J. Med. Microbiol. Ð Vol. 49 92000), 327±338 # 2000 The Pathological Society of Great Britain and Ireland ISSN 0022-2615

BACTERIAL PATHOGENESIS

The role of ®mbriae and ¯agella in the adherence of avian strains of Escherichia coli O78:K80 to tissue culture cells and tracheal and gut explants

R. M. LA RAGIONE, W. A. COOLEY and M. J. WOODWARD

Bacteriology Department, Veterinary Laboratories Agency Weybridge), New Haw, Addlestone, Surrey KT15 3NB

To investigate the role of ®mbriae and ¯agella in the pathogenesis of avian colibacillosis, isogenic insertionally inactivated mutant strains of Escherichia coli O78:K80 strain EC34195 defective in the elaboration of type-1 and curli ®mbriae and ¯agella were constructed by allelic exchange. Single and multiple non-®mbriate and non-¯agellate mutant strains were compared to the wild-type in vitro in adherence assays with a HEp-2 cell line, a mucus-secreting cell line HT2916E, a non-mucus-secreting cell line HT2919A, tracheal explant and proximal gut explant. Mutant strains defective in the elaboration of type-1 ®mbriae were signi®cantly less adherent ± in the order of 90% reduction ± than the wild-type strain in all assays. Mutant strains defective in the elaboration of ¯agella were generally as adherent as the wild-type strain except when assayed with the mucus-secreting cell line HT2916E, for which a signi®cant reduction of adherence ± of the order of 90% ± compared with the wild-type strain was observed. Mutant strains defective for the elaboration of curli ®mbriae adhered as well as the wild-type strain in all assays, except when assayed in tests with gut explant tissue for which a signi®cant reduction of adherence ± of the order of 80% ± compared with the wild-type strain was observed. Adherence to explants was to epithelial, not serous, surfaces and was 10-fold greater to tracheal than to gut explants. Together, these data support the hypothesis that type-1 ®mbriae are signi®cant factors in adherence, aided by ¯agella for penetration of mucus and curli ®mbriae for adherence to the gut.

Introduction Adherence is a necessary prerequisite for bacterial colonisation and pathogenesis. To withstand the mech- Escherichia coli is an important cause of disease in anical cleansing and bulk ¯ow associated with mammals, birds and reptiles [1]. In domestic poultry, intestinal mucosal surfaces, bacteria bind speci®cally avian colibacillosis is frequently associated with E. coli to epithelial and mucosal surfaces [8], often by strains of serotypes O78:K80, O1:K1 and O2:K1 [2]. proteinaceous surface appendages called ®mbriae. Infections commonly occur via the respiratory tract, Avian isolates of E. coli commonly express type-1, P often after a primary bacterial or viral infection or as a and curli ®mbriae and commonly possess ¯agella result of poor husbandry practices. The disease which confer motility [9]. Type-1 ®mbriae confer generally affects broilers between 3 and 10 weeks of mannose-sensitive haemagglutination, whereas P ®m- age and is associated with high morbidity and briae confer mannose-resistant haemagglutination. Both mortality. Carcass condemnation at slaughter is com- type-1 and P ®mbriae have been cited as important mon [3]. The most severe manifestation of avian factors for adherence to epithelial cell surfaces in vitro colibacillosis is septicaemia which is characterised by and in vivo [3, 10±14] and type-1 ®mbriae are also pericarditis, perihepatitis, air sacculitis and salpingitis thought to be important in the initial stages of [4±7]. infections of poultry by E. coli strains of serogroups O1 and O2 [8, 11, 15±17]. The role of curli ®mbriae in poultry infection by E. coli remains unstudied, although it has been demonstrated that in vitro they Received 15 June 1999; accepted 7 Sept. 1999. mediate binding to ®bronectin, laminin, plasminogen, Corresponding author: Professor M. J. Woodward 9e-mail: plasminogen activator proteins and type-1 collagen [email protected]). [18±22]. The role of ¯agella in the pathogenesis of 328 R. M. LA RAGIONE ET AL. avian colibacillosis is unclear, because isolates are supplemented with Xgal 9Promega) 25 mg=ml and often non-¯agellate. ampicillin 9Penbrithin, Beecham) 100 ìg=ml, was used in all cloning procedures. Minimal medium was M9 Avian strains of E. coli adhere to tracheal sections and salts medium supplemented with glucose 0.2% w=v epithelial cell lines [7, 10, 11, 15, 23, 24]. Other tissue- and 10 mM magnesium sulphate. Bacteria for tissue- culture and organ-explant approaches have been used culture adhesion and invasion assays were cultured to study the initial events of colonisation of different statically in HIB at 378C for 48 h. They were prepared pathotypes of E. coli in vitro [25±29]. Recent studies for adhesion by centrifugation at 4000 rpm for 10 min of avian colibacillosis have focused on the role of at ambient temperature and resuspended in phosphate- individual kinds of surface antigens [3, 7, 11]. By buffered saline 9PBS) to an optical density 9540 nm) of contrast, the aim of the present study was to investigate 1.2 absorbance units. Bacteria for tissue-explant the combined role of ®mbriae and ¯agella in adhesion assays were cultured as for tissue-culture pathogenesis. As a ®rst step, a reverse genetics assays and were resuspended in PBS to an optical approach was used to produce a library of non- density 9550 nm) of 0.6 absorbance units. In some ®mbriate and non-¯agellate single and multiple iso- experiments, bacteria were cultured aerobically in genic mutant strains of a clinical isolate of an avian Colonisation Factor Antigen 9CFA) Broth 9Oxoid) at strain of E. coli O78:K70. In this paper the construc- 258C for 72 h. tion of the library and its use in various tissue-culture and organ-explant adherence assays are described. Phenotypic analysis of E. coli isolates Procedures for mannose-sensitive and -resistant haem- Materials and methods agglutination, motility, agglutination reactions, anti- Bacterial isolates and media biotic resistance, ®bronectin and Congo red binding, curli ®mbriae ELISA and transmission and scanning Wild-type strains of E. coli O78:K80 were obtained electron microscopy were as described previously from the E. coli Reference Laboratory, VLA, Wey- [32, 33, 36±38]. bridge 9Table 1). Strain EC34195 was used in all mutagenesis studies. Bacterial cultures were maintained Genetic methods on Dorset's egg slopes at 208C and stored at À808Cin heart infusion broth 9HIB) supplemented with glycerol Genetic methods, including plasmid and chromosomal 30% v=v. Working cultures were maintained at 48Con DNA extraction and puri®cation, restriction endo- sheep blood 95%) agar, supplemented with antibiotics nuclease digestion, agarose gel electrophoresis, liga- as appropriate. Luria Bertani 9LB) Agar 9Oxoid), tion, preparation of electro-competent bacterial cells,

Table 1. Plasmids and bacteria used Plasmids and strains Comments Source pBLUESCRIPT Cloning vector Commercially available 9Stratagene) pCRSRIPT Direct PCR cloning vector Commercially available 9Stratagene) pBSL30 Source of strr [30] pBSL141 Source of genr [30] Source of camr Commercially available 9Stratagene) Source of kanr Commercially available 9Pharmacia) pJP5603 kanr suicide vector [31] pERFORM KpJP5603 with modi®ed MCS [32, 33] pERFORM C pJP5603 with modi®ed MCS and kanr replaced by camr [32, 33] pERFORM G pERFORM C with camr replaced by genr This study E. coli JM109 ë pir Cloning strain. Genotype: e14À 9mcrAÀ) recA 1 end A1 gyrA96thi 1 [31] ‡ q hsd R17 9rk-mk ) supE44 relA1Ä 9lac-proAB) [F'traD36 proAB lac zÄM15] ë pir E. coli S-17 pir Mobilising strain. Genotype: pro res- mod‡ RP42ÀTc::MuÀ Km::Tn7 ë pir [34] EC43297 Human urinary tract isolate This study EC54597 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC5197 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC24197 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC24297 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC24397 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC39597 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC43197 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC47897 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC30896 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC26296 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC34195 Wild-type avian E. coli O78:K80 strain [35] EC2597 Wild-type avian E. coli O78:K80 strain VLA Weybridge EC5497 Wild-type avian E. coli O78:K80 strain VLA Weybridge ADHERENCE OF E. COLI O78:K08 329 electroporation, lysis of bacterial colonies for colony from E. coli strain EC34195 of O78:K80 as a template. dot-blot hybridisation, Southern hybridisation and Amplicons were cloned into pCR-Script plasmid vector autoradiography were as described previously [39]. 9Stratagene) with E. coli K12 strain DH5á 9Gibco Probes for radiolabelled probing were either puri®ed BRL) as host. Plasmids were mapped by restriction PCR products or entire plasmids. endonuclease digestion and DNA nucleotide sequences were con®rmed as described above. Antibiotic resis- PCR tance cassettes were cloned into appropriate unique restriction sites in the centre of each gene. Insertionally Primers were designed to amplify ®mC, ¯iC, papA and inactivated genes were subcloned into appropriate csgA 9Table 2). PCR reactions were set up in 50-ìl pERFORM plasmid vectors [33] and electroporated volumes containing water, Thermo DNA polymerase into the permissive auxotrophic host E. coli K12 strain reaction buffer 9Promega), 200 ìM dNTPs 9Pharmacia), S-17 ë pir with selection for both the antibiotic 1.5 mM MgCl2, thermostable Taq polymerase 9Prome- resistance marker encoded by the resistance cassette ga) 2.5 units, 10 pmols of each primer and total and the antibiotic resistance marker conferred by the genomic DNA 1 ng from E. coli O78:K80 strain suicide vector antibiotic gene. EC34195. Reactions were overlaid with mineral oil 9Promega) 50 ìl and cycling was carried out in a Conjugation thermo-cycler 9Biometra) with the following pro- gramme: initial denaturation at 958C for 5 min followed The pERFORM vectors harbouring antibiotic resis- by 30 cycles of denaturing for 2 min at 958C, annealing tance-marked, insertionally inactivated genes were at 568C for 1.5 min, extension at 728C for 2 min, and transferred by conjugation from the permissive host thereafter by a ®nal extension at 728C for 10 min. PCR E. coli K12 strain S-17 ë pir into the non-permissive products were stored at 48C until required. E. coli O78:K80 recipient strain EC34195 by ®lter mating as described before [33] with selection made for the antibiotic resistance marker conferred by the Nucleotide sequencing and analysis cassette used to inactivate the targeted gene. The BigDye terminator cycle sequencing kit 9Perkin Elmer) was used in all reactions. Individual reaction Selection of mutant strains mixtures 20 ìl contained plasmid DNA 400 ng or PCR ampli®ed DNA 200 ng, primer 3.2 pmol and Ready Individual colonies from conjugation selective plates Reaction Mix 9Perkin Elmer) 8 ìl in Eppendorf tubes. were streaked on to fresh glucose minimal medium Reactions were overlaid with mineral oil, extensions supplemented with appropriate antibiotic 9i.e., the were performed in a thermo-cycler 9Perkin-Elmer) and antibiotic to which resistance was encoded by the prepared for analysis on a 377 automated DNA sequencer insert of the inactivated gene) and cultured overnight at following the manufacturer's instructions 9Applied 378C. Individual colonies were tested for loss of the Biosystems) Trace data were analysed and assembled antibiotic resistance conferred by the suicide vector. with the use of DNA Star software 9DNAStar). Putative double cross-over mutant strains were streaked to single colonies three times before con®rmatory Cloning and insertional inactivation of ®mbrial genotypic and phenotypic testing. Mutant strains are listed in Table 4. and ¯agellar genes Mutagenesis was carried out by allelic exchange Pathogenicity of strain EC34195 in 1-day-old [32, 33, 36] and the preparative stages for each chicks mutational event are listed in Table 3. In brief, DNA fragments encoding the ®mC, csgA and ¯iC genes, Eighteen 1-day-old SPF white Leghorn chicks were each ¯anked by immediately adjacent sequences, were separated randomly into two groups of nine birds each ampli®ed by PCR with total genomic DNA extracted and housed in separate isolators. All birds in one group

Table 2. The sequences and sources of primers used in mutagenesis studies Primer Position Accession identity Sequence 59±39 9bp) number ®mC 1 TTTCAGCTGAATGCGGATGCGACCTTCAAG 3501±3521 Z37500 ®mC 2 AAACAGCTGCATCACGCGTTGCCATATAAC 5248±5227 Z37500 csgA 1 TTTAGGCCTTATTGATCGCACACCTGACAG 2498±2518 X90754 csgA 2 AAAAGGCCTCCAAGGGTTGTGTTATCCATA 4665±4645 X90754 ¯iC 1 TTTCAGCTGCATGGCACAAGTCATTAATACC 101±122 Z36877 ¯iC 2 AAACAGCTGGATAAGCGCAGCGCATA 1861±1846 Z36877 papA 1 AACTATTCCACAGGGGCA 1834±1851 X61239 papA 2 CAGGTTGAAATTCGCAAC 2291±2308 X61239 Underlined sequences denote engineered restriction enzyme sites 9 ®mC, PvuII; csgA, StuI; ¯iC, PvuII). 330 R. M. LA RAGIONE ET AL. were dosed by intra-gastric intubation with EC34195 t li®ed. 9 c bacteria 90.1 ml containing 1 3 10 cfu) and all birds in u 2.2 6.2 1.2 r

amp the other group received a similar dose subcutaneously. ector s v Suicide a const pRLR pRLR pRLR Birds were given standard rations and water ad libitum. w Three birds were randomly selected from each group bp and killed by cervical dislocation at 24, 48 and 168 h

2078 for detailed examination at necropsy. Histological of examination of tissues was done after staining with ct marker

biotic haematoxylin and eosin and Giemsa [40]. amplicon LR2.1 LR6.1 LR1.1 produ anti a pR pR pR , istance r and

e Tissue-culture adherence and invasion assays Cloned res v e w The mucus-secreting cell line HT2916E [41] was sown Ho 2 3 105 cells in 24-well plates in Dulbecco's modi®ed Eagle's medium 9Sigma) supplemented with fetal calf 34195. bp C

bp serum 10%, non-essential amino acids 93100) 1%, bp E

n 2mML-glutamine and gentamicin 9Sigma), 50 ìg=ml. Ã 3636 4504 1121 t e

t Monolayers were incubated for 5 days and were fed on strai a at a ation e v m e alternate days. By day 5, a con¯uent mucus layer was sit f site sit fro cassett o I

I detectable on the surface of HT2916E cells. Before inacti n A RI

N use, monolayers were washed twice with Hanks's Mun gene Mun D Eco ±

± balanced salts solution 9HBSS) to remove cell debris locatio ± r r ing

r and residual gentamicin. The bacterial inoculum was kan Marker and cam str insertional 8

¯ank added 91 3 10 cfu=ml) in incomplete Dulbecco's medium 9without antibiotics or fetal calf serum) and and added in 1-ml volumes to each well. Monolayers were 1 2 6 ¯iC

licon then incubated at 378C supplemented with CO2 5% for Cloned for pRLR pRLR pRLR construct amp 2 h. The inoculum was removed and the monolayer was bp

3). washed six times with 1 ml of HBSS to remove non- adherent bacteria. The monolayer was then disrupted 1760 for 10 min with a solution of Triton 100 9Sigma) 1% F07916 of A

ct and a 12-mm magnetic stirrer. After disruption for of

licon 10 min, 10-fold serial dilutions were plated on to LB 9bp) 1747 2167 2078 produ Size amp number agar and incubated overnight at 378C to determine the count 9cfu=ml). A non-mucus-secreting cell line, PCR

a HT2919A, was used as a control [41, 42]. HEp-2 cells

lify were prepared as above except that they were grown for 9Accession 48 h in Eagle's Minimal Essential Medium 9EMEM; amp 2 ited A2

to Sigma). All assays were repeated at least twice on at imC F Csg FliC2 depos least two separate occasions. 77) used and and and 1 1 9Z368 For invasion assays, bacteria were allowed to adhere to quence mers imC se F FliC1 CsgA Pri HEp-2 or HT2916E cells for 2 h, at which time

the monolayers were washed three times with HBSS before sequence

ycin. adding EMEM containing gentamicin 100 ìg=ml. and rone om

K12 Plates were incubated at 378CinCO2 5% for 2 h and out gy chape

on washed three times with HBSS. Monolayers were then gene gene strept ried , t t al ated

2) disrupted with Triton 100 solution 1% and the number strate str car l; s of cfu was determined [37]. is imbri a FliC F Subuni Subuni protein Associ w d enico n a

1 Tracheal and proximal gut explant adhesion assay get utagenes r ¯iC a ®mC iC gene csgA m T loramph

sequencing One-day-old SPF white Leghorn chicks were killed by 9Fl ch

and cervical dislocation. Lengths 9c. 2 cm) of the trachea ned cam,

eotide and proximal gut 9duodenal loop) were removed

desig aseptically to sterile Krebs'-Ringer's solution. Fatty nucl ae e ycin; ere Cloning tissue and mesentery were removed from the gut w . ®mbri age 3 sections and tissues were cut to expose the villiated a kanam ®mbria e

l 2

mers surfaces 9c.1cm) for use in adherence assays as b kan, ype-1 Pri Con®rmatory T Append Ta y Flagell Curli à described above. Sections were gently washed twice in ADHERENCE OF E. COLI O78:K08 331 Table 4. Library of ®mbrial and ¯agellar mutant strains of E. coli O78:K80 strain EC34195 Strain Antibiotic resistance no. Genotype 9ìg=ml) Phenotype RML1 ®mC::kanr Kanamycin 925) MSHAÀ RML2 csgA::camr Chloramphenicol 910) LacyÀ, curli ELISAÀ, Congo redÀ and ®bronectinÀ RML3 ®mC::kanr Kanamycin 925) MSHAÀ and LacyÀ, curli ELISAÀ, Congo redÀ and ®bronectinÀ csgA::camr Chloramphenicol 910) RML6 ¯iC::strr Streptomycin 925) Non-motile RML7 ®mC::kanr Kanamycin 925) MSHAÀ and non-motile ¯iC::strr Streptomycin 925) RML8 csgA::camr Chloramphenicol 910) LacyÀ, curli ELISAÀ, Congo redÀ, ®bronectinÀ and non-motile ¯iC::strr Streptomycin 925) RML9 csgA::camr Kanamycin 925) LacyÀ, curli ELISAÀ, Congo redÀ ®bronectinÀ MSHAÀ and non-motile ®mC::kanr Chloramphenicol 910) ¯iC::strr Streptomycin 925) pre-warmed Krebs'-Ringer's solution and immersed isolate should express relevant surface appendages, be individually in fresh sterile Krebs'-Ringer's solution in sensitive to antibiotics and be pathogenic. Thirteen a 50-ml Falcon tube 9BDH) to which 1 ml of the isolates of E. coli O78:K80 were tested for haemag- bacterial inoculum 91 3 108 cfu=ml) was added. In- glutination 9type-1 and P ®mbriae), motility 9¯agella), cubation was at 378C with orbital rotation at 225 rpm colony morphology, Congo red and ®bronectin binding for up to 180 min. At timed intervals, sections were 9curli ®mbriae) and observed by transmission electron rinsed three times in fresh sterile pre-warmed Krebs'- microscopy for the presence of these and other surface Ringer's solution to remove all non-adherent bacteria appendages. None of the isolates elaborated mannose- and then homogenised. Serial dilutions of homogenates resistant haemagglutination, but considerable variability were plated in triplicate on to LB agar plates which in the elaboration of ¯agella and type-1 and curli were incubated at 378C overnight and bacterial counts ®mbriae was noted 9Table 5). Each isolate was tested were determined. All assays were repeated at least against a panel of 16 antibiotics 9VLA, Weybridge twice on at least two separate occasions. Enteric Bacteria Testing Section) and three antibiotic- sensitive isolates ± EC30896, EC5197 and EC34195 ± Statistical analyses which possessed type-1 ®mbriae, curli ®mbriae and ¯agella were selected for further study. Their ¯iC, ®mC Bacterial counts were converted to log10 values for and csgA genes were ampli®ed successfully by PCR statistical analysis. Repeated measures analyses of and these amplicons were cloned, mapped by restriction variance were done with the Statistica or Genstat 5 endonuclease digestion and con®rmed by DNA se- software packages. The means, SEMs and signi®cances quence analysis. PCR with pap primers failed to 9p) of strain differences were calculated. generate an amplicon for any of the isolates tested, whereas amplicons were derived successfully by PCR from a control strain 9EC43297) of E. coli isolated Results from a patient with urinary tract infection. Characterisation of avian isolates of E. coli Strain EC34195 was tested in the day-old SPF chick O78:K80 pathogenicity model. Birds were inoculated subcuta- To select one isolate of E. coli O78:K80 for study, neously or by the oral route and selected at random speci®c criteria were established, namely that the from both dosage groups for analysis at necropsy 24,

Table 5. Phenotypic characters of E. coli O78:K80 isolates Elaboration Congo Elaboration Haemagglutination of Elaboration Strain of curli Colony red Fibronectin of type-1 chicken or guinea- of Flagellar no. ®mbriae morphology binding binding ®mbriae pig erythrocytes ¯agella Motility antigen EC54597 À S ÀƇ‡‡‡51 EC5197 ‡ L ‡‡‡‡‡‡9 EC24197 ‡ L ‡‡‡‡ÀÀ± EC24297 ‡ L ‡‡‡‡ÀÀ± EC24397 ‡ L ‡‡‡‡ÀÀ± EC39597 ‡ S=L ‡‡‡‡‡‡51 EC43197 ‡ L ‡‡ÀÀ‡À51 EC47897 ‡ S=L ‡‡‡‡‡‡9 EC30896 ‡ S=L ‡‡‡‡‡‡9 EC26296 ‡ S=L ‡‡ÀÀÀÀ± EC34195 ‡ L ‡‡‡‡‡‡9 EC2597 À S ÀÆÀÀ‡‡11 EC5497 À S ÀÆÀÀ‡‡11 L, Lacy; S, smooth colonies; Æ, binding to ®bronectin at low level. 332 R. M. LA RAGIONE ET AL. 48 and 168 h after infection. Histological examination Strain RML1 9a type-1 non-®mbriate mutant, FimÀ) of tissues from diseased birds showed generalised failed to agglutinate guinea-pig erythrocytes. Strain congestion of the heart, with suppurative pericarditis RML2 9a curli non-®mbriate mutant, CrlÀ) did not and epicarditis. Examination of liver tissues revealed generate convoluted colonies, failed to bind either congestion with mild multifocal cellular in®ltration and Congo red or ®bronectin and was negative in an focal hepatocyte vacuolation. indirect curli ELISA. Strain RML6 9a non-¯agellate mutant, FlaÀ) was non-motile and failed to agglutinate in H9 antiserum. Multiple-mutant strains were tested similarly and behaved as anticipated. All mutant strains Construction of isogenic mutant strains of E. coli were shown to have the same growth rates in vitro as O78:K80 EC34195 the wild-type strain when cultured in LB broth 9data Single and multiple mutant strains were constructed in not shown). strain EC34195 as described above. Southern hybridi- sation analysis of each mutant strain and of the wild- Adherence of non-®mbriate and non-¯agellate type progenitor strain EC34195 were compared. mutant strains to tissue-culture cells Hybridisation with the native target gene PCR product showed an increase in size of the target gene Single and multiple isogenic non-®mbriate and non- commensurate with the insertion of an antibiotic ¯agellate mutant strains were compared with the resistance cassette 9Fig. 1). The antibiotic resistance isogenic wild-type strain in HEp-2, HT2916E and cassette used as probe hybridised with the target HT2919A cell-adhesion assays 9Table 6). In tests with mutant gene only, whereas detectable hybridisation HEp-2 cells, mutant strains defective in the elaboration was not observed when the pERFORM suicide plasmid of type-1 ®mbriae 9strain RML1, FimÀ; strain RML3, DNA alone was used as a probe. FimÀ CrlÀ; strain RML7, FimÀ FlaÀ; strain RML9,

Fig. 1. Southern hybridisation of E. coli O78:K80 strain EC34195 and isogenic derivatives to demonstrate inser- tional inactivation of target ®mbrial and ¯agellar genes. 9a) Lanes 1, 3, 5, wild-type strain and lanes 2, 4, 6, strain RML1 9®mC::kanr); DNA digested to completion with HindIII 91, 2), EcoRV 93, 4)orPvuII 95, 6)and hybridised against ®mC.9b)Lanes1, 3, 5, 7, wild-type strain and lanes 2, 4, 6, 8,strainRML29csgA::camr); DNA digested to completion with ScaI91, 2), PstI93, 4), ClaI95, 6)orSmaI97, 8) and hybridised against csgA.9c)Lanes1, 3, 5, 7, wild-type strain and lanes 2, 4, 6, 8 strain RML6 9¯iC::strr); DNA digested to completion with PstI91, 2), HindIII 93, 4), HpaI95, 6)andBglI97, 8) and hybridised against ¯iC. ADHERENCE OF E. COLI O78:K08 333 Table 6. Adherence of wild-type, single and multiple mutant strains of E. coli to different cell lines HEp-2y HT2916Ey HT2919Ay

Meany Signi®cance Meany Signi®cance Meany Signi®cance à { { { Cluster Strain log10 9SEM) 9p) log10 9SEM) 9p) log10 9SEM) 9p) I Wild-type 5.88 90.09) ± 5.93 90.11) ± 5.99 90.13) ± RML1 5.26 90.09) ,0.001 4.68 90.13) ,0.001 5.00 90.13) 0.013 RML2 5.91 90.10) 0.823 5.74 90.13) 0.273 6.00 90.13) 0.976 RML6 5.40 90.56) 0.527 4.73 90.13) ,0.001 5.33 90.13) 0.038 II Wild-type 6.01 90.56) ± 5.93 90.11) ± 6.18 90.06) ± RML3 5.36 90.11) 0.006 4.83 90.18) ,0.001 5.00 90.06) ,0.001 III Wild-type 6.03 90.17) ± 5.93 90.11) ± 6.13 90.03) ± RML7 5.03 90.20) 0.019 4.73 90.18) ,0.001 4.94 90.03) ,0.001 RML8 5.16 90.20) 0.030 4.84 90.18) ,0.001 5.94 90.03) 0.011 RML9 5.21 90.20) 0.035 4.75 90.18) ,0.001 4.94 90.03) ,0.001 ÃFor ease of manipulation, and to provide multiple values for wild-type strains for statistical analysis, assays were performed in three clusters 9I±III). yMean bacterial counts obtained after culture of inoculum in HIB at 378C for 48 h. {Signi®cance values calculated with reference to wild-type strains 9wild-type versus mutant).

FimÀ CrlÀ FlaÀ) showed a reduction in adherence of tively) but the curli non-®mbriate mutant, strain RML2 the order of 90% 9p , 0:001, p ˆ 0:006, p ˆ 0:019, 9CrlÀ) adhered as well as the wild-type strain. p ˆ 0:035, respectively) when compared with the wild- type strain. The curli non-®mbriate mutant strain À Invasion of HEp-2 and HT2916E cells by non- RML2 9Crl ) showed no reduction in adherence and ®mbriate and non-¯agellate mutant strains the non-¯agellate mutant strain RML6 9FlaÀ) showed a reduced adherence of c. 50% compared with the wild- Invasion assays were set up for mutant strains RML1 type strain, although this difference was not statistically 9FimÀ), RML2 9CrlÀ) and RML6 9FlaÀ) only in HEp-2 signi®cant 9p ˆ 0:527). It was of interest that the curli and HT2916E cells 9Table 7). With HEp-2 cells, non-®mbriate, non-¯agellate mutant strain RML8 invasion of strain RML1 9FimÀ) was reduced to c. 9CrlÀ, FlaÀ) was signi®cantly less adherent 9c. 90%) 0.01±0.05% of adherent bacteria compared with 0.1% than the wild-type strain 9p ˆ 0:030). for the wild-type strain, a signi®cant ®nding 9p ˆ 0:027). The reduction in invasion by strains At least 80% of the HT2916E cells secreted mucus and RML2 9CrlÀ) and RML6 9FlaÀ) was not statistically the adherence of strain RML6 9FlaÀ) to this cell line signi®cant 9p ˆ 0:285, p ˆ 0:230, respectively). About was reduced by .90% compared with the wild-type 0.005±0.01% of adherent wild-type bacteria invaded strain 9p , 0:001). Other mutant strains, lacking either HT2916E cells, and this level of invasion was less than type-1 ®mbriae or ¯agella, also showed reduced that for similar experiments with HEp-2 cells. Strains adherence of the order of 90% when compared with RML1 9FimÀ), RML2 9CrlÀ) and RML6 9FlaÀ) were the wild-type strain; again, these ®ndings were less invasive than the wild-type strain 9p , 0:001, statistically signi®cant. Adherence of strain RML2 p ˆ 0:010, p , 0:001, respectively), although the extent 9CrlÀ) was not reduced signi®cantly 9p ˆ 0:273). of invasion in these assays was directly proportional to the extent of adhesion. With the non-mucus-secreting cell line HT2919A, all mutant strains lacking type-1 ®mbriae showed reduced Development of chick tracheal and gut explant adherence 9c. 90%) compared with the wild-type adhesion models strains and this difference was statistically signi®cant. Strains RML6 9FlaÀ) and RML8 9FlaÀ CrlÀ) both Bacterial growth was not detectable when tracheal and showed c. 40% reduction in adherence compared with proximal gut samples 9before incubation with test the wild-type strain 9p ˆ 0:038, p ˆ 0:011, respec- bacteria) were homogenised and plated on LB agar

Table 7. Invasionà of HEp-2 and HT2916E cells by wild-type and single mutant strains of E. coli HEp-2 HT2916E

y y Strain Mean log10 9SEM) Signi®cance 9p) Mean log10 9SEM) Signi®cance 9p) Wild-type 3.27 90.06) ± 2.91 90.03) ± RML1 3.03 90.06) 0.027 2.25 90.03) ,0.001 RML2 3.17 90.06) 0.285 2.67 90.03) 0.010 Wild-type 2.69 90.16) ± 2.91 90.03) ± RML6 2.29 90.16) 0.230 2.03 90.03) ,0.001 ÃMean bacterial counts obtained after culture of inoculum in HIB at 378C for 48 h. ySigni®cance values calculated with reference to wild-type strains 9wild-type versus mutant). 334 R. M. LA RAGIONE ET AL. with aerobic incubation overnight at 378C. Tissues 48 h, although evidence was presented that curli non- showed signs of disaggregation after incubation for 4 h, ®mbriate mutant strains grown under these conditions and, for convenience, sampling was done 3 h after were less adherent to gut explants than the wild-type inoculation. After exposure to bacteria, tissues were strain. When inocula were prepared by growth in CFA ®xed and examined by scanning electron microscopy broth at 258C for 72 h without shaking, conditions 9Fig. 2a and b). Counts of bacteria from 10 randomly known to support expression of curli but not type-1 selected ®elds per tissue surface showed preferential ®mbriae [30, 38], adherence of strain RML2 9CrlÀ)to adherence to the ciliated epithelial rather than the both mucus-secreting and mucus-non-secreting cell serous surface 9ratio 80:1). Furthermore, bacteria lines was reduced by .80% compared with that of adhered singly and not as aggregates or clumps. the wild-type strain 9p ˆ 0:003 and p ˆ 0:026, respec- Signi®cant differences in adherence were not seen tively). Similar reductions were observed for all other between different areas of the gut or trachea when E. mutant strains prepared under these conditions 9Table coli strain EC34195 was incubated with similarly sized 9) and of particular note was the reduction in sections of tissues of proximal, mid and distal trachea, adherence to the mucus cells of .90% for mutant proximal, mild and distal gut, caecum or rectum. strains defective in the elaboration of ¯agella 9p , 0:001). Adhesion of non-®mbriate and non-¯agellate mutant strains to explant tissue Discussion The adherence to tracheal explant tissue 9Table 8) of mutant strains defective in the elaboration of type-1 Non-®mbriate and non-¯agellate mutant strains were ®mbriae 9strain RML1, FimÀ; strain RML3, FimÀ constructed in E. coli O78:K80 strain EC34195 by CrlÀ; strain RML7, FimÀ FlaÀ; strain RML9, FimÀ allelic exchange and evidence was gained that these CrlÀ FlaÀ) was reduced by c. 60±90% compared with strains possessed an antibiotic resistance cassette in the that of the wild-type strain; these differences were targeted genes and possessed the desired phenotypic signi®cant 9p , 0:001, p ˆ 0:016, p ˆ 0:036, p ˆ changes. Allelic exchange is an established technique 0:042, respectively). There was no signi®cant reduction and it was considered unlikely that other, less overt in adherence for either the curli non-®mbriate strain phenotypic changes might have resulted from the RML2 9CrlÀ) or the non-¯agellate strain RML6 9FlaÀ), mutagenesis strategy used. Considerable phenotypic although the adherence of strain RML8 9FlaÀ CrlÀ) diversity was shown by the small panel of avian, was reduced by c. 75% compared with that of the wild- colibacillosis-causing isolates of O78:K80 examined type strain, a ®nding which was signi®cant 9p ˆ 0:036). and an additional question arose as to whether strain EC34195 was typical of avian colibacillosis strains. P The adherence to gut explant tissue 9Table 8) of strains ®mbriae are elaborated by many avian isolates of E. RML1 9FimÀ) and RML2 9CrlÀ) was reduced by c. coli [17, 43], but the isolates tested in this study did not 90% and 80%, respectively, compared with that of the elaborate mannose-resistant haemagglutination and did wild-type strain 9p ˆ 0:002, p ˆ 0:005, respectively). It not possess pap genes as determined by PCR. Electron was surprising that the adherence of strains RML3, microscopy con®rmed the absence of P ®mbriae or any defective in the elaboration of both type-1 and curli other, hitherto undescribed surface elaborated structures ®mbriae, was reduced by c. 70% compared with that of and so it was reasonable to assume that differences the wild-type strain, a reduction that was not observed in adherence and invasion assays were likely statistically signi®cant 9p ˆ 0:496). In this model, the to result from the phenotypic outcome of each role of ¯agella was found to be equivocal with strains mutation. RML6 9FlaÀ), RML8 9CrlÀ, FlaÀ) and RML9 9FimÀ, CrlÀ, FlaÀ) showing only marginally less adherence All adhesion assays indicated that type-1 ®mbriae were than the wild-type strain 9p ˆ 0:086, p ˆ 0:068, of particular signi®cance in mediating adhesion of E. p ˆ 0:123, respectively), although adherence of strain coli O78:K80 to epithelial cells. This ®nding was RML7 9FimÀ FlaÀ) was reduced by 90% compared consistent with studies with E. coli of many other with that of the wild-type strain 9p ˆ 0:016). serotypes including other avian colibacillosis serotypes such as O1:K1 and O2:K1 [10, 15]. Again, evidence After incubation with bacteria for 3 h both gut and was presented that E. coli O78:K80 strain EC34195 tracheal explant tissues were shown by electron adhered to tracheal explants more effectively than to microscopy to have lost much of the mucus. That gut explants. This may re¯ect the density of receptors may have in¯uenced the ®ndings. ± largely type-1 ®mbrial adhesin, mannose-bearing glycoproteins [44, 45] ± or innate tropism for tracheal In¯uence of culture of inoculum on curli tissues which may also relate to the speci®c adhesin- ®mbriae-mediated adherence to tissue culture receptor interactions. Curli ®mbriae were not detectable by electron micro- Flagella have been cited as mediating penetration of scopy on wild-type bacteria grown in HIB at 378C for mucus before epithelial adherence mediated by type-1, ADHERENCE OF E. COLI O78:K08 335

Fig. 2. 9a) Scanning electron micrograph of tracheal tissue explant showing ciliated luminal surface after exposure to bacteria of E. coli O78:K80 strain EC34195 for 3 h. Magni®cation shown by white bar, marker. 9b) Scanning electron micrograph of tracheal tissue explant showing ®broblastic surface after exposure to bacteria of E. coli O78:K80 strain EC34195 for 3 h. Magni®cation shown by white bar, marker. 336 R. M. LA RAGIONE ET AL. Table 8. Adherence of wild-type, single and multiple mutant strains of E. coli to tracheal and gut explant tissue Tracheal tissue Gut tissue

à y à y Strain Mean log10 9SEM) Signi®cance 9p) Mean log10 9SEM) Signi®cance 9p) Wild-type 5.96 90.10) ± 4.93 90.08) ± RML1 5.10 90.10) ,0.001 3.89 90.08) 0.002 Wild-type 6.08 90.07) ± 4.93 90.08) ± RML2 6.15 90.07) 0.526 4.11 90.08) 0.005 Wild-type 5.93 90.01) ± 4.23 90.33) ± RML3 5.44 90.01) 0.016 3.85 90.33) 0.469 Wild-type 5.54 90.13) ± 4.93 90.08) ± RML6 5.50 90.13) 0.845 4.66 90.08) 0.086 Wild-type 5.10 90.27) ± 5.11 90.19) ± RML7 4.36 90.27) 0.302 4.09 90.19) 0.160 Wild-type 5.08 90.03) ± 4.83 90.06) ± RML8 4.41 90.03) 0.036 4.03 90.06) 0.068 Wild-type 5.33 90.04) ± 4.91 90.09) ± RML9 4.47 90.04) 0.042 4.24 90.09) 0.123 ÃMean counts of adherent bacteria after culture of inoculum in HIB at 378C for 48 h. ySigni®cance values calculated with reference to wild-type strains 9wild-type versus mutant).

Table 9. Adherence of wild-type and single mutant strains of E. coli to different cell lines HEp-2Ã HT2916EÃ HT2919AÃ

Mean Signi®cance Mean Signi®cance Mean Signi®cance y y y Strain log10 9SEM) 9p) log10 9SEM) 9p) log10 9SEM) 9p) Wild-type 6.71 90.046) ± 6.64 90.061) ± 6.47 90.117) ± RML1 5.97 90.046) 0.001 5.96 90.061) 0.004 5.71 90.117) 0.020 RML2 6.41 90.046) 0.020 5.87 90.061) 0.003 5.78 90.117) 0.026 RML6 6.52 90.046) 0.213 5.45 90.061) ,0.001 5.71 90.117) 0.019 ÃMean bacterial counts after culture of inoculum in CFA broth at 258C for 72 h. ySigni®cance values: see footnotes to Table 8.

and other, ®mbrial and non-®mbrial surface antigens When the bacterial inoculum was prepared in CFA [46]. The data generated in this work support that broth at 258C for 72 h, conditions which induce the concept in that non-¯agellate mutant strains adhered production of curli, but not type-1, ®mbriae [38, 48], less well to the mucus secreting cell line HT2916E differences in adherence between mutant and wild-type than did the wild-type strain. It was of interest that strains were accentuated and were statistically signi®- non-¯agellate mutant strains adhered less well to the cant. It was of interest that curli ®mbriae were not seen non-secreting cell line HT2919A, indicating that by electron microscopy on HIB-grown bacteria, factors other than ¯agella and mucus was important although differences in adherence between curliate in adherence to the HT2916-type cell lines. Whether and non-curliate derivatives were observed in gut the use of tissue explants gave a clearer understanding explant assays. Thus, it is possible that either suf®cient of the role of ¯agella is also open to debate, especially curli ®mbriae were elaborated in HIB to enable as non-¯agellate mutant strains were not signi®cantly observation of differences or contact with explants less adherent than the wild-type strain. Gut and tracheal stimulated their elaboration. Alternatively, other adhe- explant tissues from day-old chicks may lack ®mbrial sins may have been elaborated under these conditions. adhesin receptors because of immaturity [47], and that Curli ®mbriae mediate aggregation of curliate bacterial may have in¯uenced results. Electron microscopic cells [49, 50], but in this study cell clumping at the analysis of tissues after incubation with bacteria epithelial cell surface was not observed by scanning indicated that mucus was absent and it is probable electron microscopy. It was probable that differences in that mucus was lost by the sheer forces associated with bacterial counts re¯ected actual adherence values not the agitation used during incubation. In addition, that apparent reductions due to clumping, especially as agitation may have provided suf®cient contact between Triton, which disaggregates bacterial clumps, was a bacteria and tissue such that any chemotactic role component of the diluent used for the enumeration of associated with adherence was not apparent. adherent bacteria. Bacterial cultural and tissue-culture assay conditions in¯uenced bacterium±host cell inter- Curli non-®mbriate mutant strains grown in HIB at actions, ®ndings in common with those of Duguid and 378C for 48 h showed only a marginal and statistically Gillies [51], who showed that anaerobiosis induced equivocal reduction in adherence to explants, but no ®mbriation. Again, Ernst et al. 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