1 Food Microbiology Collaborating Unit, Health Protection Agency, School of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DT, UK
2 School of Biological and Chemical Sciences, University of Exeter, Exeter, Devon EX5 4PS, UK
3 Laboratory of Microbiology, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348-1692, USA
4 Department of Bacterial Diseases, Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK
5 Division of Farm Animal Science, School of Clinical Veterinary Science, University of Bristol, Langford, Bristol BS40 5DT, UK
Correspondence
T. A. Cogan
tristan.cogan{at}bristol.ac.uk
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ABSTRACT |
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INTRODUCTION |
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Salmonella enterica has the potential to elaborate numerous cell surface structures, including type 1 (SEF21), thin aggregative or curli (SEF17), SEF14, long polar (LPF) and plasmid-encoded (PEF) fimbriae, and flagella. Many of these have been shown to be important in the process of infection of the hen and in the colonization of the reproductive tissues. SEF14 fimbriae, which are only present in a few group D serovars (Turcotte & Woodward, 1993), mediate adherence to reproductive tissues (Ogunniyi et al., 1997
) and may have a role in organ invasion and persistence (Rajashekara et al., 2000
). There is some evidence that flagella are important in the subsequent invasion of internal tissues (Allen-Vercoe et al., 1999
). Type 1 and curli fimbriae in Escherichia coli, analogous to SEF21 and SEF17 of S. Enteritidis, are associated with initial persistence in the avian gastrointestinal tract (La Ragione et al., 2000
). There is little current evidence for a role for PEF and LPF in virulence or persistence in the chick model, or in laying hens. The role of any of these in the growth of Salmonella within the egg is not yet known.
Salmonella Enteritidis is able to multiply to a high level in less than 21 days, the retail shelf-life of eggs in the UK, in around 7 % of contaminated eggs (Cogan et al., 2001). Control of the proliferation of Salmonella within eggs may be achieved by their storage at lower than ambient temperature, which slows down both bacterial growth rates and changes to egg contents which facilitate Salmonella multiplication. It is believed that, although some eggs are contaminated within the yolk due to ovarian infection, in the majority of eggs infected via the tissues of the reproductive tract, Salmonella cells are deposited within the albumen close to the yolk membrane (Humphrey et al., 1989
; Mawer et al., 1989
) or on the vitelline membrane (Gast & Holt, 2000
). As Salmonella cannot proliferate in the albumen of fresh eggs (Baron et al., 1997
), bacteria must penetrate the vitelline membrane and gain access to the yolk contents in order to grow. The vitelline membrane of the hen's egg comprises a collagenous matrix overlaid with a layer of glycoproteins (Bellairs et al., 1963
). Glucose within the albumen reacts with these proteins, causing a progressive increase in the porosity of this membrane, during the storage of eggs at temperatures above 6 °C. This results in the release of yolk contents into the albumen and growth of bacterial contaminants after approximately 21 days at 20 °C (Clay & Board, 1991
). In fresh eggs, however, the yolk membrane presents an obstacle to bacterial invasion of the yolk.
It is possible that survival within, and motility through the albumen, as well as adherence to the vitelline membrane, may be characteristics of those Salmonella serovars that multiply in eggs, such as S. Enteritidis. To test this hypothesis, we set out to determine the multiplication of S. Enteritidis wild-types and mutants defective for motility (aflagellate) and adherence (afimbriate) in an egg model that we developed previously (Cogan et al., 2001).
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METHODS |
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Eggs were obtained from a local caged-production unit, from hens that had not been vaccinated against Salmonella. The eggs were inspected on arrival at the laboratory and cracked or dirty eggs discarded. Shells were surface sterilized with 70 % ethanol that was allowed to evaporate. A small hole was punched in the pointed end of the egg with a sterile pin and 200 µl of bacterial suspension was introduced into the albumen. The hole was then sealed with quick-drying adhesive. Eggs were subsequently stored at a constant temperature of 20 °C for 8 days. The number of Salmonella c.f.u. in the egg contents was determined using previously published techniques (Humphrey et al., 1991). Eggs were surface sterilized with ethanol, aseptically cracked and placed in a sterile plastic bag, then the contents were homogenized using a stomacher. Decimal dilutions were made of the homogenate and 0·5 ml aliquots spread onto xylose/lysine/deoxycholate agar plates, which had been dried at 60 °C for 30 min. The inoculated plates were then incubated at 37 °C for 24 h. Twenty eggs were inoculated with each bacterial strain in each experiment. Experiments were performed three times.
Determination of level of curli and flagella expression using a direct binding ELISA.
Monoclonal antibodies (mAbs) were a gift from M. Dibb-Fuller (Veterinary Laboratories Agency) and were produced against purified curli fimbriae and flagella as described by Hotani (1971) and Thorns et al. (1992)
. A modification of the method of Dibb-Fuller et al. (1999)
was used for the ELISA. Cells were grown at 20 °C in colonization factor antigen broth (CFA) containing 0·5 g l1 ovotransferrin (apo-ovotransferrin, Sigma), the iron-binding protein present in albumen, at pH 9·2, to produce cells that were iron-restricted. Cells were harvested after 36 h growth, 4 h before maximum optical density was reached, to examine late-exponential-phase expression, or after 48 h for stationary phase. Cultures were centrifuged at 3500 g for 5 min and the pellet resuspended in 0·1 M carbonate buffer, pH 9·6, to an OD540 of 1·2. This suspension was coated onto Maxisorb microtitre plates (Merck) and dried overnight at 37 °C. The plates were washed four times with 0·1 M PBS containing 0·05 % Tween 20 (PBS-Tween) at this point, and after each subsequent incubation step. Wells were blocked with 200 µl PBS containing 3 % dried milk and incubated at 37 °C for 1 h. One hundred microlitres of mAb suspended in PBS containing 0·1 % dried milk was added to each well and plates incubated at 37 °C for a further 1 h. Goat anti-mouse horseradish peroxidase-conjugated antibody was added to the wells and incubated for 1 h, followed by the addition of tetramethylbenzidine substrate (TMB). The reaction was stopped with 10 % sulphuric acid and absorbance read at 450 nm. For each antigen, the absorbance of the well containing the strain deficient for that antigen was used as the blank.
Motility tests.
To determine whether the bacterial strains were motile in albumen, cells were grown on BA for 24 h at 37 °C. A small amount of colony mass was then picked from this plate with a needle and inoculated into 20 ml fresh egg albumen in a Petri dish, so that cells were introduced at the edge of the plate. A 10 mm diameter disc of BA was placed into the albumen at the centre of the dish and plates were kept at 20 °C for 8 days and examined daily. Five replicate experiments were performed with each strain. Colonies appearing on the central BA disc were recorded and subsequently typed biochemically, serotypically and by antibiotic resistance, as appropriate, to ensure that they were of the isolate that had originally been introduced into the albumen.
Motility was also recorded after growth in CFA containing 0·5 g ovotransferrin l1, at pH 9·2, for 48 h at 20 °C. Cells were viewed as a wet mount by phase-contrast microscopy and the percentage of motile cells in five fields of view recorded.
Statistical analysis of results.
Results from individual experiments were pooled so that the number of eggs in which growth occurred was considered as a proportion of the total number originally inoculated with each strain of Salmonella. The standard error was calculated from this overall proportion. The number of eggs that would have originally received no inoculum was calculated from the Poisson distribution of the inoculum sizes, found by plating aliquots on to BA. The proportion of eggs showing no growth was adjusted using this figure so that uninoculated eggs were not included in the data analysis (Cogan et al., 2001). Differences between the numbers of eggs in which growth of Salmonella occurred were analysed using a chi-squared test, with P values of less than 0·05 regarded as significant. The fit of the distribution of the inoculum size to a Poisson distribution was checked using a chi-squared test. ELISAs for flagella or fimbrial expression were performed on three occasions and differences analysed using a one-tailed t-test, with P values of less than 0·01 considered as significant, in order to look for large differences in levels of expression.
Differences in the frequency of high-level growth (>106 c.f.u. ml1) or levels of fimbrial expression, between groups of strains, were examined using a Student's t-test.
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RESULTS |
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Sixty eggs were infected and enumerated for each Salmonella isolate. Four significantly (P<0·05) different frequencies of yolk invasion were exhibited by the wild-type isolates (Fig. 1). Serovars Gallinarum and Pullorum did not multiply to >106 c.f.u. ml1 in any of the eggs tested, indicating that no invasion of the yolk had occurred. Six per cent of eggs showed yolk invasion when inoculated with the PT6 isolates. This was a significantly (P<0·05) lower proportion than the PT8, 13a and 23 isolates, which invaded and multiplied in the yolk of between 9 and 15 % of the eggs, as did two of the PT4 isolates (E, C6B). The highest frequency of yolk invasion was shown by the two Typhimurium and one of the Enteritidis PT4 strains (S1400), which invaded the yolks of more than 24 % of eggs, a significantly (P<0·05) higher proportion than the PT8, 13a and 23 strains.
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Motility and expression of flagella
Under the high pH, iron-restricted conditions tested, the direct binding ELISA showed that flagella were expressed by all of the Enteritidis and Typhimurium strains, but not by serovars Gallinarum and Pullorum (data not shown). The albumen motility test and microscopy indicated that Gallinarum and Pullorum strains were not motile. Among the mutants, the two fliC mutants and the motAB mutants were not motile (Table 2). All other strains were motile in albumen.
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DISCUSSION |
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Curli production is not vital for the growth of Salmonella in eggs, but is an important factor in permitting a high frequency of yolk invasion. The agfA mutant, which is deficient in the production of curli, showed the lowest frequency of yolk invasion of all the motile but afimbriate mutants tested. Both PT6 strains were also unable to produce curli and showed a decreased frequency of yolk invasion. Curli have been shown to attach to matrix glycoproteins such as fibronectin (Collinson et al., 1991). It is therefore possible that this fimbrial type could be responsible for mediating bacterial attachment to the yolk membrane, which is made up of glycoprotein and collagenous matrix layers (Bellairs et al., 1963
). Attachment to the membrane may then facilitate yolk invasion, or proliferation of cells on the surface of the yolk. This is an area for further study.
It is noted that all of the mutants showed lower levels of yolk invasion than the parental strain, although these differences were not always significant at the 5 % level. This difference could be explained by the lower levels of flagella and/or curli expression seen in these strains. The expression of different types of fimbriae may be linked, so a mutation in one fimbrial type would also affect expression of curli. This hypothesis is supported by the results of the ELISA for SEF17, which show significant decreases in expression of these structures in some of the fimbrial mutants. Flagella expression and motility were also significantly altered in some of the fimbrial mutants. Walker et al. (1999) observed such pleiotropic effects on various bacterial surface structures caused by the disruption of fimbrial expression. The rpoS mutant, EAV53, showed growth in only 5 % of eggs; levels of both SEF17 and flagella, and the percentage of motile cells, were significantly lower in this strain than in S1400 (P<0·01), suggesting that
S may be a regulator of flagella expression as well as of SEF17.
In this work the wild-type strain S1400 showed growth to a high level in 24 % of eggs after 8 days storage at 20 °C while the two other strains of S. Enteritidis PT4 (E, C6B) grew to a high level in only 915 % of eggs stored for the same time at the same temperature. The fact that different PT4 isolates behaved differently in their growth in eggs may be an important factor in understanding the emergence of PT4 as an egg-associated pathogen. Rabsch et al. (2001) suggested that the Enteritidis pandemic was caused by the expansion of a more virulent or successful PT4 clone. Strain S1400 has an advantage over the other PT4 strains tested in the present study as it produced curli fimbriae under both rich and poor nutrient conditions (Figs 4 and 5
). The curli fimbriae would allow the microbe to attach to surfaces in a wider range of conditions than other strains. Bacteria attached to surfaces are known to be more stress tolerant than free cells (Rowbury, 1995
; Humphrey et al., 1997
). The role this behaviour may have had in the emergence of PT4 as an egg-associated pathogen merits further investigation.
The expression of curli during the late exponential phase of growth was found to correlate with a high frequency of growth in eggs. Starvation is known to induce expression of this fimbrial type and expression of the agf genes is controlled by the sigma factor RpoS, produced during entry into stationary phase (Moreno et al., 2000). This is supported by the work presented here, which showed a lack of curli expression in both the agfA and rpoS mutants under all of the conditions tested (Fig. 3
). Growth-phase-independent curli expression has been seen under conditions such as reduced oxygen tension (Gerstel & Römling 2001
) and is known to be controlled by multiple pathways. Results presented in this report show that curli production in some strains is growth-phase independent, and can be induced in some strains under low iron, high pH conditions. Within most of the albumen, iron availability is restricted, so cells would have a stationary-phase phenotype. As bacteria move closer to the yolk, they move up a nutrient concentration gradient (Garcia et al., 1983
). This would relieve growth restriction and result in cells being able to actively grow once they are in close proximity to the yolk. Thus, the strains that have an advantage in eggs are those able to express curli under growth, rather than starvation, conditions.
The two S. Typhimurium strains also showed a high level of yolk invasion. This is at odds with the fact that Typhimurium is not highly associated with egg-borne salmonellosis. Keller et al. (1997) showed that although Typhimurium was able to colonize the ovaries of laying hens, it was not found in eggs post-lay. It is thus possible that Typhimurium does not survive as well as Enteritidis in the forming egg, and that this explains the infrequent incidence of human Typhimurium infection via eggs.
Neither of the Enteritidis PT6 strains elaborated curli to a detectable level under the conditions tested. Both also showed yolk invasion in a smaller proportion of eggs than the other Enteritidis strains, supporting the conclusion that curli are important in invasion of the yolk. As both PT4 and PT6 are closely related and belong to the same clonal lineage (Olsen et al., 1994), it is surprising that the PT6 isolates used here were poorly or non-curliated. A similar phenomenon has been observed in E. coli (La Ragione et al., 1999
), where non-curliation of O78 : K80 strains was associated with the presence of an insertion sequence in the agf operon. The basis of poor curliation in PT6 is the subject of ongoing work.
It has previously been suggested that the principal role of storage temperatures below 20 °C was to slow changes to the yolk membrane that facilitate the growth of Salmonella spp. (Humphrey, 1990). Data in this paper indicate additional temperature effects. Curli are not produced at temperatures below 20 °C (Walker et al., 1999
), which may reduce the growth potential of S. enterica serovars in eggs. Although the genes for curli production appear to be ubiquitous within the genus Salmonella, the fact that expression during exponential-phase growth correlated with a high frequency of yolk invasion provides a useful test for identifying strains with a greater potential to multiply in a large proportion of eggs. This work suggests that Salmonella strains can be categorized, relative to their ability to multiply to high numbers in eggs over 8 days storage, as belonging to one of four groups: (1) motile strains which express curli during exponential-phase growth are able to multiply in the highest proportion of eggs; (2) other motile, curliated strains multiply in a lower proportion of eggs; (3) motile non-curliated strains in the lowest proportion. Non-motile strains (group 4), deposited outside of the yolk, are unable to multiply in any eggs.
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ACKNOWLEDGEMENTS |
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Received 26 September 2003;
accepted 22 December 2003.
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