1 Unité de Génomique des Microorganismes Pathogènes and CNRS URA 2171, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris, France
2 Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic
3 Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, 97070 Würzburg, Germany
4 Research Institute of Veterinary Medicine, Hlinkova 1/A, 040 01 Kosice, Slovakia
5 Plate-Forme 4 Intégration et Analyse Génomique, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris, France
Correspondence
Carmen Buchrieser
cbuch{at}pasteur.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence of the completely sequenced BAC insert (C4/1) is AJ829704.
A list of non-detectable ORFs of E. coli strain MG1655 in strain Colinfant in the order in which the ORFs appear in the MG1655 chromosome, together with a list of detectable virulence-associated or PAI-localized genes of pathogenic E. coli may be found in Supplementary Table S1 with the online version of this paper at http://mic.sgmjournals.org. A list of genes of the flexible E. coli genome complement identified in E. coli strain A0 34/86 by the combination of DNA array hybridization, Multiplex PCR and partial genome sequencing may be found in Supplementary Table S2.
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INTRODUCTION |
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Recently, detailed characterization of the genetic organization of pathogenic and commensal E. coli strains was initiated by successive publication of E. coli genome sequences from two non-pathogenic K-12 strains, MG1655 and W3110, two enterohaemorrhagic E. coli (EHEC) isolates, O157 Sakai and EDL933, and one uropathogenic E. coli (UPEC) isolate, CFT073 (Blattner et al., 1997; Hayashi et al., 2001
; Perna et al., 2001
; Welch et al., 2002
). Early comparisons of these genomes have already revealed that horizontal gene transfer in E. coli is far more extensive than anticipated. Compared to E. coli MG1655, the genome of the EHEC strain EDL933 was found to contain 1387 additional genes encoded in strain-specific clusters of diverse sizes (Perna et al., 2001
). Further comparisons with the genome of the UPEC strain CFT073 revealed that the three completely sequenced strains share only 2996 (39·2 %) of the identified 7638 E. coli genes (Welch, 2001
), the rest belonging to the flexible gene pool moving horizontally along a highly conserved framework of the core E. coli genome (Ochman & Jones, 2000
; Welch, 2001
). Given this variation in chromosome size, different E. coli strains can contain well over a megabase of unique DNA, accounting for specific traits distinguishing the members of the species.
In this study, we have focused on the characterization of E. coli strain A0 34/86 of serotype O83 : K24 : H31, which was initially isolated from porcine faeces. This strain has been safely and effectively used in Czech paediatric clinics over the past three decades for prophylactic and therapeutic colonization of the intestine of several thousand premature and newborn infants at risk of nosocomial infection and diarrhoea (Lodinova et al., 1967, 1980
; Lodinova-Zadnikova et al., 1998
). Colonization with E. coli A0 34/86 significantly reduces the occurrence of gastrointestinal infection and death associated with nosocomial infection in preterm newborns, most likely by displacing intestinal pathogens in infected carriers and by assisting the re-establishment of a normal gut flora (Lodinova et al., 1980
; Lodinova-Zadnikova et al., 1995
, 1998
). Colonization by E. coli A0 34/86 further results in important stimulation of local antibody formation in the gut and saliva of colonized infants (Lodinova-Zadnikova et al., 1991
). Moreover, subjects colonized by this strain early after birth appear to be significantly less prone to repeated infections and development of allergies later in life (Lodinova-Zadnikova et al., 2003
). E. coli strain A0 34/86 is a type 1 fimbriated strain which does not produce thermolabile or thermostable enterotoxins (Lodinova et al., 1980
) but produces
-haemolysin (HlyA), which is considered a virulence factor of UPEC and EPEC.
Given its clinical safety and efficacy record, the A0 34/86 strain has been approved for routine use in the Czech and Slovak Republics as a live oral vaccine preparation for infants. Aiming at identifying factors accounting for its beneficial effects, in particular those conferring its high colonization capacity, and in view of the potential use of this strain as a live vector for oral delivery of mucosal vaccines, we report here the first analysis of its genome, as obtained by hybridization to DNA arrays, bacterial artificial chromosome (BAC) and shotgun library construction and sequencing, and in vivo screening for genes potentially accounting for intestinal colonization capacity.
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METHODS |
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Genome comparison by DNADNA hybridization.
Total genomic DNA of E. coli A0 34/86 was [33P]dATP-labelled and used to probe Panorama E. coli Gene arrays (Sigma-Genosys) and the Pathoarray, as described previously (Dobrindt et al., 2003). Hybridization experiments were repeated four times, using independently labelled DNA probes; the DNA from E. coli strain MG1655 served as positive control and that of the Staphylococcus aureus strain Wood 46 as negative control, respectively. Normalization of spot intensities and array data analysis were performed as described earlier (Dobrindt et al., 2003
), and the ORFs were recorded as lacking/not detectable if the signal : noise ratio was below 1·0 in at least three of the four hybridization experiments.
Multiplex PCR.
The multiplex PCR assay for the presence of virulence-associated genes of extraintestinal pathogenic E. coli (ExPEC) was performed as previously described (Johnson & Stell, 2000).
PFGE.
Genomic DNA for PFGE analysis was prepared in agarose plugs, as previously described (Buchrieser et al., 1994), cleaved by I-CeuI (New England Biolabs) and separated on a CHEF-Dr III system (Bio-Rad) at 12 °C in 0·5x TBE buffer, at 6·5 kV cm2 and with pulse times increasing from 5 to 50 s over a period of 22 h.
BAC and shotgun library construction.
The BAC library was prepared by cloning HindIII partially digested and size-separated genomic DNA of E. coli A0 34/86 into the pBeloBAC11 vector (CmR), as previously described (Buchrieser et al., 1999). The size distribution of inserts ranged between 40 and 100 kb, with a mean size of 70 kb, as judged from PFGE analysis of a representative sample of randomly picked BAC plasmids digested by NotI. Shotgun libraries of chromosomal and/or BAC C4/1 DNA sheared to fragments of 13 kb by nebulization were constructed in the pcDNA2.1 vector (Invitrogen), as previously described (Buchrieser et al., 1999
).
DNA sequencing and sequence analysis.
Sequencing reactions were performed on purified plasmid DNA using the Taq BigDye Terminator cycle sequencing kit (Applied Biosystems), the ABI PRISM 377 automatic sequencer and the 3700 capillary DNA sequencer (Applied Biosystems). Sequencing reads were assembled using the PHRED (Ewing & Green, 1998) and PHRAP software (P. Green, unpublished data). Editing was performed with CONSED (Gordon et al., 1998
) and annotation was performed using the ARTEMIS (Rutherford et al., 2000
) and CAAT-box (Frangeul et al., 2004
) software tools. BLAST (Altschul et al., 1997
) and FASTA (Pearson, 1990
; Pearson & Lipman, 1988
) sequence similarity searches were performed using public databases (NCBI). The end sequences of BAC library clones were compared to the genome of E. coli K-12 using the Colibri server at the Institut Pasteur (http://genolist.pasteur.fr/Colibri/). The sequence of the completely sequenced BAC insert (C4/1) is available under the GenBank/EMBL accession number AJ829704. The contigs assembled from the shotgun and BAC sequences are available from our website at http://www.pasteur.fr/gmp/sitegmp/Escherichia_coli_A034-86.
Gut persistence assay in newborn piglets.
Piglets (three to four per group) were orally inoculated immediately after birth and prior to colostrum ingestion by depositing 2 ml of bacterial suspension, in a viscous slurry with dried whey, at the root of the piglet tongue using a syringe without a needle. The piglets were then placed with the sow and reared conventionally for the duration of the experiment. Samples of piglet colon content were withdrawn from individual animals at indicated times, diluted in a 10-fold series in PBS and plated on appropriate selective agar media for determination of bacterial counts (c.f.u.) per gram of faeces.
Persistence of the E. coli A0 34/86 (ZKRL+) strain in the piglet gut was assessed upon single-dose inoculation by 109 c.f.u. On specified days the counts of -haemolytic and nalidixic acid-resistant (HlyA+ and NalR) bacteria in colon content samples were determined by plating on LB agar supplemented with 5 % sheep blood and nalidixic acid (100 µg ml1). It was verified that prior to piglet colonization the flora of the sow was free of HlyA+ and NalR bacteria and that the counts of HlyA+ and NalR on LB plates correlated well with total counts of NalR E. coli from simultaneous platings on MacConkey agar containing nalidixic acid (100 µg ml1). The identity of the HlyA+ and NalR bacteria was further verified by PCR analysis of randomly picked colonies using the primers specific for the hlyA and hlyB intergenic region of E. coli (5'-CTAATGCGGGCAGAGAAATAAAGT-3' and 5'-ATAAAGACGGCAGGGTAACACAC-3'). Screening for BAC clones which conferred enhanced persistence in the piglet gut to the K-12 laboratory E. coli strain DH10B was performed by inoculation of piglets with three different pools of BAC clones, designated A, B and A+B. Pool A contained an equivalent mix of 80 BAC clones, which were overlapping with respect to the E. coli K-12 genome sequence; pool B contained 21 equivalently represented BAC clones with end sequences absent from the E. coli K-12 genome sequence and absent from pool A; pool A+B was an equivalent mixture of pools A and B. The pools of selected BAC clones were prepared by mixing equal volumes of overnight 2 ml cultures of selected BAC library clones grown individually in LB medium with chloramphenicol (12·5 µg ml1). The pooled cultures were pelleted and stored frozen at 76 °C until use for animal inoculation. Cells of E. coli DH10B strain carrying the empty pBeloBAC11 vector were used as a negative control inoculum. To verify that representation of the individual BAC clones in the initial pools was unbiased and that the inocula were not dominated by one or more BAC clones, serial dilutions of pools A and B were plated on LB media with chloramphenicol (12·5 µg ml1), and BAC DNA was extracted from 96 randomly picked colonies per pool. After digestion with NotI, the diversity of BAC clones in the pool was first assessed by PFGE analysis, and the identity of the individual BAC clones was verified by determining the terminal sequences of the inserts. Newborn piglets (three to four per group) were inoculated with 1011 c.f.u. of the indicated pools of BAC clones, as described above, and the representation of the individual BACs within the colon content of individual piglets was assessed at days 4 and 6 after inoculation. Reisolated BAC plasmids were analysed using PFGE and partial fragment sequencing, or PCR with primer pairs specific for selected BAC clones.
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RESULTS AND DISCUSSION |
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Analysis of the genome content of E. coli A0 34/86 by hybridization to DNA arrays
To obtain a first characterization of the genome of E. coli A0 34/86, we compared it to that of E. coli K-12 strain MG1655 by hybridization to Panorama E. coli gene arrays and to the E. coli Pathoarray. The latter contains probes specific for the typical virulence-associated genes of ExPEC, intestinal pathogenic E. coli (IPEC) and Shigella species. It also contains genes known to contribute to the fitness and adaptability of enteric bacteria and which may also be involved in the virulence of ExPEC and/or IPEC, or which are present on mobile genetic elements (Dobrindt et al., 2003). This analysis revealed that 410 ORFs of E. coli MG1655 (10 %) were not detectable in the A0 34/86 strain (Table 1
, Fig. 1
; see also Supplementary Table S1, available with the online version of this paper at http://mic.sgmjournals.org, which lists the 410 E. coli K-12 genes absent in A0 34/86 and their functions, where known). The majority (245) of the missing ORFs encode hypothetical, unclassified or unknown gene products. The E. coli A0 34/86 genome also exhibits diversity in ORFs representing mobile genetic elements, i.e. the prophages CP4-6, DLP12, e14, Rac, Qin, CP4-44, CPS-53, CPZ-55, CP4-57 and KpLEC, which are present in E. coli MG1655 but absent or variable in E. coli A0 34/86.
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Characterization of E. coli A0 34/86 flexible genome complement by partial sequencing and mapping of BAC clones on the core genome of E. coli K-12
An exhaustive BAC library of the A0 34/86 genome was prepared and characterized by sequencing of BAC insert termini. A total of 3936 BAC clones of E. coli A0 34/86 chromosomal DNA in the pBeloBAC11 vector was obtained. The BAC insert sizes ranged between 40 and 100 kb, with a mean insert size of 70 kb, as assessed from PFGE analysis of a representative sample of NotI-digested BAC DNA (data not shown). The BAC library obtained thus represented approximately 3050-fold coverage of the A0 34/86 genome.
A subset of 341 randomly picked BAC clones corresponding to approximately fivefold coverage of the genome, with 7·1 % of the clones (25 BACs) being represented twice, was subjected to sequencing of insert termini. These DNA sequences were analysed for similarity at the protein and nucleotide levels using the GenBank database. Out of the 682 BAC insert termini sequences, 537 could be mapped to the E. coli K-12 (MG1655) genome sequence. Furthermore, 51 BAC end sequences exhibited homology to genes encoding hypothetical proteins of unknown function present in the genomes of UPEC CFT073 and/or EHEC O157 : H7 strains. As summarized in Table 2, 48 BAC end sequences exhibited a significant homology to known virulence-associated genes, including genes encoding the K1 capsule and LPS synthesis systems and fimbrial adhesins (fimAH, F17), toxins (hlyA, cnf) and microcin (mch), confirming the detection of these genes by the Pathoarray hybridization experiments (Table 2, Supplementary Table S2). In contrast, 27 (5 %) did not match any sequence in the public databases. These BAC clones hence appear to carry inserts containing genomic DNA specific to E. coli A0 34/86.
A minimal overlapping set of BAC clones already represents a powerful tool for comparative and functional genomics. The 341 BAC clones were thus positioned on the map of the E. coli K-12 chromosome (Blattner et al., 1997) and an ordered physical map was established (Fig. 2
). Of these clones, 318 covered altogether
94 % of the E. coli K-12 genome, while 23 of the BACs (7 %) could not be positioned on the map because there was no homology of their insert termini sequences to the K-12 genomic sequence. Moreover, the 70 BAC clones having a single insert terminal sequence matching the K-12 chromosome could be exploited to roughly locate at least some of the A0 34/86-specific (non-K-12) DNA on the framework of the E. coli K-12 core genome. As further shown in Fig. 2
, inserts of this A0 34/86-specific genetic material appear to be scattered around the core E. coli genome in at least 10 major regions.
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To identify the E. coli A0 34/86-specific sequences with respect to the already known E. coli genome sequences, we masked the E. coli K-12, E. coli O157 : H7 and E. coli CFT073 sequences, using Cross-match (http:/bozeman.mbt.washington.edu/). When comparing the A0 34/86 sequences with those of the E. coli K-12 genome, a total of 227 kb (19 %) of DNA sequence (134 contigs and 186 single sequences) was identified as present in E. coli A0 34/86 but absent from E. coli K-12 (Table 3). Sixty-seven of these 227 kb sequences were present in E. coli O157 : H7, determining 160 kb (103 contigs and 142 single sequences) as sequences specific to A0 34/86 (Table 3
). Further comparison of A0 34/86 sequences with the merged K-12 and E. coli CFT073 (UPEC) genomes, resulted in 81 kb (50 contigs and 64 single sequences) of A0 34/86-specific sequence (Table 3
). Finally, about 64 kb of DNA sequence (38 contigs and 56 single sequences) scored as A0 34/86-specific when compared to all three completely sequenced genomes of E. coli (K-12, O157, CFT073), as given in Table 3
. Furthermore, 32 kb out of the 64 kb of these A0 34/86-specific sequences were found by comparison to public databases to have been previously identified in other E. coli strains (Table 2
, Supplementary Table S2). Altogether, 32 kb of the A0 34/86 DNA sequences distributed in 18 contigs (14 kb) and 29 single sequences (18 kb) did not match any sequences in public databases and thus are new E. coli sequences, some of which might code particular functions of this strain.
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Agglutination of the bacteria by latex particles containing the Pap receptor and by specific agglutination with an anti-PrfA serum showed that the pap pilus operon is expressed by E. coli A0 34/86, at least in vitro (Table 4). Moreover, agglutination with anti-FocA serum revealed that the F1C fimbriae are also expressed by the A0 34/86 strain (Table 4
). It has been shown that P fimbriae, which bind to galactose-
-1,4-galactose molecules on uroepithelial cells (Baga et al., 1985
), are implicated in increased persistence of E. coli in the colon (Wold et al., 1992
). Thus they may be contributing to the intestine colonization capacity of the A0 34/86 strain.
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In a similar way to iron-acquisition systems, the capacity to produce bacteriocins may also contribute to the colonization capacity and competitiveness of E. coli strains. The mchEFXI genes encoding the microcin H47 secretion and immunity determinant (Rodriguez et al., 1999) are present in the A0 34/86 strain. Moreover, production of H47 microcin/colicin activity by strain A0 34/86 was detected in vitro when using an E. coli DH5
indicator strain (Table 4
). Furthermore, the cvaA and cvaB genes required for secretion of the small proteinaceous colicin V were present in strain A0 34/86 (Gilson et al., 1990
). Production of colicin and microcin may also contribute to the persistence and competitiveness of E. coli A0 34/86 in the gastrointestinal environment.
The E. coli strain A0 34/86 is further known to produce the RTX (repeat in toxin) -haemolysin HlyA, and the activity of the cytotoxic necrotizing factor 1 (CNF1) in A0 34/86 cellular lysates was revealed by using a HeLa cell-based assay (Table 4
). The presence and expression of these different adhesin and toxin genes, usually present in UPEC, makes A0 34/86 resemble a uropathogenic rather than a commensal isolate. Furthermore, the results of a triplex-PCR typing method (Clermont et al., 2000
) support this finding. Commensal strains belong mainly to the phylogenetic lineage A of E. coli, whereas the A0 34/86 strain belongs, according to the triplex-PCR method, to the B2 group (data not shown), which comprises mainly extraintestinal pathogenic strains and, in particular, uropathogenic isolates.
In vivo selection for E. coli A0 34/86 gut persistence factors
The availability of an ordered BAC library opened the way to in vivo screening for genes which could be involved in the gut colonization capacity of the E. coli A0 34/86 strain and which could potentially confer an enhanced persistence in the gut on the BAC carrier strain DH10B (rough, derivative of K-12). Screening experiments were hence performed in the natural porcine host of the A0 34/86 strain using the model of bacterial survival in the gut of newborn piglets. To approximate the prophylactic colonization of newborn infants under the competitive pressure of maternal flora and of the nosocomial strains in the clinics, the animals were reared conventionally with the sow, following inoculation by the bacterial suspensions.
First, the colonization capacity of the E. coli A0 34/86 strain in newborn piglets was characterized, using a NalR variant of A0 34/86 (ZKRL+) that could be unambiguously traced in samples of piglet colon content by plating on blood agar and MacConkey plates containing nalidixic acid (100 µg ml1). As documented by the typical result shown in Fig. 3(a), upon a single oral inoculation of newborn animals by 109 c.f.u. of E. coli A0 34/86 (NalR), the strain rapidly colonized the piglet gut, reaching
1010 c.f.u. per gram colon content within 24 h, and persisted at >108 c.f.u. g1 for a week after a single inoculation. Moreover, the A0 34/86 strain was still detectable in the piglet colon content (
104 c.f.u. g1) 4 weeks after a single inoculation, despite the strong competitive pressure of the intestinal flora of the sow, which was uncontrollably ingested by the piglets with sow excrement over the duration of the experiment. It is also noteworthy that control piglets inoculated with buffer became rapidly contaminated and colonized by the A0 34/86 (NalR) strain to quite high levels, when reared together with the inoculated animals (Fig. 3a
). These results confirmed the high colonization capacity of E. coli A0 34/86 in newborn piglets, and showed that this animal model can be used for in vivo selection of BAC clones contributing to the high capacity of the A0 34/86 strain to persist in the gut.
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Complete sequence determination and analysis of BAC clone C4/1
To characterize the potential colonization factor genes of A0 34/86 carried by BAC C4/1, its entire sequence (55·5 kb) was determined. As illustrated in Fig. 4, the insert of BAC C4/1 carries several gene clusters that might be contributing to the intestinal persistence of E. coli. These comprise the fim gene cluster, which is present in both pathogenic and commensal E. coli strains and encodes production of type I fimbriae, which largely account for the adhesive properties of the family Enterobacteriaceae. Interestingly, the sequence of the tip adhesin, FimH, of A0 34/86 appeared to be identical to that of the highly adhesive pathogenic CFT073 strain, suggesting that the A0 34/86 fimbriae may be contributing to the enhanced retention on intestinal mucosa of the DH10B clone carrying the BAC C4/1.
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The rest of the BAC C4/1 insert consisted of a 25 kb segment almost identical to a chromosomal region of the uropathogenic isolate CFT073, containing the hypothetical yji genes, the yjj osmotic adaptation genes and several chemotaxis and transporter protein-coding genes. These also appear to be partly present in the K-12 strain MG1655 and are largely present in isolates of the EHEC E. coli O157 : H7. More interestingly, a predicted type I restriction and modification system was encoded on this segment of the C4/1 BAC, with the hsdSMR (ecoE specificity, data not shown) genes similar to that of the EHEC (O157 : H7) and UPEC (CFT073) strains. The ecoE genes were expressed by BAC C4/1 in vitro, and conferred on the DH10B carrier strain the capacity to restrict differently methylated phages (Table 4
). As the DH10B carrier strain has all E. coli K-12 restriction modification systems (hsdRMS, mcr and mrr) disabled for the purpose of stable propagation of large heterologous DNA segments, the ecoE restriction modification system expressed by C4/1 BAC could contribute to the enhanced persistence of the C4/1 clone in the animal intestine by bringing about an enhanced protection of DH10B against bacteriophage attack. However, to confirm a role for the C4/1-encoded genes in intestinal colonization, defined mutants of A034/86 and corresponding trans-complemented strains will be constructed and then characterized in the porcine model.
Conclusions
Genomic characterization of E. coli strain A0 34/86 revealed the presence of a broad range of genes classified so far as enterobacterial virulence factors. However, these factors may instead contribute to the fitness and colonization capacity of this commensal strain. The detected genomic islands may provide a selective advantage under specific environmental conditions, and might contribute to the high colonization capacity of this strain in humans and piglets. Our results are in line with the recent observations of Dobrindt and colleagues that a rather high proportion of ORFs encoding putative virulence-associated or PAI-localized genes are also present in non-pathogenic commensal and probiotic E. coli isolates (Grozdanov et al., 2004). However, in contrast to E. coli A0 34/86, the probiotic E. coli strain Nissle lacks virulence factors such as
-haemolysin, P-fimbrial adhesins and the semirough lipopolysaccharide phenotype but, like E. coli A0 34/86, it does express microcins, different iron-uptake systems, adhesins and proteases, which thus may support its survival and its successful colonization of the human gut and not be virulence factors as defined for other E. coli strains (Grozdanov et al., 2004
). These results clearly suggest that for the species E. coli the terms virulence, fitness' and colonization factor are overlapping, and that the relationship between virulence and commensalism in E. coli is far from being fully understood. It is tempting to speculate that expression of these virulence-associated factors may account for the capacity of the A0 34/86 strain to outcompete pathogens and to immunize the host by stimulating the previously observed strong and polyclonal secretory IgA response (Lodinova & Jouja, 1977
; Lodinova-Zadnikova et al., 1991
), which in turn might explain its clinical efficacy when used as a live oral vaccine against nosocomial infections and diarrhoeal disease of newborn infants.
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ACKNOWLEDGEMENTS |
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Received 8 July 2004;
revised 29 September 2004;
accepted 12 October 2004.
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