Division of Botany & Zoology, Australian National University, Canberra, ACT 0200, Australia1
Tel: +61 2 6125 3552. Fax: +61 2 6125 5573. e-mail: David.Gordon{at}anu.edu.au
Keywords: population genetics, virulence factors, enteric bacteria, ecological structure
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Overview |
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Background |
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Less effort has been made to determine the degree to which genetic variation is partitioned among populations of a species. Earlier work with E. coli demonstrated that the same clone, or at least the same multi-locus genotype, could be isolated from widely separated geographical localities (Selander et al., 1987 ). These results suggested that little spatial structure existed in species like E. coli. However, the frequency of studies concerned with among-population variation has increased in recent years. Most of these studies have concerned soil bacteria and they have, almost universally, demonstrated a significant degree of population differentiation (Haubold & Rainey, 1996
; Wernegreen et al., 1997
; Silva et al., 1998
; Bouzar et al., 1999
).
The mini-review is organized in four sections. First, the rationale behind efforts to trace the source of bacterial contamination in the environment and the goals of these efforts are outlined, together with the assumptions that are implicit in these goals. The second section discusses the validity of these assumptions in light of studies concerning the population genetics of E. coli and other enteric species. The third section summarizes recent data concerning the distribution of virulence factors among the clonal lineages of E. coli. The implications of these data, as they concern tracing the source of environmental bacterial contamination, are discussed. The mini-review closes with suggestions of alternative approaches to tracing the source of environmental bacterial contamination.
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Tracing the source of environmental E. coli contamination |
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In an effort to manage the problem of elevated coliform counts, programmes are under way in many parts of the world to develop methods which will allow the source of the faecal contamination to be determined. For example, can elevated coliform counts be attributed to human-derived or animal-derived faecal contamination, or can the geographical source of the contamination be identified? Much of this work focuses on E. coli, as it is the dominant member of the aerobic flora of humans and the cause of a significant fraction of human bacterial disease (Siitonen, 1994 ). The techniques in use include examining antibiotic resistance profiles and genotyping bacteria using ribotyping or PCR-based methods such as rep-PCR (Parveen et al., 1997
, 1999
; Dombek et al., 2000
). The aim of these methods is to obtain a fingerprint of the environmental isolate that can be then be classified as derived from a particular host group or locality.
Regardless of the bacterial species being monitored, the success of these efforts depends on several assumptions being valid. (1) The species shows geographical structure. That is, the clonal composition of populations differs among localities. (2) The species exhibits some degree of host specificity. That is, it is more likely that particular clones will be isolated from one host species, or group of species, than another. (3) The clonal composition of the species isolated from soil and water represents the clonal composition of the species in the host populations responsible for the faecal inputs to the environment. (4) The clonal composition of populations is stable through time. That is, that the same clones can be recovered from the same locality or host populations for extended periods.
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Geographical structure |
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Geographical structure seems to explain more of the observed genetic variation in other enteric species than it does for E. coli. For isolates collected from Australian mammals, spatial effects accounted for 12% of the allelic variation in Citrobacter freundii, 17% in Hafnia alvei and 22% in Klebsiella pneumoniae, compared to 5% in E. coli (Gordon & Lee, 1999 ).
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Host specificity |
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Other species of enteric bacteria, such as Enterobacter cloacae, H. alvei and Klebsiella oxytoca appear to exhibit a greater degree of host specificity (Fig. 1), although this is not always reflected in the genetic structure of the species (Gordon & FitzGibbon, 1999
). For example, K. oxytoca is far more likely to be recovered from a bat (Vespertilionidae) than a member of any other family of Australian mammal (Fig. 1
). Yet, host taxonomic family explained none of the observed genetic variation in K. oxytoca (Gordon & Lee, 1999
).
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Primary versus secondary habitats |
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Linkage disequilibrium is a hallmark of a clonal genetic structure and simply means that alleles at one locus are non-randomly associated with alleles at other loci (Maynard Smith et al., 1993 ). Virtually all studies of E. coli isolated from hosts have demonstrated linkage disequilibrium. Pupo & Richardson (1995)
sampled E. coli from the inflow to a sewage treatment plant serving a population of about 16000 people and identified 159 haplotypes. Although the authors observed linkage disequilibrium for some pairwise locus comparisons, a multi-locus analysis of their data failed to reveal linkage disequilibrium among the 159 haplotypes (Gordon 1997
). Whittam (1989)
studied E. coli isolated from domestic birds and the litter on which the birds were raised. Only 10% of the 113 distinct clones isolated were recovered from both habitats. Further, the clones isolated from the environment were genetically distinct from those isolated from the host population. These studies suggest that the clonal composition of E. coli communities changes substantially during the transition from the host to the external environment (Whittam, 1989
).
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Temporal variation within populations |
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Temporal changes in the genetic composition of E. coli communities occur at scales beyond that of an individual host. Significant variation over a 4 month period was observed in the composition of E. coli sampled from the inflow to a sewage treatment plant (Pupo & Richardson, 1995 ). No obvious environmental factor (pH, temperature) appeared to account for this variation. In another study, significant changes were observed over a 6 month period in the clonal composition of E. coli isolated from feral house mice (Gordon, 1997
). Some clones were observed in every sample, others were recovered intermittently, while others were only recovered over part of the study. Not all of this variation could be solely attributed to stochastic variation in the relative abundance of strains. A statistically significant decline (30%) in the frequency of colicinogenic isolates and an increase in the frequency of colicin-resistant isolates was observed over the 6 month sampling period (Gordon et al., 1998
).
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Is E. coli the appropriate coliform to examine? |
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Most of the structure observed in E. coli populations appears to occur at the individual host level. Differences among individuals of the same species living in close proximity, such as members of the same human family, baboon troop or bird flock, can account for 2560% of the observed diversity (Caugant et al., 1984 ; Routman et al., 1985
; Whittam, 1989
).
Perhaps the most significant problem with using E. coli to trace coliform contamination is that there appears to be substantial changes in E. coli community composition during the transition from host to external environment. Indeed, the very limited evidence available suggests that there is little similarity between the dominant E. coli community of the host population and the community in the environment where the faeces of that host population accumulate. Such differences were observed in a largely closed system, where the bird population was restricted to a single area and there was little opportunity for outside contamination of the litter on which the birds were raised (Whittam, 1989 ). Therefore, it is likely that changes that are even more significant will be observed in environments that are more complex. For example, when comparing the composition of the inputs to a sewage system and the bacterial community entering the treatment plant after having travelled through kilometres of sewage pipe.
Assigning an environmental isolate to a particular source population not only requires the presence of geographical structure, but that the clonal composition of populations is stable over significant time scales. This assumption appears to be unwarranted. The evidence indicates that there is little temporal stability in the clonal composition of an E. coli population. The temporal variation seems to occur at every level, the individual host, host population and locality. Changes in the clonal composition of populations appear to occur within time frames measured in weeks, rather than months or years.
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E. coli genetic structure and virulence factors |
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E. coli isolates can also be classified as commensal (non-disease causing) or as those that are capable of causing disease in their host. The majority of commensal E. coli clones, those isolated from the faeces of healthy humans, belong to the clusters A and B1 (Picard et al., 1999 ). Although there is a lack of studies characterizing E. coli isolated from non-domesticated animals, the available evidence suggests that most commensal isolates from domestic animals are also members of the A or B1 clusters (Goullet & Picard, 1986
). Isolates capable of causing disease may be further categorized as those responsible for extra-intestinal diseases, such as pyelonephritis or neonatal meningitis, and those occasioning intestinal diseases, such as haemorrhagic colitis. Isolates taken from patients with extra-intestinal disease are grossly overrepresented among the B2 cluster of E. coli (Picard et al., 1999
). However, it has been estimated that B2 strains represent only about 5% of the strains isolated from the faeces of healthy humans (Picard et al., 1999
). Cluster D strains also seem to be under-represented in the faeces of asymptomatic humans (Picard et al., 1999
).
The situation for strains accountable for intestinal infection is somewhat different. Whilst all the intestinal disease-causing strains are highly clonal, they can occur among any of the four major clusters of E. coli (Fig. 2) (Pupo et al., 1997
; Reid et al., 2000
). Recent studies have suggested that clones causing intestinal disease have evolved several times in different lineages of E. coli and this explains why these clonal groups are present in all four clusters (Reid et al., 2000
).
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Sampling considerations |
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There is another dimension to this sampling problem, at least when it concerns E. coli. The dominant strains isolated from the faeces of asymptomatic hosts are mostly from genetic clusters A and B1. These strains represent only a portion of the diversity to be found in E. coli and are less likely to be of clinical significance. Cluster B2 and D strains, those mostly likely to be the cause of extra-intestinal disease, are rarely isolated as the dominant members of the E. coli flora of a host. Where do the B2 and D strains typically persist? Do they occur at low frequencies in the gut of their hosts? If this is the case then conventional sampling regimes are not detecting the clones of potential clinical significance.
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Alternative approaches for tracing the source of coliform contamination |
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Such a PCR assay could be applied directly to faecal material. Serial dilution of the faecal sample followed by PCR of the diluted samples would allow the relative frequency of a particular virulence profile to be determined. Such a sampling approach would not allow the profile of individual isolates to be determined, rather a host profile would be produced. The advantage of such an approach is that it would allow the detection of low-frequency strains. The use of such an assay would of course depend on knowing the frequency of individual virulence factors and their linkage relationships in E. coli, especially the dominant commensal strains of clusters A and B1. This knowledge would enable one to calculate the probability of obtaining a particular virulence profile. If the sample had a virulence profile that suggested the presence of a high-risk strain, and the probability of obtaining this profile by chance was low, then this would suggest that a virulent strain was present in the sample.
Virulence finger-printing has the advantage of detecting clones of potential clinical significance. However, it may have no advantages over conventional fingerprinting techniques given that E. coli appears to exhibit little spatial structure or host specificity and populations show a high degree of temporal variation in their clonal composition. Cluster B2 and D strains, those most likely to cause disease, represent a fraction of the diversity to be found in E. coli. Furthermore, it is possible that these strains exhibit a greater degree of among-population differentiation. An additional advantage to focusing on clinically significant strains is that it may eliminate much of the noise introduced by examining the far more abundant commensal strains that are of little clinical significance.
Other enterics, such as C. freundii or H. alvei, are at least as genetically diverse as E. coli and appear, generally, to exhibit a greater degree of host or spatial structure (Gordon & Lee, 1999 ). They also exhibit a greater degree of host specificity (Gordon & FitzGibbon, 1999
). Although they are much less frequently responsible for human disease than E. coli, perhaps they might be more appropriate for tracing the source of coliform contamination. Whilst there are no data available, these species may show more temporal stability in their host populations and undergo less dramatic changes in their clonal community composition during the transition from the host to external environment. They are also easily isolated using selective plating methods.
This mini-review has highlighted the results of some of the recent studies concerning the population genetics of bacteria. It has endeavored to discuss the results of these studies in the context of a problem perceived to be of potential significance to human health. There are many other aspects of the population genetics and ecology of bacteria that are of equal applied significance. There is every indication that bacteria will continue to be important pathogens of animals and plants, and much evidence to suggest that their significance as disease agents is increasing. The control of infectious diseases depends on, among other aspects, a sound understanding of their population ecology and genetics.
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