1 Department of Immunology, Elizabeth Macarthur Agriculture Institute, Woodbridge Rd, Menangle, NSW 2568, Sydney, Australia
2 Center for Advanced Food Research, University of Western Sydney, Hawkesbury Campus, Penrith South DC, NSW 1797, Australia
3 School of Botany and Zoology, Australia National University, Canberra, ACT 0200, Australia
Correspondence
James J.-C. Chin
james.chin{at}agric.nsw.gov.au
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ABSTRACT |
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MLEE and virulence factor screening results for all E. coli isolates examined are available as supplementary data with the online version of this paper at http://mic.sgmjournals.org.
Present address: NSW Agriculture, PMB 8, Camden, NSW 2570, Sydney, Australia.
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INTRODUCTION |
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Most strains of E. coli are harmless commensal of mammals (Hartl & Dykhuizen, 1984; Selander et al., 1987
); however, some strains are capable of causing either intestinal or extra-intestinal disease (Ørskov & Ørskov, 1992
). Strains responsible for intestinal diseases are thought to cause much of their pathology in the small intestine (Grauke et al., 2002
). This pathology is due, in part, to the ability of the strains to adhere to gut epithelial cells (Law, 2000
). Some commensal E. coli isolates can also adhere to gut epithelial cells (Chin & Mullbacher, 2003
; Chin, 2003
).
Probiotic treatment is a disease-management strategy based on the use of bacterial strains incapable of causing disease in their host, but which can prevent the establishment of pathogenic bacteria (Ouwehand et al., 1999; Kailasapathy & Chin, 2000
). Pathogens may be prevented from establishing a population via a number of mechanisms. The probiotic strain may pre-empt attachment sites in the gut, produce antimicrobial compounds that kill the invading pathogen, or outcompete the pathogen for nutrients (Todoriki et al., 2001
; Gismondo et al., 1999
). Strains of the same species are more likely to have a similar ecological niche than are strains of different species. Most intestinal disease in pigs is caused by pathogenic strains of E. coli (Osek, 1999
). Thus E. coli is a likely candidate for use as a probiotic in pigs. However, selecting a suitable strain or suite of strains for use in a probiotic formulation requires finding strains that are incapable of causing disease but which are able to establish populations in the small intestine of pigs.
The results of mathematical studies modelling the intestine as a plug-flow reactor have suggested that the ability of E. coli to establish populations in the large and small intestine should be different (Ballyk & Smith, 1999). This difference results from the higher velocities that occur in the small intestine as compared to the colon (Chivers & Langer, 1994
). The models suggest that E. coli is unlikely to establish a population in the small intestine unless it is capable of wall growth. If incapable of wall growth, E. coli will be more likely to establish a population in the colon. Thus, the theoretical studies suggest that E. coli inhabiting different gut regions may well be genetically distinct. This is particularly important in the selection of commensal strains as candidate probiotic bacteria.
The goal of this study was to determine if strains of E. coli inhabiting different regions of the gut represent genetically distinct populations. E. coli isolates were obtained from the major gut regions, duodenum, ileum, colon and faeces, of pigs. The isolates were characterized using the technique of multi-locus enzyme electrophoresis (MLEE), screened for the presence/absence of a number of genes implicated as virulence factors and assigned to one of the four main groups of E. coli found in the E. coli Reference collection (ECOR) (Ochman & Selander, 1984). The virulence gene assays were also conducted with a view to investigating the safety factor of commensal isolates if they were to be used as probiotic bacteria.
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METHODS |
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Virulence factor screening.
A total of 18 virulence factor genes were assessed using modifications of the multiplex PCR protocols described by Paton & Paton (1998) and Johnson & Stell (2000)
. Each reaction was carried out using a 25 µl mixture containing 2·5 µl 10x buffer, MgCl2 at a final concentration of 1·5 mM, 20 pmol of each primer, each deoxynucleoside triphosphate (Astral Scientific) at a concentration of 400 µM, 2·5 U HotStarTaq polymerase (Qiagen) and 2 µl boiled bacterial cell lysate. The PCR was performed with a PC960 Air Cooled Thermal Cycler (Corbett research). The 18 virulence factor genes were: PAI, iha, papA, fimH, sfa/focDE, kpsMT II, ibeA, hlyA, gafD, fyuA, ompT, cdtB, traT, kpsMT K5 (group 1); stx1, stx2, eaeA and ehxA (group 2). The PCR procedure for group 1 genes involved the following conditions: 1 cycle of 15 min at 95 °C followed by 30 s at 94 °C; 30 s at 63 °C and 3 min at 68 °C for 25 cycles; and a final extension step of 10 min at 72 °C. The steps involved in group 2 PCR conditions were as follows: denaturing for 3 min at 95 °C followed by 10 cycles of 1 min at 95 °C, 2 min at 72 °C; 1 cycle of 1 min at 95 °C, 2 min at 72 °C; 1 cycle of 1 min at 95 °C, 2 min at 63 °C, 1·5 min at 72 °C; 1 cycle of 1 min at 95 °C, 2 min at 63 °C, 1·5 min at 72 °C; 1 cycle of 1 min at 95 °C, 2 min at 62 °C, 1·5 min at 72 °C; 10 cycles of 1 min at 95 °C, 2 min at 61 °C, 1·5 min at 72 °C; 10 cycles of 1 min at 95 °C, 2 min at 60 °C, 2·5 min at 72 °C.
ECOR assignment.
The method of Clermont et al. (2000) was used to assign the E. coli isolates to one of the four main groups of E. coli identified in the ECOR collection (Ochman & Selander, 1984
; Herzer et al., 1990
). The groups are designated A, B1, B2 and D. The Clermont method is based on a multiplex PCR protocol that determines the presence or absence of two genes (chuA and yjaA) and an anonymous DNA fragment (TSPE4.C2). The presence/absence of the three PCR products is used in the manner of a dichotomous key to assign an unknown isolate to one of the ECOR groups. A fraction of ECOR group A strains are negative for the three DNA products (Clermont et al., 2000
). Strains failing to produce any PCR products were repeated twice more using newly prepared template DNA. Any strain consistently negative for all three PCR products was identified using the BBL Crystal Enteric/Nonfermenter Identification System (Becton Dickinson) according to the manufacturer's protocol. Any strain identified as E. coli and negative for the three DNA products expected from the Clermont method was considered to represent an ECOR group A strain (Gordon & Cowling, 2003
).
Allozyme analysis.
All E. coli isolates were purity streaked onto Luria Broth agar plates from MacConkey plates. A single colony was inoculated into 10 ml Luria Broth (LB) culture and grown overnight with agitation at 37 °C. Cells were harvested by centrifugation, resuspended in 5 ml wash buffer (100 mM Tris, 1 mM EDTA pH 6·8) and harvested by centrifugation. Pellets were resuspended in 600 µl extraction buffer (0·005 mM NADP, 7·2 mM mercaptoethanol) and sonicated with a Branson 200 Sonifier for 10 s using two bursts of 5 s each. Lysed cell culture was centrifuged at 20 000 g for 5 min at 4 °C. Supernatants were stored at 80 °C until further use. Titan-III cellulose acetate plates (Helena Laboratories) were used for enzyme electrophoresis, which was carried out at a constant voltage (200 V) for between 20 and 40 min. A total of 10 enzyme systems were tested, based on similar work involving E. coli carried out in other laboratories (Gordon & Lee, 1999). The following enzyme systems were used: SDH (shikimate dehydrogenase, EC 1.1.1.25), AK (adenylate kinase, EC 2.7.4.3), ADH (alcohol dehydrogenase, EC 1.1.1.1), MR (menadione reductase, EC 1.6.99.2), G6PDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49), MDH (malate dehydrogenase (EC 1.1.1.37), ME (malic enzyme, EC 1.1.1.40), IDH (isocitrate dehydrogenase, EC 1.1.1.42), PGD (6-phosphogluconate dehydrogenase, EC 1.1.1.44) and PGM (phosphoglucomutase, EC 5.4.2.2). ADH, MDH, PGD, PGI, PGM were run in 0·01 M Tris/glycine (TG) buffer, pH 8·5; all others were run in 0·1 M Tris/EDTA/maleate/MgCl2 (TEM) buffer, pH 7·2.
All strains were run at least twice against strains giving similar band mobility in order to confirm their genotype. When no enzyme activity was detected at a particular locus, a null value was assigned to that locus. Nulls were included in the calculations of the genetic distances between strains and diversity estimates but were discarded when determining the total number of alleles per locus. Occasionally two protein bands were detected at a locus in some strains. These cases were treated as representing a different allele when calculating genetic diversity but not for the calculations of the number of alleles.
Statistical analysis.
The genetic diversity of a locus was calculated as
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Molecular analysis of variance (AMOVA) (Excoffier et al., 1992) was used to determine how much of the variation among strains was explained by a population-level effect (pig and gut region). AMOVA is built on the principles of classic analysis of variance. However, the data analysed with AMOVA is the matrix of pairwise genetic distances (the number of loci at which two strains differ). The estimated variance components are expressed as the proportion of the total variance explained by the population-level effect (
st).
st statistics have an interpretation equivalent to the classic single-locus F statistics of Wright (1943)
, which are used for assessing the extent of population differentiation. Although AMOVA is based on analysis of variance, F statistics cannot be used to assess significance levels. Instead a randomization approach is used, whereby strains are randomly assigned to populations and
st recalculated. This procedure is repeated 99 times and the probability of obtaining a
st value as extreme as that observed is determined. The AMOVA analysis was done using the software WINAMOVA 1.5. Principal components analysis was used to reduce the dimensions of the data matrix and the first three axes were used as the response variables in a standard nested analysis of variance.
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RESULTS |
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Strains designated B1 had a significantly greater median number of virulence factors (2 per strain) than did A strains (1 per strain) (KruskallWallis test: =21·2, P<0·001). This was due to the significantly greater prevalence of fimH, fyuA and ompT in B1 strains (contingency table analysis: fimH,
=17·5, P<0·001; fyuA,
=28·9, P<0·001; ompT,
14·1, P<0·001).
Variation within and among pigs
Nested AMOVA based on the allozyme and virulence factor screening results revealed that variation among pigs accounted for 6 % (AMOVA, P<0·001) of the observed genetic variation in this collection of strains. Variation among gut regions (within pigs) accounted for, on average, 27 % (AMOVA, P<0·001) of the variation observed. In two pigs (166 and 181) no statistically significant (P>0·05) differentiation among the E. coli populations inhabiting the different gut regions could be detected (AMOVA). In the pigs where significant differentiation was observed, the extent of population differentiation among gut regions was: pig 129, 42 %; pig 130, 30 %; pig 167, 46 %; pig 172, 31 %; pig 173, 25 % and pig 180, 24 %. Excluding the virulence factor data from the AMOVA analysis had little effect on the outcome of the analysis (6 % among pigs and 26 % among gut regions nested within pigs). The first three principal components accounted for 12 %, 11 % and 10 %, respectively, of the variation in the MLEE and virulence factor data matrix. For the first and third principal components, significant (P<0·01) among pigs and among gut region (nested within pigs) variation was detected using standard analysis of variance methods (results not presented). AMOVA was used to calculate the amount of genetic differentiation occurring among gut regions combining the data for all pigs. The resulting population differentiation statistics were used in a UPGMA analysis to illustrate the genetic similarity of strains from the different gut regions (Fig. 1). Overall, E. coli isolated from the duodenum and ileum were genetically similar and distinct from strains isolated from the colon and faeces. Strains from the colon and faeces were, on average, more distinct from each other compared to strains from the two regions of the small intestine.
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DISCUSSION |
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The results of this study endorse the concept that different gut regions represent different ecological niches for E. coli. The mean amount of among-gut region differentiation was 27 %. This value is comparable to the degree of population differentiation observed (29 %) among the E. coli populations inhabiting different human hosts (Gordon, 2001; Gordon et al., 2002
). It far exceeds the values of 25 % differentiation observed among populations isolated from different geographical localities (Whittam et al., 1983
; Gordon, 1997
; Gordon & Lee, 1999
; Gordon et al., 2002
). It also exceeds the 5 % differentiation observed in E. coli populations isolated from different mammalian taxonomic families (Gordon & Lee, 1999
).
The differentiation that is observed among the E. coli populations inhabiting different gut regions may simply be a consequence of the dynamics of strain colonization and extinction within an individual pig. That is, different regions of the gut are colonized at random by strains and once a region has been occupied other strains are prevented from establishing. However, the observation that ECOR group A strains are more likely to be isolated from the upper GIT and B1 strains from the faeces indicates that the colonization of different regions of the GIT is not a random process. In addition, the results of the AMOVA analysis (Fig. 1) show that the E. coli populations in the duodenum and ileum are very similar to another, but divergent from the populations inhabiting the lower GIT. These outcomes suggest that strains inhabiting different regions of the GIT may possess unique suites of traits that lead to niche specialization with respect to the different environments that exist along the length of the GIT. Further work is required to identify these traits and to determine how they lead to habitat specialization.
Are the commensal strains isolated from pigs in this study capable of causing disease? They seem to be unlikely to cause extra-intestinal disease as most strains that do so belong to the B2 and D ECOR groups and carry virulence genes such as papA and hlyA (Johnson & Stell, 2000). The strains in this study were negative for most virulence genes associated with extra-intestinal infection and were found to be either B1 or A strains. It also appears that they are unlikely to be capable of causing intestinal disease because they lack many virulence gene combinations found in E. coli isolated from clinical cases of both neonatal and postweaning diarrhoea (Wu et al., 2003
). Although stx1 and eaeA are thought to be virulence factors for the VTEC group of diarrhoeic E. coli, these genes were not found to co-occur in a strain and their co-occurrence is thought to be necessary for their virulence to be expressed (Whittam, 1998
; Law, 2000
). The adherence characteristics of some of the commensal isolates have also been analysed (T. Chapman & J. J.-C. Chin, unpublished results) and a number of these have displayed a high capacity to bind to enterocytes in culture. These properties together with the lack of virulence genes increase the probability that commensal E. coli from the upper and lower GIT of healthy pigs may be suitable as a probiotic consortium for the rapid colonization of the intestines of newborn piglets.
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ACKNOWLEDGEMENTS |
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Received 27 August 2003;
revised 11 February 2004;
accepted 5 March 2004.
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