1 INRA Centre de Tours, UR 86, Pathologie Bactérienne, 37380 Nouzilly, France
2 Children's Hospital Los Angeles and University of Southern California Keck School of Medicine, Los Angeles, CA 90027, USA
3 Laboratorio de Referencia de E. coli, Departamento de Microbiología y Parasitología, Faculdad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain
Correspondence
Pierre Germon
germon{at}tours.inra.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY248744.
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INTRODUCTION |
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APEC strains belong predominantly to serogroups O1, O2, O5, O8, O18 and O78 (Blanco et al., 1997). The presence of several putative virulence genes has been positively linked to the pathogenicity of APEC strains. Indeed, several adhesins and the tsh gene are more frequently found in pathogenic isolates (Dozois et al., 1992
, 1996
; Stordeur et al., 2002
). The presence of different toxins has also been reported but with different prevalences (Blanco et al., 1997
; Parreira & Gyles, 2002
; Reingold et al., 1999
). Gene inactivation studies confirmed a role in the pathogenicity of APEC for the F1 (type 1) and P fimbrial adhesins (Dozois et al., 1992
, 1996
; La Ragione et al., 2000a
, b
; Marc et al., 1998
; Pourbakhsh et al., 1997
), for the K1-antigen mediating resistance to phagocytosis (Mellata et al., 2003a
), for the aerobactin high-affinity iron transport system (Lafont et al., 1987
) and for the temperature-sensitive haemagglutinin Tsh (Dozois et al., 2000
; Provence & Curtiss, 1994
). Recently, Parreira & Gyles (2003)
showed that a vacuolating toxin, Vat, was involved in the virulence of an APEC isolate. Another gene whose precise function remains to be elucidated is the iss gene (Binns et al., 1979
); this gene is presumed to play a role in serum resistance but, if so, its role seems to be minor (Mellata et al., 2003a
).
Different associations of virulence genes have been detected and could reflect the existence of different pathotypes (Delicato et al., 2003). Indeed, not a single virulence gene is found in any of the APEC strains that is absent from all non-pathogenic strains. This might indicate the use of different virulence mechanisms by different putative pathotypes (Delicato et al., 2003
; La Ragione & Woodward, 2002
).
More generally, pathogenic E. coli are separated into those responsible for intestinal infections and those responsible for extraintestinal infections. The latter class, referred to as ExPEC (extraintestinal pathogenic E. coli) (Russo & Johnson, 2000) includes strains causing urinary tract infections (uropathogenic E. coli or UPEC), septicaemia in humans or meningitis in human neonates (neonatal meningitis E. coli or NMEC) as well as APEC strains.
Several studies have unravelled common traits between APEC and human isolates. Using different typing methods early studies showed clonal relationships among isolates from human and animal infections, including chicken (Achtman et al., 1986; Chérifi et al., 1994
). Virulence determinants common to APEC and ExPEC have been identified. They include the aerobactin iron transport system, the K1 capsule and type 1 and P fimbriae (Bahrani-Mougeot et al., 2002
; Dho-Moulin & Fairbrother, 1999
; Gunther et al., 2002
; Hoffman et al., 1999
; Mobley et al., 1993
; Pourbakhsh et al., 1997
; Torres et al., 2001
). Vandemaele and colleagues also found a high level of homology between papG sequences, the P pilus adhesin gene from APEC isolates and papG sequences from mammalian E. coli isolates, including human isolates (Vandemaele et al., 2003
; Johnson et al., 1997
).
Additionally, Johnson et al. (2003) isolated E. coli strains from retail chicken products that had virulence profiles similar to ExPEC isolates. These authors concluded that these strains represent potential human pathogens.
Among the functional similarities, the finding that type 1 pili are expressed in the early steps of avian colibacillosis and that P pili are expressed in the later steps is reminiscent of the finding that the UPEC strain J96 expresses either type 1 pili or P-pili, but not both at the same time, the expression of type 1 pili being inhibited by the PapB protein (Mobley et al., 1993; Pourbakhsh et al., 1997
).
Another functional similarity comes from data showing that the K1 antigen could play a similar role in the virulence of APEC and NMEC strains. Indeed, Kim et al. (2003) demonstrated that a K1 derivative of the NMEC strain E44 showed higher binding and internalization rates than the parent strain, but decreased intracellular survival. Similarly, a K1 derivative of strain BEN 2908 showed a slightly greater association with phagocytes, but a decreased survival in phagocytes and heterophils (Mellata et al., 2003b
) than the wild-type strain.
An important step in the process of infection by NMEC is the entry into the bloodstream and, once the bacteraemia has reached a certain threshold, the invasion of the central nervous system after traversal of the brain microvascular endothelial cells (BMEC) (Huang & Jong, 2001; Kim, 2001
). It has been shown that efficient penetration of E. coli across the bloodbrain barrier is mediated by multiple factors. Mutants in several genetic determinants, including ibe genes, aslA, ompA, traJ and cnf1, were found to be significantly less invasive in BMEC monolayers in vitro and in a newborn rat model of haematogenous E. coli meningitis (Kim, 2001
). One of these genes, ibeA, encodes a 50 kDa protein (456 aa) whose exact function is still unknown. ibeA is located on a 20·3 kb island inserted between yjiD and yjiE proposed to contain four operons, some of which are potentially involved in energy metabolism (Huang et al., 2001a
). An NMEC strain deleted for ibeA has been shown to be defective in invasion of BMEC both in vitro and in vivo (Huang et al., 1995
). A receptor for IbeA has been identified on the surface of both human (45 kDa) and bovine (55 kDa) BMEC (Huang et al., 1995
, 2001a
, b
; Prasadarao et al., 1999
) and has been shown to be an albumin-like protein. IbeA could therefore be involved in receptorligand-mediated invasion of BMEC.
In the view of the multiple properties shared by APEC and ExPEC strains, we decided to investigate the presence of ibeA in APEC strains. After analysing the prevalence of ibeA in a collection of E. coli strains isolated from healthy or sick chickens, a mutant was constructed and its capacity to invade BMEC and to induce colibacillosis were tested.
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METHODS |
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Bacteria were grown in LuriaBertani (LB) broth at 37 °C with agitation unless otherwise stated. E. coli strain HB101 was routinely used for plasmid cloning. Ampicillin (100 µg ml1), kanamycin (25 µg ml1), nalidixic acid (30 µg ml1) and tetracycline (10 µg ml1) were used when required. The determination of O serogroups was carried out as previously described employing all available O (O1O181) antisera (Blanco et al., 1998). All antisera were obtained and absorbed with the corresponding cross-reacting antigens to remove non-specific agglutinins. The O antisera were produced in the Laboratorio de Referencia de E. coli [Lugo, Spain (http://www.lugo.usc.es/ecoli)].
DNA and genetic manipulations.
DNA manipulations and bacteria transformations were carried out as described by Sambrook et al. (1989). Restriction endonucleases and modification enzymes (New-England Biolabs) were used according to the manufacturer's instructions. DNA fragments were purified from agarose gels using the DNA purification kit from MachereyNagel (Germany).
Primers used in this study are described in Table 1. PCR reactions were run with an iCycler apparatus (Bio-Rad) using 0·5 U Taq DNA polymerase from Promega in 1x buffer, 200 µM each dNTP, 0·8 µM each primer and 10 ng chromosomal DNA in a 50 µl reaction volume. PCR conditions were as follows : 95 °C for 5 min followed by 29 cycles consisting of 30 s at 95 °C, 30 s at 52 °C and 1 min per kb at 72 °C with a final extension of 10 min at 72 °C.
An ibeA derivative of strain BEN 2908 was constructed by inactivating the ibeA gene by the insertion of the suicide vector pCVD442 (Donnenberg & Kaper, 1991) and designated BEN 2755. The complemented strain was obtained after transformation of strain BEN 2755 with plasmid pUC23A (Huang et al., 2001b
).
DNA sequencing and analysis.
Nucleotide sequences were determined using ABI Prism Big Dye Terminators (PE biosystems) according to the manufacturer's instructions and a Perkin Elmer 9600 thermal cycler. Samples were run on an automated sequencer (ABI377; Perkin Elmer).
Dot-blot hybridization.
For dot-blot analysis, DNA was isolated from a 1 ml overnight bacterial culture in LB grown at 37 °C with agitation. Cells were resuspended in 100 µl distilled water and boiled for 5 min. Five microlitres of each lysate was spotted on nitrocellulose membranes (Roche). The ibeA gene was detected using a PCR fragment generated with primers ibeA-F and ibeA-R and the BEN 2908 chromosome as a probe. Labelling of the probe, hybridization and detection of the hybridized fragments were performed using the ECL Direct Nucleic Acid Labelling and Detection System (Amersham Pharmacia).
Serum bactericidal assay.
Bacterial survival in chicken serum was determined as described previously (Dozois et al., 2000) with an initial inoculum of 100 µl containing 107 c.f.u. incubated in 900 µl fresh normal chicken serum. The serum/bacteria suspensions were incubated at 37 °C for 3 h and counts of viable cells were estimated at the 1 and 3 h time points.
Invasion assays.
E. coli invasion assays were performed as described previously (Huang et al., 1995). Briefly, confluent human BMEC in 24-well plates were incubated with 107 E. coli (m.o.i. 100) in experimental medium (1 : 1 mixture of M199/Ham's F-12 containing 5 % heat-inactivated fetal bovine serum) for 90 min at 37 °C. The monolayers were washed with HBSS and then incubated in experimental medium containing gentamicin (100 µg ml1) for 1 h to kill extracellular bacteria. The monolayers were washed again and lysed with 0·5 % Triton X-100. The released intracellular bacteria were enumerated by plating on sheep blood agar plates. The actual inoculum size was determined by colony plate count for every experiment. Each assay was conducted in triplicate and repeated at least three times. Bacterial viability was not affected by 0·5 % Triton X-100 treatment. The MIC of gentamicin for all strains used was 1 µg ml1. Cell viability was routinely verified by trypan blue staining assay. Results were expressed as percentage invasion [100x(no. of intracellular bacteria recovered)/(no. of bacteria inoculated)].
Strain E44, whose invasion properties toward BMEC have been described by Prasadarao et al. (1996), was used as a control.
In vivo virulence assays.
All birds used in this study were specific-pathogen-free. Pathogens tested for were Salmonella pullorum, Salmonella gallinarum, Mycoplasma gallisepticum, Mycoplasma synoviae, avian leucosis virus, pseudoadenovirus, adenovirus, Newcastle disease virus, Marek disease virus, influenza virus, reovirus, infectious bronchitis virus and infectious anaemia virus.
The pathogenicity for day-old chicks of all 268 strains was assessed by inoculating five 1-day-old chicks subcutaneously with 108 c.f.u. as described by Dho-Moulin & Lafont (1982).
The in vivo colonization assay was conducted as described by Dozois et al. (2000). Briefly, 3·5-week-old White Leghorn specific-pathogen-free chicken were inoculated into the air sacs with 107 c.f.u. of a bacterial inoculum consisting of a diluted (in 0·9 % NaCl) overnight culture of E. coli grown in 20 ml LB broth in a 100 ml flask without agitation. Inocula were adjusted by measuring OD600 and bacteria were counted after inoculation. Two groups of 22 animals were inoculated with either strain BEN 2908 or BEN 2755 (
ibeA). Blood samples were collected after 24 and 48 h. Animals were then euthanized, necropsied and a piece of liver, one of the colonized organs, was collected. After homogenization in saline, serial dilutions were plated on Drigalski plates supplemented with nalidixic acid. Clinical signs of colibacillosis, including laboured breathing, cough, inactivity and prostration, were observed. For statistical analysis using the MannWhitney rank test, values were arranged in increasing numbers, dead animals being given the highest rank.
Statistical analysis.
Statistical analysis of the results from colonization and invasion assays were done by applying a MannWhitney test. Exact P-values were calculated with the software StatXact (version 5.0 Cytel Software, Cambridge, MA, USA) and P<0·05 was considered significant.
The significance in distribution differences of the ibeA gene was analysed with a Chi-square test. Exact P-values were again calculated with StatXact (version 5.0) and P<0·05 was considered significant.
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RESULTS |
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Prevalence of ibeA in a collection of 268 avian E. coli strains
An 800 bp internal fragment of ibeA was amplified from the BEN 2908 chromosome with primers ibeA-F/ibeA-R and used as a probe to detect the ibeA gene by dot-blot hybridization in a collection of 268 E. coli strains of avian origin. This collection contains 213 pathogenic strains and 55 non-pathogenic strains isolated from the gut of healthy birds and non-lethal for day-old chicks. As a control for day-old chick experiments, chicks were inoculated with 108 c.f.u. of a non-pathogenic control strain (BEN 2269) whose properties have already been described elsewhere (Dho-Moulin & Lafont, 1982). As expected, no chick died in these control experiments. The serogroup of the 268 strains was determined to allow further analysis.
A total of 53 strains, all belonging to the group of pathogenic strains, were ibeA+, which represents 26 % of the pathogenic strains (Table 2). ibeA is therefore significantly more likely to be present in pathogenic strains (P<0·0001). Moreover, there was a negative association of ibeA with strains of the O78 serogroup: among these 59 strains, only one was ibeA+, indicating that O78 strains are significantly less likely to carry the ibeA gene (P<0·0001). On the contrary, the frequency of ibeA+ strains is significantly higher (P<0·0001) in O2 (49·1 %), O18 (70 %) and O88 (100 %) strains compared to the frequency of ibeA+ strains in the entire collection (19·8 %).
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Inactivation of ibeA and invasion studies
To test whether the deletion of ibeA would have any influence on the pathogenicity of strain BEN 2908, an ibeA derivative of BEN 2908, BEN 2755 (ibeA), was constructed using the method described by Donnenberg & Kaper (1991)
. The ibeA deletion was confirmed by PCR using primers ibeA-5F/ibeA-5R (Fig. 1
, lanes 5 and 6). The growth rate of mutant BEN 2755 (
ibeA) was identical to that of BEN 2908 grown in LB broth at 37 °C with agitation (data not shown). Other phenotypic traits of BEN 2755 (
ibeA) were checked to ensure that the construction of the ibeA mutant had not modified the basic properties of the strain. Indeed, BEN 2755 (
ibeA) was shown to still express the O2 and K1 antigens, and its resistance to serum-mediated killing was unaffected (data not shown).
Since the ibeA gene had been described as being involved in the invasion of BMEC by the NMEC strain E44, we tested whether BEN 2908 had a similar activity and, if so, whether this activity was modified by the ibeA deletion. The invasion activity of strains BEN 2908 and BEN 2755 (ibeA) was compared to that of strain E44 in an in vitro invasion assay using human BMEC.
Strain BEN 2908 expressed an invasion activity which was approximately 10 times higher than that of strain E44 (2·5 % versus 0·25 %) (Fig. 2). The results obtained for this latter strain (0·25 % invasion) were in agreement with those obtained previously (0·4 %; Prasadarao et al., 1996
). Strain BEN 2269, a non-pathogenic E. coli strain of avian origin, was unable to invade BMEC.
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Infection studies
The virulence of BEN 2755 (ibeA) was tested in an in vivo model by inoculating 3·5-week-old chickens into the air sacs. One group of 22 chickens was inoculated with 107 c.f.u. of strain BEN 2908 and a second group of 22 with 107 c.f.u. of strain BEN 2755 (
ibeA). As a control, three animals were inoculated with a non-pathogenic E. coli strain of avian origin (BEN 2269) and did not show any sign of colibacillosis (data not shown). Six out of 22 chickens died when inoculated with BEN 2908, whereas only one died when inoculated with BEN 2755 (
ibeA). Birds inoculated with the parent strain BEN 2908 and with the BEN 2755 (
ibeA) mutant showed clinical signs of colibacillosis and bacteria were recovered from the liver and the blood at 48 h post-inoculation (Fig. 3
). However, bacterial counts in the blood and in the liver were reduced in chickens inoculated with the mutant BEN 2755 (
ibeA) compared to chicken inoculated with strain BEN 2908 (median=7640 vs 1100 in the liver; 333 vs 20 in the blood; Fig. 3
). A statistical analysis was performed using the MannWhitney test as described in Methods. Chickens inoculated with the mutant BEN 2755 (
ibeA) had significantly fewer bacteria in the blood and in the liver (P<0,05), indicating that the mutant BEN 2755 (
ibeA) was less virulent than the wild-type strain BEN 2908.
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DISCUSSION |
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We showed in this study that, in a collection of strains isolated from chicken, ibeA is present in the genome of 26 % of the pathogenic isolates but absent from the genome of non-pathogenic isolates. The prevalence of ibeA in APEC strains (26 %) is not very different from the frequencies observed in isolates associated with neonatal meningitis (4033 %) or vaginal isolates (32 %) (Johnson et al., 2001a; Obata-Yasuoka et al., 2002
). However, this distribution is very different from that observed in the entire E. coli Reference Collection (ECOR collection) where only 3 % of the strains are ibeA+ (Johnson et al., 2001a
). The reason for this might be that strains from the ECOR collection are mainly of human faecal origin and that ibeA is less likely to be found in such isolates (Bingen et al., 1997
; Johnson et al., 2001a
). Our analysis also allowed comparison of the prevalence of ibeA with the serogroup of strains. It is interesting to point out that ibeA is positively associated with O88, O18 and O2 strains. Among the O18 strains, most were ibeA+ and pathogenic for day-old chicks. This compares with the high percentage of O18 NMEC isolates that were shown to be ibeA+ (Johnson et al., 2001b
). On the contrary, ibeA is negatively associated with O78 strains; indeed, only one out of 59 O78 strains carried ibeA.
We also showed that ibeA is involved in the invasion of HBMEC by the APEC strain BEN 2908.This strain exhibited a very high invasion capacity compared to that of E44 which is considered a model strain for the study of E. coli neonatal meningitis. Inactivating ibeA significantly reduced the invasion capacity of BEN 2908, indicating that ibeA is partly required for invasion. The fact that the ibeA mutant retains a high invasion capacity also indicates that factors other than ibeA probably mediate invasion by BEN 2908. In strain E44, several genes such as ompA, aslA and ibeB, have also been shown to participate in the invasion of BMEC. One question raised by the in vitro results is that of host specificity. A receptor for IbeA, whose N-terminal part shares some homology with serum albumin, has been described in both human and bovine BMEC. The ability of BEN 2908 to invade human BMEC therefore suggests that the receptor for IbeA is shared between human and avian host cells. Another possibility is that BEN 2908 has a ligand, yet to be discovered, that is recognized by both human and avian host cell receptors.
In addition, ibeA was shown to be involved at some stage in the pathogenesis of colibacillosis. The BEN 2908 strain routinely used in the laboratory as a reference APEC strain was shown to be ibeA+. From Southern blot and sequencing data (not shown) we demonstrated that the ibeA locus shares a similar organization in the human pathogenic strain RS218 and the APEC strain BEN 2908. Indeed, we found a significant reduction in the virulence of the ibeA mutant in chicken.
The precise role of ibeA in the pathogenicity of APEC and NMEC remains to be elucidated. From our in vivo results, it is not clear at which particular step in the infection ibeA might play a role. One hypothesis, suggested by our in vitro invasion data, would be that ibeA participates in the traversal of epithelia, such as the air sac epithelium or the pulmonary epithelium. In both cases the inactivation of ibeA could reduce the entry of bacteria into the bloodstream and explain the reduced bacteraemia observed when chickens are inoculated with the ibeA mutant.
Three properties shared with ExPEC isolates have been shown in this paper. First, ibeA, a virulence gene that had only been described in human isolates so far, is present in a significant number of APEC strains; second, strain BEN 2908 is able to invade HBMEC cells in a process that is partly dependent on ibeA; third, the BEN 2755 (ibeA) mutant is significantly less virulent in 3·5-week-old chickens than the parent strain BEN 2908.
The possible relationship between APEC and NMEC, and more generally between APEC and ExPEC, remains to be investigated more thoroughly. For example, PFGE or RAPD analysis of a large collection of E. coli strains will indicate a possible lineage and would help the positioning of APEC strains inside the four phylogenetic groups described for ExPEC (Ochman & Selander, 1984; Picard et al., 1999
).
In conclusion, we have shown that ibeA is present in the genome of a significant number of APEC strains, but absent from the genome of non-pathogenic strains. Challenge studies indicated a significant reduction of the virulence of the ibeA mutant. Based on prevalence studies and virulence attenuation of a mutant, conclusions have been drawn for several of the virulence genes identified to date for APEC strains (Blanco et al., 1997; Delicato et al., 2003
; Dozois et al., 1992
, 2000
; Parreira & Gyles, 2003
). It is therefore reasonable to assume that ibeA, when present, plays a role in the pathogenicity of APEC strains.
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ACKNOWLEDGEMENTS |
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Received 3 December 2004;
revised 3 January 2005;
accepted 11 January 2005.
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