExPEC enter the colon following ingestion of APECcontaminated food. This contention is based on the similarities between human ExPEC and APEC in terms of genomic sequences, virulence genotypes, serogroups, phylogenetic groups, antimicrobial susceptibilities, diseases caused, and a documented history of transmission of E. coli and their plasmids from poultry to humans (Levy et al 1976; Linton et al 1977; Ojeniyi 1989; van den bogaard et al 2001; Rodriguez Siek et al 2005b; Johnson et al 2007). To date, we have shown that some APEC are more like human ExPEC than human ExPEC are like each other (Johnson et al 2007), APEC-like organisms enter the human food chain on contaminated poultry (Johnson et al 2009), APEC cause urinary tract infections and neonatal meningitis in models of human disease (Tivendale et al 2010; unpublished data), and APEC plasmids are transmissible to human ExPEC and enable E. coli to grow in human urine (Skyberg et al 2006) and cross the blood brain barrier (a trait required for a bacterium to cause meningitis) (Johnson et al 2010). Thus, we still cannot eliminate the possibility that APEC-contaminated poultry is a food-borne source of human ExPEC.

Altekruse, S. F. , Elvinger, F., DebRoy, C., Pierson, F.W., Eifert, J.D. and Sriranganathan, N. (2002) Pathogenic and fecal Escherichia coli strains from turkeys in a commercial operation. Avian Dis. 46, 562-9.

Arne, P., Marc, D., Bree, A., Schouler, C. and Dho-Moulin, M. (2000) Increased tracheal colonization in chickens without impairing pathogenic properties of avian pathogenic Escherichia coli MT78 with a fimH deletion. Avian Dis. 44, 343-55.

Babai, R., Stern,B.E., Hacker, J., and Ron, E.Z. (2000) New fimbrial gene cluster of Sfimbrial adhesin family. Infect. Immun. 68, 5901-7.

Barnes, J., Nolan, L.K., and Vaillancourt, J.P. (2008) Colibacillosis In Diseases of Poultry

Blackwell pp 691-732 A. F. Y.M. Saif, J. Glisson, I. McDonald, L.K. Nolan, and D. Swayne (eds)

de Brito, B.G., Gaziri, L.C., and Vidotto, M.C. (2003) Virulence factors and clonal relationships among Escherichia coli strains isolated from broiler chickens with cellulitis. Infect. Immun. 71, 4175-7.

Cernat, R., Lazăr, V., Balotescu, C., Cotar, A., Coipan, E., Cojocaru, C. (2002a) Clonal analysis of some multiple antibiotic resistant E. coli strains isolated from river and polluted waters. Bacteriol Virusol Parazitol Epidemiol. 47, 179-84.

Cernat, R., Lazăr, V., Balotescu, C., Cotar, A., Coipan, E., Cojocaru, C. (2002b) Distribution and diversity of conjugative plasmids among some multiple antibiotic resistant E. coli strains isolated from river waters. Bacteriol Virusol Parazitol Epidemiol. 47, 147-53.

Delicato, E.R., de Brito, B.G., Konopatzki, A.P., Gaziri, L.C., and Vidotto, M.C. (2002) Occurrence of the temperature-sensitive hemagglutinin among avian Escherichia coli. Avian Dis. 46, 713-6.

Delicato, E.R., de Brito, B.G., Gaziri, L.C., and Vidotto, M.C. (2003) Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis. Vet. Microbiol. 94, 97-103.

Dho, M. and Lafont, J.P. (1984) Adhesive properties and iron uptake ability in Escherichia coli lethal and nonlethal for chicks. Avian Dis. 28, 1016-25.

Dozois, C.M., Daigle, F. and Curtiss, 3rd , R. (2003) Identification of pathogen-specific and conserved genes expressed in vivo by an avian pathogenic Escherichia coli strain. Proc. Natl. Acad. Sci. U S A 100, 247-52.

Ewers, C., Janssen, T., Kiessling, S., Philipp, H.C. and Wieler, L.H. (2004) Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet. Microbiol. 104, 91-101.

Gophna, U., Barlev, M., Seijffers, R., Oelschlager, T.A., Hacker, J. and Ron, E.Z. (2001a)

Curli Fibers Mediate Internalization of Escherichia coli by Eukaryotic Cells. Inf. Immun. 69, 2659-2665.

Gophna, U., Oelschlaeger, T.A., Hacker, J. and Ron, E.Z. (2001b) Yersinia HPI in septicemic Escherichia coli strains isolated from diverse hosts. FEMS. Microbiol. Lett. 196, 57-60.

Janssen,T., Schwarz, C., Preikschat, P., Voss, M., Philipp, H-C., and Wieler, L.H. (2001)

Virulence-associated genes in avian pathogenic Escherichia coli (APEC) isolated from internal organs of poultry having died from colibacillosis. Int. J. Med. Micr. 291, 371-378.

Johnson, T.J., Siek, K.E., Johnson, S.J. and Nolan, L.K. (2005) DNA sequence and comparative genomics of pAPEC-O2-R, an avian pathogenic Escherichia coli transmissible R plasmid. Antimicrob Agents Chemother. 49, 4681-8

Johnson,T.J., Siek, K.E., Johnson, S.J. and Nolan, L.K. (2006a) DNA sequence of a ColV plasmid and prevalence of selected plasmid-encoded virulence genes among avian Escherichia coli strains. J. Bacteriol. 188, 745-758.

Johnson, T.J., Johnson, S.J. and Nolan, L.K. (2006b) Complete DNA sequence of a ColBM plasmid from avian pathogenic Escherichia coli suggests that it evolved from closely related ColV virulence plasmids. J. Bacteriol. 188, 5975-5983.

Johnson, T.J., Wannemeuhler, Y.M., Scaccianoce, J.A., Johnson, S.J., Nolan, L.K. (2006c) Complete DNA sequence, comparative genomics, and prevalence of an IncHI2 plasmid occurring among extraintestinal pathogenic Escherichia coli isolates. Antimicrob Agents Chemother. 50, 3929-33.

Johnson, T.J., Wannemuehler, Y., Doetkott, C., Johnson, S.J., Rosenberger, S.C., Nolan, L.K. (2008) Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J Clin Microbiol. 46, 3987-96.

Johnson, T.J., Logue, C.M., Wannemuehler, Y., Kariyawasam, S., Doetkott,C., DebRoy, C., White, D.G. and Nolan, L.K. (2009) Examination of the source and extended virulence genotypes of Escherichia coli contaminating retail poultry meat. Foodborne Pathog. Dis. 6, 657-67.

Johnson, T.J., Jordan, D., Kariyawasam, S., Stell, A.L., Bell, N.P., Wannemuehler, Y.M., Alarcón, C.F., Li, G., Tivendale, K.A., Logue, C.M., Nolan, L.K. (2010) Sequence analysis and characterization of a transferable hybrid plasmid encoding multidrug resistance and enabling zoonotic potential for extraintestinal Escherichia coli. Infect Immun. 78, 1931-42.

Kottom, T.J., Nolan, L.K., Robinson, M., Brown, J., Gustad, T., Horne, S.M. and Giddings, C.W. (1997) Further characterization of a complement-sensitive mutant of a virulent avian Escherichia coli isolate. Avian Dis. 41, 817-23.

Lafont, J.P., Dho, M., D´Hauteville, H.M., Bree, A. and Sansonetti, P.J. (1987) Presence and expression of aerobactin genes in virulent avian strains of Escherichia coli. Infect. Immun. 55, 193-7.

La Ragione, R.M. and Woodward, M.J. (2002) Virulence factors of Escherichia coli serotypes associated with avian colisepticaemia. Res.Vet.Sci. 73, 27-35.

Levy, S.B., FitzGerald, G.B. and Macone, A.B. (1976) Spread of antibiotic-resistant plasmids from chicken to chicken and from chicken to man. Nature 260, 40-42.

Li, G., Tivendale, K.A., Liu, P., Feng, Y., Wannemuehler, Y., Cai, W., Mangiamele, P., Johnson, T.J., Constantinidou, C., Penn, C.W., Nolan, L.K. (2011). Transcriptome analysis of avian pathogenic Escherichia coli O1 in chicken serum reveals adaptive responses to systemic infection. Infect Immun. 79, 1951-60.

Linton, A.H., Howe, K., Bennett, P.M., Richmond, M.H. and Whiteside, E.J. (1977) The colonization of the human gut by antibiotic resistant Escherichia coli from chickens

J. Appl. Bact. 43, 465-469.

Lymberopoulos, M.H., Houle, S., Daigle, F., Leveille, S., Bree, A., Moulin-Schouleur, M., Johnson, J.R. and Dozois, C.M. (2006) Characterization of Stg fimbriae from an avian pathogenic Escherichia coli O78:K80 strain and assessment of their contribution to colonization of the chicken respiratory tract. J. Bacteriol. 188, 6449-59.

Lynne, A.M., Foley, S.L., Nolan, L.K. (2006) Immune response to recombinant Escherichia coli Iss protein in poultry. Avian Dis. 50, 273-6.

Maurer, J.J., Brown, T.P., Steffens, W.L., and Thayer, S.G. (1998) The occurrence of ambient temperature-regulated adhesins, curli, and the temperature-sensitive hemagglutinin tsh among avian Escherichia coli. Avian Dis. 42, 106-18.

Mellata, M., Dho-Moulin, M., Dozois, C.M., Curtiss, 3rd, R., Lehoux, B., and Fairbrother, J.M. (2003) Role of avian pathogenic Escherichia coli virulence factors in bacterial interaction with chicken heterophils and macrophages. Infect. Immun. 71, 494-503.

Morales, C., Lee, M.D., Hofacre, C., and Maurer, J.J. (2004) Detection of a novel virulence gene and a Salmonella virulence homologue among Escherichia coli isolated from broiler chickens. Foodborne Pathog. Dis. 1, 160-5.

McPeake, S.J., Smyth, J.A., and Ball, H.J. (2005) Characterisation of avian pathogenic Escherichia coli (APEC) associated with colisepticaemia compared to faecal isolates from healthy birds. Vet. Microbiol. 110, 245- 53.

Nagai, S., Yagihashi, T., and Ishihama, A. (1998) An avian pathogenic Escherichia coli strain produces a hemolysin, the expression of which is dependent on cyclic AMP receptor protein gene function. Vet. Microbiol. 60, 227-38.

Naveh, M.W., Zusman, T., Skutelsky, E., and Ron, E.Z. (1984) Adherence pili in avian strains of Escherichia coli: effect on pathogenicity. Avian Dis. 28, 651-661.

Nolan, L.K., Horne, S.M., Giddings, C.W., Foley, S.L., Johnson, T.J., Lynne, A.M. and Skyberg, J.A. (2003) Resistance to serum complement, iss, and virulence of avian Escherichia coli. Vet. Res. Commun. 27, 101- 10.

Ojeniyi , A.A. (1989) Direct transmission of Escherichia coli from poultry to humans. Epidemiol. Infect. 103, 513-22.

Parreira, V., Arns, C., and Yano, T. (1998) Virulence factors of avian Escherichia coli associated with swollen head syndrome. Avian Path. 27, 148-154.

Parreira, V.R., and Gyles, C.L. (2002) Shiga toxin genes in avian Escherichia coli Vet. Microbiol. 87, 341-52.

Reingold, J., Starr, N., Maurer, J., and Lee, M.D. (1999) Identification of a new Escherichia coli She haemolysin homolog in avian E. coli. Vet. Microbiol. 66, 125-34.

Rodriguez-Siek, K.E., Giddings, C.W., Doetkott, C., Johnson, T.J. and Nolan, L.K. (2005a) Characterizing the APEC pathotype. Vet. Res. 36, 241-256.

Rodriguez-Siek, K.E., Giddings, C.W., Doetkott, C., Johnson, T.J., Fakhr, M.K. and Nolan, L.K. (2005b) Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis. Microbiol. 151, 2097-2110.

Roe, M.T., Vega, E., Pillai, S.D. (2003) Antimicrobial resistance markers of class 1and class 2 integron-bearing Escherichia coli from irrigation water and sediments. Emerg Infect Dis. 9, 822-6.

Rosario, C.C., Lopez, A.C., Tellez, I.G., Navarro, O.A., Anderson, R.C., and Eslava, C.C. (2004) Serotyping and virulence genes detection in Escherichia coli isolated from fertile and infertile eggs, dead-in-shell embryos, and chickens with yolk sac infection. Avian Dis. 48, 791-802.

Russo, T.A. and Johnson, J.R. (2003) Medical and economic impact of extraintestinal infections due to Escherichia coli: focus on an increasingly important endemic problem. Microbes Infect. 5, 449-56.

Sabri, M., Leveille, S. and Dozois, C.M. (2006) A SitABCD homologue from an avian pathogenic Escherichia coli strain mediates transport of iron and manganese and resistance to hydrogen peroxide. Microbiol. 152, 745-758

Salvadori, M.R., Yamada, A.T., and Yano, T. (2001a) Morphological and intracellular alterations induced by cytotoxin VT2y produced by Escherichia coli isolated from chickens with swollen head syndrome. FEMS Microbiol. Lett. 197, 79-84.

Salvadori, M.R., Yano, T., Carvalho, H.E., Parreira, V.R., and Gyles, C.L. (2001b) Vacuolating cytotoxin produced by avian pathogenic Escherichia coli. Avian Dis. 45,43-51.

Skyberg, J.A., Johnson, T.J., Johnson, J.R., Clabots, C., Logue, C.M., Nolan, L.K. (2006) Acquisition of avian pathogenic Escherichia coli plasmids by a commensal E. coli isolate enhances its abilities to kill chicken embryos, grow in human urine and colonize the murine kidney. Infect Immun. 74, 6287- 92.

Skyberg, J.A., Johnson, T.J., Nolan, L.K. (2008) Mutational and transcriptional analyses of avian pathogenic Escherichia coli ColV plasmid. BMC Microbiol. 29, 24.

Smets, B.F. and Barkay, T. (2005) Horizontal gene transfer: perspectives at a crossroads of scientific disciplines. Nat.Rev.Microbiol. 3, 675-678.

Tivendale, K.A., Logue, C.M., Kariyawasam, S., Jordan, D., Hussein, A., Li, G., Wannemuehler, Y., and Nolan, L.K. (2010) Avian-pathogenic Escherichia coli strains are similar to neonatal meningitis E. coli strains and are able to cause meningitis in the rat model of human disease. Infect. Immun. 78, 3412-9.

van den Bogaard, A.E., London, N., Driessen, C., and Stobberingh, E.E. (2001) Antibiotic resistance of faecal Escherichia coli in poultry, poultry farmers and poultry slaughterers. J. Antimicrob. Chemother. 47, 763-771.

Vandekerchove, D., Vandemaele, F., Adriaensen, C., Zaleska, M., Hernalsteens, J.P., Baets, L.D., Butaye, P., Immerseel, F.V., Wattiau,P., Laevens, H., Mast, J., Goddeeris, B. and Pasmans, F. (2005) Virulenceassociated traits in avian Escherichia coli: Comparison between isolates from colibacillosis-affected and clinically healthy layer flocks. Vet.Microbiol. 108, 75-87.

Wittig, W., Prager, R., Tietze, E., Seltmann, G. and Tschape, H. (1988) Aerobactin-positive Escherichia coli as causative agents of extraintestinal infections among animals. Arch.exper.Vet.med. 42, 221-29.

Wyborn, N.R., Clark, A., Roberts, R.E., Jamieson, S.J., Tzokov, S., Bullough, P.A., Stillman, T.J., Artymiuk, P.J., Galen, J.E., Zhao, L., Levine, M.M., and Green, J. (2004) Properties of haemolysin E (HlyE) from a pathogenic Escherichia coli avian isolate and studies of HlyE export. Microbiol. 150, 1495- 505.

Yang, H., Chen, S., White, D.G., Zhao, S., McDermott, P., Walker, R. and Meng, J. (2004)

Characterization of multiple-antimicrobialresistant Escherichia coli isolates from diseased chickens and swine in China. J. Clin. Microbiol. 42, 3483-9.

Yerushalmi, Z., Smorodinsky, N.I., Naveh, M.W. and Ron, E.Z. (1990) Adherence pili of avian strains of Escherichia coli O78. Infect. Immun. 58, 1129-31.

Zhao, S., Maurer, J.J., Hubert, S., De Villena, J.F., McDermott, P.F., Meng, J., Ayers, S., English, L., and White, D.G. (2005) Antimicrobial susceptibility and molecular characterization of avian pathogenic Escherichia coli isolates. Vet. Microbiol. 107, 215-24.