Microbiology (2003), 149, 3575–3586 DOI 10.1099/mic.0.26486-0 The distribution and genetic structure of Escherichia coli in Australian vertebrates: host and geographic effects David M. Gordon1 and Ann Cowling2 School of Botany and Zoology1 and Statistical Consulting Unit2, Australian National University, Canberra, ACT 0200, Australia Correspondence David Gordon [email protected] Received 14 May 2003 Revised 16 September 2003 Accepted 18 September 2003 Escherichia coli was isolated from more than 2300 non-domesticated vertebrate hosts living in Australia. E. coli was most prevalent in mammals, less prevalent in birds and uncommon in fish, frogs and reptiles. Mammals were unlikely to harbour E. coli if they lived in regions with a desert climate and less likely to have E. coli if they lived in the tropics than if they lived in semi-arid or temperate regions. In mammals, the likelihood of isolating E. coli from an individual depended on the diet of the host and E. coli was less prevalent in carnivores than in herbivores or omnivores. In both birds and mammals, the probability of isolating E. coli increased with the body mass of the host. Hosts living in close proximity to human habitation were more likely to harbour E. coli than hosts living away from people. The relative abundance of E. coli groups A, B1, B2 and D strains in mammals depended on climate, host diet and body mass. Group A strains were uncommon, but were isolated from both ectothermic and endothermic vertebrates. Group B1 strains could also be isolated from any vertebrate group, but were predominant in ectothermic vertebrates, birds and carnivorous mammals. Group B2 strains were unlikely to be isolated from ectotherms and were most abundant in omnivorous and herbivorous mammals. Group D strains were rare in ectotherms and uncommon in endotherms, but were equally abundant in birds and mammals. The results of this study suggest that, at the species level, the ecological niche of E. coli is mammals with hindgut modifications to enable microbial fermentation, or in the absence of a modified hindgut, E. coli can only establish a population in ‘large-bodied’ hosts. The non-random distribution of E. coli genotypes among the different host groups indicates that strains of the four E. coli groups may differ in their ecological niches and life-history characteristics. INTRODUCTION There is an extensive empirical and theoretical framework regarding the ecological niche and life history characteristics of plant and animal species. However, there have been few attempts to characterize the ecological niche or life history traits of micro-organisms. Even for relatively well known groups of bacteria, such as the Enterobacteriaceae, there is little consensus as to whether a particular species is animalassociated, plant-associated or free-living (Leclerc et al., 2001). Escherichia coli is considered to be a commensal of the lower gastro-intestinal tract of mammals (Hartl & Dykhuizen, 1984; Selander et al., 1987). However, in a study of E. coli isolated from non-domesticated Australian mammals, Gordon & FitzGibbon (1999) showed that the probability of isolating E. coli from an individual depended on the taxonomic family of the host and on the geographic locality from which the host was collected. In an investigation of the genetic structure of E. coli isolated from Australian mammals using the technique of multi-locus enzyme 0002-6486 G 2003 SGM electrophoresis, Gordon & Lee (1999) demonstrated that host taxonomic family and geographic locality explained a small but significant amount of the observed genetic variation. However, there have been few studies that have examined the distribution and genetic structure of E. coli isolated from non-mammalian vertebrates. We present the results of a survey that examined nonmammalian vertebrates and expanded the database available for mammals. This study is based on the examination of faecal samples collected from over 2300 individuals representing more than 350 species that encompass all major vertebrate groups occurring in Australia. The results of analyses investigating the host and geographic factors that influence the probability of isolating E. coli from a host individual are presented. In addition, the E. coli strains isolated were assigned to one of the four groups of E. coli described in the E. coli Reference (ECOR) Collection (Ochman & Selander, 1984) using the recently developed PCR-based method of Clermont et al. (2000). We show that the same host and geographic factors that are significant Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 Printed in Great Britain 3575 D. M. Gordon and A. Cowling predictors of the presence of E. coli in a host species also predict the distribution of strains of the different E. coli groups among host species. METHODS Bacterial isolation and identification. Depending on the host species being sampled, bacteria were isolated from freshly deposited faeces or directly from faecal material in the rectum or cloacae of the host. Faecal collection was carried out using a swab/transport tube system containing Aimes transport medium. Bacteria were isolated by dilution-streaking the sample onto a MacConkey agar plate. After incubation, a single colony with the appropriate morphology was dilution-streaked onto a MacConkey agar plate and, after a further round of incubation, dilution-streaked onto another MacConkey agar plate, and then onto an LB agar plate. All lactosepositive isolates were tested for growth on a Simmons citrate agar plate and for indole production. Freezer cultures were produced by incubating the isolate overnight in LB broth, after which 1 ml of the overnight culture was mixed with 30 ml glycerol and stored at 280 uC. All incubations were carried out at 35 uC. Logistic regression was used to account for the presence/absence of E. coli in each faecal sample in a nested analysis using the taxonomic classification of the host (Subclass, Order, Family, Genus, Species) as explanatory variables. To test the significance of the four highest taxonomic levels, the ratio of the mean deviance of that level to the mean deviance of the level below were compared to the F distribution with the appropriate degrees of freedom. To test the significance of Species, the usual x2 test of deviance was used. Logistic regressions were fitted to presence/absence data for E. coli using host diet, log10(body mass), climate where the host was collected and Species as explanatory variables for mammals. Whilst for birds the explanatory variables were human association, diet, body mass and Species. In logistic regression, the order of fitting explanatory variables affects their significance in the model. Therefore, variables other than Species were fitted using a forward stepwise procedure: the explanatory variable with the highest mean deviance was fitted first, followed by the variable with the second highest mean deviance, and so on. Species was fitted last. Interactions of the explanatory variables, except Species, were also tested. The statistical significance of each variable was tested using a x2 test for the change in deviance. Predicted values were obtained using the final models, which included only significant explanatory variables. Bacterial genotyping. All strains with a phenotype consistent with that of E. coli (Lac+ Cit2 Ind+) were genotyped. The recently developed 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 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. After three attempts, any strain negative for all three PCR products was identified using the BBL Enteric/Nonfermenter ID kit in conjunction with the BBL crystal System Electronic code book. If the strain was identified as E. coli it was considered to be an ECOR A strain. The presence of one or more PCR products of the correct size was considered to be further evidence of the identity of an isolate as E. coli. For the samples from lizards and frogs, to test for differences between species in the prevalence of E. coli, binomial general linear models were fitted and the significance of the Species effect was tested. Multinomial models were used to assess whether diet, climate and body mass affected the distribution of E. coli groups (A, B1, B2, D) in mammals. Significance was assessed using a x2 test for the change in deviance. RESULTS Definition of prevalence Statistical analyses. The study design was unbalanced for two Prevalence is usually defined as the fraction of hosts in which a particular species of parasite is present. The isolation procedure used in this study has the ability to detect E. coli only when it is abundant in a host. The dilution streaking method used for the primary isolation of bacteria in the faecal sample typically results in about 100 well isolated colonies growing on the MacConkey agar plate. Consequently, E. coli will only be detected if it represents, on average, more than 1 % of the total number of bacterial cells capable of growth on this medium. MacConkey medium is non-selective with respect to members of the Enterobacteriaceae (Ewing, 1986), but it inhibits the growth of Gram-positive and many Gramnegative species, to the extent that about 92 % of the bacteria recovered from mammals were members of the Enterobacteriaceae (Gordon & FitzGibbon, 1999). Relative to other members of the Enterobacteriaceae, E. coli has a distinct colony morphology and colour on MacConkey agar and consequently is unlikely to be overlooked. Finally, one person processed over 95 % of the samples, so that variability among observers was not a factor. reasons. First, several families and many genera do not occur in every climate region of Australia (Strahan, 1995). Second, the study samples were acquired in an ad hoc manner so that the number of samples from each species varied considerably. Thus, in this study prevalence of E. coli is defined as the fraction of hosts in which E. coli was a dominant member of the Enterobacteriaceae community in that host. Host and climate data. The taxonomic classifications used in this study are those presented by Cogger (1992), Strahan (1995) and Simpson & Day (1996). Values for the ‘typical’ body mass (g) of each host species were taken from the literature. The diet of a particular species can vary significantly with season and with habitat. Consequently for mammals, each host species was assigned to one broad diet category. The diet categories include carnivorous (invertebrate and vertebrate prey), herbivorous (predominantly vegetation) and omnivorous (invertebrates, vertebrates, fungi, pollen, nectar, seeds and vegetation). For birds, the diet categories were carnivorous (vertebrates), frugivorous (fruit), insectivorous, frugivorous/insectivorous, nectarivorous, nectarivorous/insectivorous, granivorous (seeds) and omnivorous. The climate [desert, semi-arid (grassland), temperate and tropical] of each host locality was determined using the classification developed by the Australian Bureau of Meteorology (www.bom.gov.au), which is based on long-term temperature and rainfall data. 3576 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 Microbiology 149 E. coli distribution and genetic structure The prevalence of E. coli in vertebrates Prevalence in mammals. The prevalence of E. coli among host species varied from 0 to 100 % (Table 1). Overall, E. coli was detected in 56 % of the 1063 mammalian hosts examined. Taxonomic rank accounted for 42 % of the total deviance in the presence/absence of E. coli in each faecal sample. The presence of E. coli in a faecal sample is largely explained by the taxonomic levels of Order, Genus and Species, whilst Subclass and Family were not significant (Table 2). Mammal species live in different climates and differ in their body mass and diet. Each of these factors is a statistically significant predictor of the presence of E. coli in a host Table 1. Prevalence of E. coli for selected Australian mammal species (n>6) Order Monotremata Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Dasyuromorphia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Diprotodontia Peramelemorphia Peramelemorphia Chiroptera Chiroptera Chiroptera Chiroptera Chiroptera Rodentia Rodentia Rodentia Rodentia Rodentia Rodentia Rodentia Rodentia Rodentia Rodentia Rodentia http://mic.sgmjournals.org Family Species Sample size Prevalence (%) Tachyglossidae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Dasyuridae Burramyidae Macropodidae Macropodidae Macropodidae Macropodidae Petauridae Phalangeridae Phalangeridae Phascolarctidae Potoroidae Potoroidae Vombatidae Vombatidae Peramelidae Peramelidae Vespertilionidae Vespertilionidae Vespertilionidae Vespertilionidae Vespertilionidae Muridae Muridae Muridae Muridae Muridae Muridae Muridae Muridae Muridae Muridae Muridae Tachyglossus aculeatus Antechinus flavipes Antechinus minor Antechinus stuartii Antechinus swainsonii Dasycercus cristicauda Dasyurus geoffroii Dasyurus hallucatus Dasyurus maculatus Dasyurus viverrinus Ningaui ridei Parantechinus bilarni Sarcophilus harrisii Burramys parvus Macropus eugenii Macropus giganteus Macropus robustus Onychogalea fraenata Petaurus gracilis Trichosurus caninus Trichosurus vulpecula Phascolarctos cinereus Bettongia penicillata Potorous tridactylus Lasiorhinus krefftii Lasiorhinus latifrons Isoodon obesulus Perameles nasuta Chalinolobus gouldii Chalinolobus morio Nyctophilus geoffroyi Vespadelus darlingtoni Vespadelus vulturnus Mastacomys fuscus Mus musculus Notomys alexis Notomys mitchellii Pseudomys desertor Pseudomys hermannsburgensis Pseudomys patrius Rattus fuscipes Rattus lutreolus Rattus rattus Zyzomys argurus 9 23 8 26 18 19 9 34 14 42 9 9 89 11 13 24 26 11 10 60 52 18 15 11 10 7 8 8 8 7 24 12 16 33 10 40 7 21 35 8 84 20 7 14 44 22 0 23 22 16 89 76 79 74 0 0 79 36 92 96 96 82 40 100 75 78 93 82 80 86 75 75 25 0 25 42 13 33 80 3 100 0 0 37 75 65 100 29 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 3577 D. M. Gordon and A. Cowling Source of variation Subclass Order|S Family|O, S Genus|F, O, S Species|G, F, O, S Residual Total d.f. 2 3 10 28 48 971 1062 Deviance 82?77 75?65 51?93 227?50 175?56 842?51 1455?92 F 1?64 4?86 0?64 2?22 P value 0?333 0?025 0?767 0?007 0?000 Prevalence of E. coli Table 2. Analysis of deviance for the presence/absence of E. coli in each faecal sample from mammals with respect to the taxonomic rank of the host 1.0 0.9 0.8 0.7 11 6 0.0 _0.1 0 4 18 1 9 2 5 16 3 8 7 14 10 1 2 log individual (Table 3). The inclusion of any of the possible second- and third-order interaction terms (e.g. Diet*Climate) did not significantly improve the explanatory power of the statistical model. Inclusion of host species as a random effect in the analysis did not alter the significance of diet, climate and body mass as predictors of the prevalence of E. coli. Climate, diet and body mass accounted for 65 % of the among-species variation in prevalence. The probability of isolating E. coli from a host individual will depend on the climate in which the host lives, its diet and body mass. Overall, E. coli is unlikely to be isolated from hosts living in the desert, whilst hosts living in the tropics are less likely to harbour E. coli than hosts living in temperate or semi-arid environments. E. coli is less likely to be isolated from carnivores than herbivores and most likely to be isolated from omnivores. The probability of isolating E. coli from a mammal increases with the body mass of the host. The relationship between the prevalence of E. coli and host body mass is illustrated for the carnivores (Fig. 1). The observed prevalence of E. coli in the echidna (Tachyglossus aculeatus) is clearly lower than that predicted by its body mass and this discrepancy probably reflects the echidna’s highly specialized diet of ants and termites. Dasycercus cristicauda is a desert-dwelling species and this explains the low incidence of E. coli in this species. Table 3. Analysis of deviance for the prevalence of E. coli among mammalian host individuals The data are unbalanced so terms are added to the model sequentially beginning with body mass. Source of variation d.f. Deviance P value log10(Body mass) Climate Diet Species Residual Total 1 3 2 88 968 1062 322?95 51?88 26?80 214?69 839?60 1455?92 0?000 0?000 0?000 0?000 3578 15 13 17 0.6 0.5 0.4 0.3 0.2 0.1 12 10 3 [Body Mass (g)] 4 5 Fig. 1. The relationship between the prevalence of E. coli in carnivorous Australian mammals and the log10[body mass (g)] of the host species. The solid line depicts the relationship between body mass and prevalence as determined using the data for 209 individuals belonging to the Order Dasyuromorphia. Points represent the observed prevalence for carnivorous species where n>5. The circles denote species in the Chiroptera: 1, Chalinolobus gouldii; 2, Nyctophilus geoffroyi; 3, Scotorepens balstoni; 4, Vespadelus darlingtoni; 5, Vespadelus vulturnus. Squares denote species in the Dasyuromorpha: 6, Antechinus bellus; 7, Antechinus flavipes; 8, Antechinus stuartii; 9, Antechinus swainsonii; 10, Dasycercus cristicauda; 11, Dasyurus geoffroii; 12, Dasyurus hallucatus; 13, Dasyurus viverrinus; 14, Parantechninus bilarni; 15, Sarcophilus harrisii; 16, Sminthopsis murina. Triangles denote species in the Order Monotremata: 17, Ornithorhynchus anatinus; 18, Tachyglossus aculeatus. Prevalence in birds. Prevalence among host species varied from 0 to 100 %. Prevalence data for those species for which more than six individuals were examined are presented in Table 4. E. coli was detected in 23 % of the 634 bird hosts examined. Taxonomic rank accounted for 37 % of the total deviance in the presence/absence of E. coli in each faecal sample (Table 5). The presence/ absence of E. coli is explained largely by the taxonomic rank of Genus. Taxonomic Order, Family and Species were not significant explanatory variables (Table 5). A significant number of the birds examined were sampled from localities where they lived in close association with humans, whilst others were collected from localities away from significant levels of human habitation. Diet varies substantially among species and although not as extensive as in mammals, among-species variation in body mass is considerable. Most of the birds sampled were collected from temperate regions and consequently climate could not be included as a factor in the statistical analyses. Human association, diet and body mass were found to be significant predictors of the prevalence of E. coli in birds Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 Microbiology 149 E. coli distribution and genetic structure Table 4. Prevalence of E. coli for selected Australian bird species (n>6) Order Anseriformes Ciconiiformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Passeriformes Family Species Sample size Prevalence (%) Anatidae Accipitridae Artamidae Dicruridae Dicruridae Fringillidae Maluridae Maluridae Meliphagidae Meliphagidae Meliphagidae Meliphagidae Meliphagidae Meliphagidae Meliphagidae Meliphagidae Muscicapidae Oriolidae Pachycephalidae Pardalotidae Pardalotidae Pardalotidae Passeridae Passeridae Passeridae Petroicidae Ptilinorhynchidae Chenonetta jubata Falco berigora Gymnorhina tibicen Grallina cyanoleuca Rhipidura fulginosa Carduelis carduelis Amytornis barbatus Malurus cyaneus Acanthagenys rufogularis Lichenostomus chrysops Lichenostomus fuscus Lichenostomus penicillattus Manorina melanophrys Melithreptus brevirostris Philemon corniculatus Phylidonyris novaehollandiae Turdus merula Oriolus sagittatus Pachycephala rufiventris Acanthiza lineata Acanthiza pusilla Sericornis frontalis Neochmia temporalis Passer domesticus Taeniopygia guttata Eopsaltria australis Ptilonorhynchus violaceus 10 37 22 8 15 7 7 38 7 7 27 19 12 10 10 15 7 8 13 7 17 23 12 7 7 10 32 70 16 50 25 47 0 0 34 14 14 19 32 8 0 20 7 71 0 0 0 6 26 8 86 14 8 6 (Table 6). Whilst human association, diet and body mass were significant predictors of prevalence, these factors accounted for only 21 % of the among-species variation in prevalence. Birds living in close association with humans were more likely to have E. coli than birds living away from human habitation. The probability of a host harbouring E. coli increased with its body mass. The prevalence of E. coli was lowest in exclusively seed- or fruit-eating species and higher in those species that include nectar, insects or vertebrates in their diet. Prevalence in reptiles. E. coli was isolated from 33 % of the crocodile hosts examined (n=33), 4 % of the turtles (n=56), 2 % of the snakes (n=44) and 10 % of the lizards (n=314). Prevalence data for individual species where at least six hosts were examined are presented in Table 7. E. coli is unlikely to be isolated from most species of lizard, although prevalence did vary significantly among Table 6. Analysis of deviance for the prevalence of E. coli among bird host individuals Table 5. Analysis of deviance for the presence/absence of E. coli in each faecal sample from birds with respect to the taxonomic rank of the host The data are unbalanced so terms are added to the model sequentially beginning with human association. Source of variation d.f. Deviance F P value Source of variation d.f. Deviance P value Order|S Family|O, S Genus|F, O, S Species|G, F, O, S Residual Total 8 26 56 38 505 633 23?76 79?60 113?29 34?15 431?01 681?81 0?97 1?51 2?25 0?480 0?099 0?005 0?648 Human association log10(Body mass) Diet Species Residual Total 1 1 7 119 504 633 32?96 7?64 13?99 201?40 425?20 681?81 0?000 0?006 0?051 0?000 http://mic.sgmjournals.org Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 3579 D. M. Gordon and A. Cowling Table 7. Prevalence of E. coli for selected Australian reptile species (n>6) Order Crocodilia Crocodilia Squamata Squamata Squamata Squamata Squamata Squamata Squamata Squamata Squamata Squamata Squamata Squamata Squamata Squamata Testudines Testudines Testudines Family Species Sample size Prevalence (%) Crocodylidae Crocodylidae Agamidae Scincidae Scincidae Scincidae Scincidae Scincidae Scincidae Scincidae Scincidae Scincidae Scincidae Gekkonidae Elapidae Elapidae Chelidae Chelidae Chelidae Crocodylus johnstoni Crocodylus porosus Ctenophorus isolepes Ctenotus taeniolatus Ctenotus pantherinus Egernia saxatilis Eulamprus heatwolei Eulamprus tympanum Lampropholis delicata Lampropholis guichenoti Niveoscincus microlepidotus Pseudemoia entecasteauxii Tiliqua rugosa Oedura leseuri Pseudechis porphyriacus Rhinoplocephalus nigrescens Chelodina sp Elseya dentata Emydura victoriea 23 10 8 9 7 12 29 20 8 23 7 11 44 11 16 10 9 21 21 26 50 0 0 0 17 3 0 12 22 0 0 32 0 0 0 0 5 0 species (x2=35?3, d.f.=11, P=0?000). E. coli was relatively common in four species: Lampropholis delicata, Lampropholis guuichenoti, Egernia saxatilis and Tiliqua rugosa. The two Lampropholis species are small skinks and all individuals examined of these two species were collected from a cemetery located in central Sydney, New South Wales. Egernia saxatilis is a moderately sized skink that includes a significant amount of fruits and flowers in its diet, whilst Tiliqua rugosa is a large skink that is unusual in that it feeds almost exclusively on vegetation as an adult. Prevalence in frogs. E. coli was isolated from 12 % of the frogs examined (n=106). However, 10 of the 13 isolates of E. coli were recovered from two species of large tree frog (Litoria infrafrenata and Litoria caerulea) collected in the suburbs of Cairns, Queensland. Both Litoria species often live in or around houses and sheds. Prevalence in fish. E. coli was isolated from 10 % of the fish examined (n=138). No differences in the prevalence of E. coli could be detected among the four species for which there were reasonable samples (n>12) (x2=3?6, d.f.=3, P=0?30). The distribution of A, B1, B2 and D strains in vertebrates A supplementary data file for all strains examined for this study can be obtained as part of the online version of this paper at http://mic.sgmjournals.org. The supplementary file contains the results of the Clermont et al. (2000) method for the assignment of E. coli strains to one of the four groups (A, B1, B2, D) identified in the ECOR collection, together with data concerning the diet and 3580 body mass of the host of origin, as well as the climate in which the host was collected. Distribution of E. coli groups in mammals. Of the 497 E. coli isolates characterized for their group membership 15 % were members of group A, 33 % were group B1, 35 % were group B2 and 17 % were group D strains. Host diet and the climate where the host was collected were found to account for a significant amount of the variation in the distribution of groups among hosts (Table 8). Host body mass also explained a portion of the variation, but the nature of the relationship between the distribution of the E. coli groups and body mass depended upon host diet (Table 8). Climate, host diet and body mass accounted for 40 % of the among-species variation in the distributions of A, B1, B2 and D strains. However, Table 8. Analysis of deviance for the distribution of strains of the four groups (A, B1, B2, D) of E. coli among mammalian host individuals The data are unbalanced so terms are added to the model sequentially beginning with diet. Source of variation d.f. Deviance P value Diet Climate log10(Body mass) Diet*log10(Body mass) Species Residual Total 6 9 3 6 183 1320 1527 66?70 41?44 6?89 88?19 299?42 842?83 1345?47 0?000 0?000 0?075 0?001 0?000 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 Microbiology 149 E. coli distribution and genetic structure Relative frequency of E. coli groups (%) 100 80 60 40 20 0 A B1 B2 D Desert A B1 B2 D Semi-arid A B1 B2 D Temperate Fig. 2. The predicted probability of observing E. coli groups A, B1, B2 and D in different climates assuming a 150 g omnivorous host. A B1 B2 D Tropical even after accounting for these effects, differences among host species still accounted for a significant fraction of the observed variation (Table 8). The distribution of A, B1, B2 and D strains in hosts from different climates is illustrated using the predictions of the statistical model adjusted to reflect rodent hosts (omnivores weighing 120 g), a group present in most Australian biomes (Fig. 2). Group A strains are predicted to predominate in hosts living in desert regions and strains from the other groups are expected to be uncommon. Group A strains are predicted to occur in hosts living in temperate and tropical regions, but are anticipated to be uncommon in hosts living in semi-arid environments. Group B2 strains are expected to be less frequently isolated from hosts living in the tropics than from hosts living in semi-arid or temperate climates. The predicted relationship between the frequencies of A, B1, B2 and D strains and body mass is illustrated for each diet class for hosts living in a temperate climate (Fig. 3). In omnivores B2 strains predominated. The frequency of B2 strains increased and the frequency of B1 strains declined as host body mass increased. Group A and D strains were relatively rare and their frequency showed little change with host body mass. In herbivores, B2 strains were most common in hosts weighing less than 6 kg, but as body mass increased above 6 kg, B2 strains became much less common, with B1 strains becoming correspondingly more common. There was a slight decline in the frequency of group A strains with increasing body mass. In carnivores, B1 strains were http://mic.sgmjournals.org 0.8 0.6 0.4 0.2 0.0 0.5 Relative frequency of E. coli groups The presence/absence of the three PCR products generated using the Clermont method can be used to assign a strain to one of the four E. coli groups. Alternatively, the Clermont method gives rise to eight possible genotypes based on the possible combinations of the presence or absence of the three PCR products. (We note that the genotype chuA2 yjaA+ and TSPE4.C2+ was never observed.) When the analysis presented in Table 8 was repeated, analysing the frequencies of the seven observed genotypes rather than A, B1, B2 and D frequencies, the same outcomes were observed. All factors included in the model presented in Table 8 are statistically significant and there are only small changes in the probability values. This outcome demonstrates that E. coli genotypes are non-randomly distributed with respect to climate, host diet and host body mass. (a) 1.0 1 1.5 2.5 2 3 3.5 4 (b) 1.0 0.8 0.6 0.4 0.2 0.0 2 2.5 3 3.5 4 4.5 5 3.5 4 (c) 1.0 0.8 0.6 0.4 0.2 0.0 1 1.5 3 2.5 2 log10 [Body mass (g)] Fig. 3. Predicted probability of observing E. coli groups A, B1, B2 and D for (a) carnivorous, (b) herbivorous and (c) omnivorous mammalian hosts at different body masses (log10) in temperate climates. Tick marks at the top of the graph show body masses of host species used in the study. (a) A, solid line; B1, dashdot line; B2, dashed line; D, dotted line. (b, c) A, dashed line; B1, dash-dot line; B2, solid line; D, dotted line. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 3581 D. M. Gordon and A. Cowling Table 9. Frequency of occurrence of the four groups (A, B1, B2, D) of E. coli in vertebrates Host group No. of strains Fish Frogs Turtles Snakes and lizards Crocodiles Birds Mammals 12 13 4 33 10 134 497 E. coli group (%) A B1 B2 D 0 7?7 25 15?1 20 8?2 15?5 91?7 84?6 50 69?7 70 49?2 32?7 8?3 0 25 6?1 10 22?3 35?3 0 7?7 0 9?1 0 20?1 16?5 the most common. There was an increase in the frequency of group A strains with host body mass and a corresponding decline in the frequency of B1, B2 and D strains. Distribution of E. coli groups in non-mammalian vertebrates. The relative frequencies of strains of the four groups recovered from fish, crocodiles, turtles, snakes and lizards as well as birds and mammals are presented in Table 9. Too few strains were isolated from the ectothermic vertebrates or birds to allow for any meaningful statistical analysis. Distribution of E. coli groups among hosts groups. The frequency of strains in the four groups isolated from the different vertebrate host groups is depicted in Fig. 4. For this analysis, the ectothermic vertebrates were pooled as a single host group. The relative frequencies of the four groups were similar in mammalian herbivores and omnivores, but differed from that found in carnivores. The distributions of A, B1, B2 and D strains were similar in birds and carnivorous mammals. Ectothermic vertebrates had a very different distribution of the four groups compared to endothermic vertebrates. DISCUSSION The repeatability of the results The patterns observed in this study appear to be temporally stable and independent of the method used to isolate the bacteria. The majority of mammalian hosts were sampled over two periods, 1993–1997 and 2000–2002. The same basic patterns were observed for the prevalence of E. coli in each set of samples. E. coli group A strains are overrepresented in Tasmanian devils (Sarcophilus harrisii) and this pattern was observed in two sets of samples collected 8 years apart from two different localities (data not presented). Pupo et al. (2000b) reported on the results of a study of E. coli isolated from mammals collected from one locality in eastern Australia. The majority of their samples were from a single species of native rat, but they did sample a number of other species. Although the method they used to isolate bacteria was quite different from the one used in the present study, they also observed that E. coli was less commonly isolated from bats and carnivorous marsupials than from omnivores or herbivores. Climate and the prevalence of E. coli E. coli is less likely to be isolated from hosts living in desert or tropical climates than hosts living in semi-arid or temperate climates. This pattern of variation was consistent across host orders, and does not appear to be a host effect as the prevalence of E. coli in species of the same genus differed depending on climatic zone. Why E. coli is less prevalent in mammals living in the tropics is unknown. Maximum summer temperatures in the desert regions of central Australia are typically in excess of 35 uC, the relative humidity is less than 25 % and median rainfall is less than 20 mm a month. It seems likely these conditions will adversely affect the survival of E. coli in the external environment and may, in turn, limit the transmission of E. coli among host individuals. Alternatively, the near absence of E. coli in the faeces of desert-dwelling mammals may be a consequence of host biology. Mammals living in arid regions have adaptations that enable them to conserve water. Whilst water loss through defecation represents a small fraction of an animal’s water balance, morphological modifications in the colon to maximize water retention do occur (Stevens, 1988; Woodall & Skinner, 1993; Murray et al., 1995). It may be that the lower water content of material in the distal colon adversely affects the survival of E. coli cells in this region and as a result cell densities are reduced to such an extent that E. coli is unlikely to be detected in faeces. Relative frequency of E. coli groups (%) 100 3582 80 60 40 20 0 A B1 B2 D A B1 B2 D A B1 B2 D A B1 B2 D A B1 B2 D Birds Mammalian Mammalian Mammalian Fish, frogs, carnivores omnivores herbivores reptiles Fig. 4. The observed frequency of E. coli groups A, B1, B2 and D in different vertebrate host groups. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 Microbiology 149 E. coli distribution and genetic structure Host effects and the prevalence of E. coli The likelihood of a host harbouring E. coli will depend primarily on three factors: first, the frequency with which a host individual is exposed to E. coli. Second, the probability that an exposure event will result in the establishment of a population. Third, the mean length of time the E. coli population can persist in the host, or alternatively the rate at which the host loses its population of E. coli. If one assumes a cohort of new-born hosts uninfected with E. coli, then the change with age in the number of hosts with and without E. coli is described by the following differential equations: If contact rate is not the sole factor responsible for the variation in prevalence observed among species, then this would suggest that the among-species variation may, in part, be due to the ability of E. coli to establish a population in the gastro-intestinal tract of the host. dN ~{bNzdE{kN da dE ~zbN{dE{kE da where N is the number of individuals in the cohort without E. coli and E the number of hosts harbouring E. coli. The transmission rate, b, represents the rate at which a host is exposed to E. coli and the probability that exposure will result in the establishment of a population. The E. coli population is lost from a host individual at the rate d. Individuals are lost from the cohort due to death, which occurs at the rate m. The total number of individuals in the cohort is given as N+E. The equilibrium prevalence of the host harbouring E. coli is: %E ~ bz kd bz kd z1 Thus, in a cohort of hosts the prevalence of E. coli will depend on both the transmission rate and the rate at which a host loses its population of E. coli. Where, for a given transmission rate, the prevalence will decline as the residency time (1/d) of the E. coli population in an individual declines. The observation that birds are more likely to harbour E. coli if they are commensally associated with humans than if they are not indicates that exposure rates do vary among hosts. These higher levels of exposure may be a consequence of many species of birds, such as house sparrows (Passer domesticus) and starlings (Turdus merula) feeding on household food scraps or in compost heaps that are likely to contain E. coli. In addition, these higher exposure levels may also be due to elevated levels of environmental contamination by E. coli due to the faeces of domestic pets and livestock, hosts in which E. coli is prevalent. The observation that E. coli was more prevalent in frogs and lizards living in association with humans provides support for elevated levels of E. coli in the environment, as these species feed primarily on insects or other invertebrates. In mammals, variation in prevalence due to exposure would be expected; however, hosts in which E. coli is normally uncommon (bats and small marsupial carnivores) and which live in urban or suburban habitats were not sampled. http://mic.sgmjournals.org E. coli is not common in most species of birds or carnivorous mammals, many of which feed largely or exclusively on insects. Why E. coli is rare in these hosts is unknown. Is E. coli uncommon in insectivores because these hosts are seldom exposed to E. coli? This seems to be unlikely for two reasons. First, E. coli can be isolated from insects (unpublished data) and insectivores eat many insects per day. Second, it is not immediately obvious why grazers such as kangaroos that feed predominantly on grasses or browsers such as wallabies that feed on the foliage of trees and shrubs would be more frequently exposed to E. coli in their diet. Yet, E. coli is prevalent in kangaroos and wallabies. The gut morphology of the host, diet, food retention times and body size, are all inter-related factors that correlate with host phylogeny (Hume, 1999). Animals represent an energyrich, easily digested food source. Consequently carnivorous mammals have relatively simple guts. The digestive tract is dominated by the small intestine, and the colon is short and often poorly differentiated from the small intestine. Amongst Australian carnivores, a caecum is usually lacking or is poorly developed (Hume, 1999). Short intestines, coupled with high quality food, result in rapid gut transit times. Furthermore, gut transit times increase with body size and vary from about 1 hour for the 18 g dasyurid Sminthopsis crassicaudata to 13 hours for the 1000 g dasyurid, Dasyurus viverrinus (Hume, 1999). By contrast, plant tissue represents an energy-poor food source that is difficult to digest. Microbial fermentation enables mammals consuming plant tissue to increase the nutritional value of the material they ingest, but it requires the presence of a fermentation chamber. In kangaroos and wallabies (Macropodidae), as well as the rat kangaroos (Potoroidae), the foregut provides the primary site of microbial fermentation, although these species also have a caecum (Hume, 1999). The hindgut is the site of microbial fermentation in the balance of Australian herbivores (Hume, 1999). In the majority of small (<10 kg) herbivores the caecum forms the fermentation chamber, whilst in the larger species fermentation takes place in the colon (Hume, 1999). Retention times are significantly longer in herbivores as compared to carnivores and, as in carnivores, increase with body mass. For example, food transit times are about 35 h in the 1 kg common ringtail possum (Pseudocheirus peregrinus) and about 100 h in the 10 kg koala (Phascolarctos cinereus) (Hume, 1999). The digestive tract of Australian omnivores is intermediate between that of the carnivores and the herbivores (Hume, 1999). In the small, primarily insectivorous, mountain pygmy possum the caecum is very small and the colon short, whilst the sugar glider (Petaurus breviceps) has a Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 3583 D. M. Gordon and A. Cowling well-developed caecum. The bandicoots have a modestsized caecum and colon. All Australian rodents possess a caecum (Chivers & Langer, 1994). Food transit rates in omnivores fall between those of carnivores and herbivores (Hume, 1999). Several mathematical models have been developed with the purpose of exploring the dynamics of bacterial populations in the gastro-intestinal tract. These models have investigated the dynamics of bacterial establishment using chemostat models (Topiwala & Hamer, 1971; Baltzis & Fredrickson, 1983; Freter et al., 1986; Smith & Waltman, 1995; Stemmons & Smith, 2000; Ballyk et al., 2001) and plug flow reactor models (Ballyk & Smith, 1999; Ballyk et al., 2001; Jones et al., 2002). The chemostat models describe a physical system closely resembling that of a caecum, while the plug flow reactor models describe a typical carnivore gut. These models have shown that establishment is more likely when the bacterial population exhibits wall growth than when no wall growth can occur. Nutrient concentration is also a factor and there is a threshold concentration of nutrients entering the model gut, below which the bacterial population cannot establish and above which the population will establish and persist. Further, the rate at which material moves through the gut (transit time) can also determine whether a bacterial population can establish, where decreasing transit times lead to populations being less likely to establish. Consequently, the conditions for establishment are predicted to be more restrictive for the small intestine than for the hindgut, due to the faster transit times experienced in the small intestine. The quantity and type of nutrients entering the lower gastro-intestinal tract will vary with host diet. Gut turnover rates and transit times will also vary with diet, gut morphology and body mass. The theoretical models suggest that these factors may well influence the likelihood of E. coli being able to establish a population in a host and hence determine the prevalence of E. coli in a particular host species. Further experimental and theoretical work is required to determine if one of the factors of host diet, gut morphology or body mass (food transit rate) is likely to dominate the dynamics of bacterial establishment in the gastro-intestinal tract. To what extent residence time of an E. coli population varies with diet, gut morphology or body mass is unknown. There have been few studies that have examined the persistence time of an E. coli strain in a host and these studies have largely been restricted to humans (Sears et al., 1950, 1956; Sears & Brownlee, 1952; Shooter et al., 1977; Caugant et al., 1981). The results of these studies indicate that a strain may persist for months if not years. However, the data also suggest that eventually the resident strain is inevitably lost. The factors responsible for the turnover of strains in a host are unknown. Long-term longitudinal studies of the persistence of E. coli in different host species are required. 3584 From the results of this survey of the distribution of E. coli in Australian vertebrates suggest that, as a species, E. coli is adapted to mammals with hindgut microbial fermentation chambers, or in the absence of a hindgut fermentation chamber the host must be ‘large’. The contrast between rodents and carnivores highlights the apparent importance of a hindgut fermentation chamber. E. coli is common in most rodent species, all of which possess a caecum. E. coli is uncommon in bats and many marsupial carnivores, all of which lack a caecum. Yet, many species of rodent are at least as small as bats and smaller than many of the species of marsupial carnivore in which E. coli is uncommon. The observation that the prevalence of E. coli in carnivores increases with host body mass indicates that a ‘large’ body mass can compensate for the lack of a hindgut fermentation chamber. The distribution of ECOR groups in vertebrates The PCR-based Clermont method can produce eight genotypes, seven of which were observed. An analysis similar to that presented in Table 8 showed that these genotypes are non-randomly distributed with respect to climate, host diet and body mass. Does this non-random distribution of genotypes correspond to a non-random distribution of the E. coli groups identified in the ECOR collection? For this question to be answered in the affirmative requires two assumptions to be valid. One is that the ECOR collection is broadly representative of the genetic diversity to be found in E. coli worldwide and that the great majority of strains can be classified into one of the four ECOR groups (A, B1, B2 and D). Recently, Thierry Wirth and colleagues (personal communication) investigated the genetic structure of E. coli using the technique of multi-locus sequence typing, MLST (Maiden et al., 1998). The MLST study was based on over 460 clinical and faecal isolates from humans and 41 species of animal predominantly collected from Africa, Australia, Europe and North America. Analyses of the MLST results demonstrated that the ECOR collection is largely representative of the MLST diversity to be found in faecal isolates of E. coli and confirmed the existence of four genetic groups that correspond to the groups defined in the ECOR collection. The MLST results revealed that recombination among the four groups does occur. Strains are observed which are a ‘mix’ of group A and B1 genetic backgrounds. In addition, some strains are highly heterogeneous and appear to be a mix of A, B1, B2 and D genetic backgrounds. However, three-quarters of the highly heterogeneous strains were clinical isolates associated with intestinal disease. Further details of the Wirth and colleagues MLST study are available at www.mpiib-berlin.mpg.de. Given that the great majority of E. coli strains can be assigned to one of four groups corresponding to those identified in the ECOR collection, then the other assumption is that the Clermont method accurately assigns a strain to an ECOR group. The Thierry Wirth MLST study included 15 of the Australian strains genotyped in the present study. In all but one case there was agreement between group Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 18:26:16 Microbiology 149 E. coli distribution and genetic structure assignment based on the Clermont method and the phylogenic assignment based on the MLST data (Thierry Wirth, personal communication). Johnson et al. (2001) demonstrated that a range of virulence factors associated with extra-intestinal disease vary in their occurrence and their frequency among the four groups of E. coli. All of the isolates examined in the present study have been screened for 30 virulence factors associated with intestinal and extraintestinal disease (unpublished data). The results of the virulence factor screening are in close agreement with the E. coli group assignment of the strains. Consequently, in the discussion to follow we assume that E. coli consists of four, largely distinct, genetic groups and that the group assignments based on the Clermont method are accurate for more than 90 % of the isolates. E. coli groups A, B1, B2 and D are non-randomly distributed among different mammalian hosts. Diet, gut morphology and body mass are all significant predictors of the distribution of the groups among mammalian host. Birds and carnivorous mammals have relatively simple tube-like gastro-intestinal tracts and in both host groups B1 strains predominate. The herbivorous and omnivorous animals all possess a caecum and in these hosts, group B2 strains predominate. The effects of gut morphology and body mass on the distribution of the E. coli groups supports the conclusion that the observed among-species variation in prevalence is in part due to gut morphology and dynamics and is not solely a consequence of among-species differences in exposure to E. coli. The observed distribution of strains from the different E. coli groups suggests that these groups may have different life-history tactics and ecological niches. A and B1 strains appear to be ‘generalists’ as they can be recovered from any vertebrate group. Group B2 and D strains seem to be ‘specialists’ as they are largely restricted to endothermic vertebrates. Group B2 strains appear to be the most specialized, as they predominate in mammals with a hindgut fermentation chamber. Several factors may be responsible for the different distributions of A, B1, B2 and D strains among host groups. The observed distributions may reflect among-group differences in the ability of strains to establish a population, as appears to be the case for B2 and D strains in ectotherms. Competitive interactions among strains within a host may also play a role. Are group B1 strains primarily adapted to vertebrates with simple hindguts or are they competitively inferior to group B2 strains in hosts with hindgut fermentation? Finally, these patterns may reflect differences in the ‘typical’ transmission dynamics of strains among host individuals. Strains of the different E. coli groups differ substantially in the types of disease they are capable of causing in humans. The majority of extra-intestinal E. coli infections in humans are caused by group B2 and D strains (Picard et al., 1999; Johnson et al., 2001). Most strains responsible for intestinal E. coli infections in mammals are derived from strains related to groups A and B1 and to a lesser extent D (Pupo et al., 1997; http://mic.sgmjournals.org Rolland et al., 1998; Pupo et al., 2000a). The source of strains responsible for extra-intestinal infections is thought to be the faecal E. coli population of the infected individual, whilst food and water are the main source of strains responsible for intestinal infection. Host habitat, diet, ‘typical’ body temperature and gut morphology are all factors that influence the prevalence of E. coli and the distribution of the four E. coli groups among host individuals. However, the observation that even after accounting for these factors, host species still explains a significant amount of the among-host variation indicates that other factors may be influencing the distribution of E. coli. Variation in prevalence due to host species effects appears to be particularly significant in birds, where the factors of human association, host diet and body mass account for only 21 % of the among-species variation. To what extent the unexplained variation in birds is a consequence of stochastic factors as compared to unknown ecological factors will require further study. By contrast, in mammals, climate, host diet and body mass accounted for 65 % of the among-species variation in the occurrence of E. coli. In mammals, the unexplained variation may be due to the fact that even closely related species living in the same climate will usually exhibit different microhabitat or diet preferences. Similar species may also differ in their behaviour, for example, in their social organization. The results of this survey of the distribution of E. coli in Australian vertebrates indicates that as a species, E. coli is adapted to mammals with hindgut microbial fermentation chambers. In the absence of a hindgut fermentation chamber, E. coli is more likely to establish a population in ‘large’ hosts than in ‘small’ hosts. Within the species, strains from the different genetic groups of E. coli appear to have contrasting life-history tactics and ecological niches, although the precise nature of these differences is unknown, as is the nature of the adaptations that result in these differences. ACKNOWLEDGEMENTS This study could not have been undertaken without the assistance of the many people who took extra time and effort during their own field studies to collect the samples on which this study is based. Their help was essential to the success of this study and is very much appreciated. 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