Survival of Escherichia coli 0157 in a soil protozoan:implications for disease

John Barker a, Tom J. Humphrey b, Michael W.R. Brown a;*a Pharmaceutical Sciences Institute, School of Life and Health Sciences, Aston University, Birmingham B4 7ET, UK

b Public Health Laboratory Service, Food Microbiology Research Unit, Church Lane, Heavitree, Exeter EX2 5AD, UK

Received 1 November 1998; received in revised form 6 January 1999; accepted 12 January 1999

Abstract

Intra-protozoal growth of bacterial pathogens has been associated with increased environmental survival, virulence andresistance to biocides and antibiotics. Using laboratory microcosms we have shown that Escherichia coli 0157 survives andreplicates in a common environmental protozoan, Acanthamoeba polyphaga. As protozoa are widely distributed in soils andeffluents, they may constitute an important environmental reservoir for transmission of E. coli 0157 and otherpathogens. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights re-served.

Keywords: E. coli 0157; Protozoan; Acanthamoeba polyphaga ; Intracellular growth; Environmental survival ; Vero-cytotoxin

1. Introduction

The importance of protozoa in soil, sewage andwater ecosystems has been recognised for severaldecades and the relevance of predatory protozoa inthe control of bacterial populations is widely ac-knowledged [1]. Yet, the potential role of protozoaas reservoirs of human/animal pathogens has onlyrecently received attention [2^4]. Bacterial pathogensthat multiply/survive in amoebae and animal/humancells include: Legionella spp., Listeria, opportunistmycobacteria, and coliforms [2^7]. Intra-protozoalgrowth has been associated not only with enhancedenvironmental survival [8] but also with increased

virulence [9] and greatly increased resistance to bio-cides [10] and antibiotics [11]. Legionella spp. havebeen found in all phases of sewage treatment andpopulation numbers do not signi¢cantly declinethrough the treatment process [12]. Survival couldbe assisted by internalisation within protozoan hosts.

Vero-cytotoxin-producing Escherichia coli0157:H7 strains have caused major outbreaks ofhaemolytic uraemic syndrome in Britain and NorthAmerica [13]. The infection is highly transmissibleand may be acquired after ingestion of less than100 bacterial cells [14]. The organism readily infectscattle herds and it can be found in soil [15]. Mudcontaminated with E. coli 0157 may have been re-sponsible for nine cases of E. coli 0157 infection thatoccurred at the Glastonbury Festival in Somerset,UK in 1997 [16]. An environmental source may bean important reason for the continuing E. coli 0157

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* Corresponding author. Tel. : +44 (121) 359 3611, ext. 4164;Fax: +44 (359) 0733; E-mail: M.R.W.Brown@aston.ac.uk

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re-infection of cattle herds. The role of protozoa inthe environmental survival of E. coli 0157 has notbeen studied although the organism can grow in cat-tle manure slurry for several weeks [17]. There isgrowing concern about the survival of pathogens insewage sludges disposed of on land now that greenlaws limit sea dumping. Soils contaminated with or-ganic matter and sewage waste contain greatly in-creased numbers of protozoa such as acanthamoebae[1,18]. Thus it is highly likely that E. coli 0157 in soiland slurry will be preyed on by free-living amoebaewhich could be potential vectors for the spread ofthis pathogen. Using laboratory microcosms we havestudied the potential for a ubiquitous free-living pro-tozoan, Acanthamoeba polyphaga to support thegrowth of E. coli 0157:H7 (vero-cytotoxin-produc-ing).

2. Materials and methods

A well characterised strain of A. polyphaga wasobtained from T. Rowbotham, Leeds Public HealthLaboratory, UK [10]. It was grown axenically at35³C in peptone-yeast-extract-glucose broth as shal-low monolayers in 75 cm2 tissue culture £asks asdescribed previously [10,11]. After 3 days incubationat 35³C the exponentially growing amoebae wereharvested by centrifugation (400Ug, 5 min), washedtwice and resuspended in sterile amoeba-saline [19]to give densities of ca. 105 amoebae ml31. For someassays, suspensions of washed trophozoites (105

ml31) were lysed by ultrasonic disintegration andsterilised by passing through a 0.2 Wm membrane¢lter.

E. coli 0157:H7 (vero-cytotoxin-producing) associ-ated with an outbreak of haemolytic uraemic syn-drome was obtained from the Centre for AppliedMicrobiology, Porton Down, UK. Broth-grown (Ox-oid No 2, Oxoid Ltd, UK) stationary phase cells ofE. coli 0157 were used throughout. The bacteria werewashed (3U) and resuspended in amoeba-saline.

Co-cultures with ¢nal densities of A. polyphagatrophozoites (ca. 105 cells ml31) and E. coli (ca.106 cfu ml31) were prepared in tissue culture £asks.E. coli microcosms in amoeba-lysate and amoeba-saline were also prepared. Static incubation was car-ried out in the dark at 4 and 25³C for 35 days.

Bacterial survival was assessed weekly by platingten-fold dilutions onto MacConkey agar (OxoidLtd, UK). For some experiments internalisationand survival of E. coli was determined after priorstaining of the bacterial cells with a BacLight Live/Dead stain (Molecular Probes, Leiden, The Nether-lands) which stains live cells green and cells withdamaged membranes, red. Internalisation and repli-cation was una¡ected by the staining procedure.

3. Results and discussion

Stationary phase E. coli 0157 multiplied in the co-cultures containing Acanthamoeba trophozoites in-creased in number by s 1.0 log cycle after 4 daysat 25³C (Fig. 1). Thereafter the bacterial count de-creased by 6 0.5 log cycle and then remained con-stant. Lysed Acanthamoeba trophozoites provided asubstrate for rapid growth of the E. coli 0157: after

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Fig. 1. Viable counts of stationary phase E. coli 0157 (vero-cyto-toxin-producing) in amoeba-saline microcosms at 25³C cells (E)and with either metabolic products of lysed A. polyphaga (O), orin co-culture with A. polyphaga (R). Bars represent the standarderrors of the means of three replicate experiments.

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4 days incubation at 25³C the colony count increasedby ca. 1 log cycle. Over the next 3 days the countdecreased by 6 0.5 log cycle but then remained con-stant for the remaining 28 days. E. coli 0157 survivedin the amoeba-saline microcosms, without added nu-trients, at 25³C, showing about a 1 to 2 log cyclereduction in the viable count over the 35 day incu-bation period (Fig. 1). As an additional control, ex-ponential phase E. coli 0157 was inoculated intoamoeba-saline microcosms, without added nutrients,at 25³C. These cells rapidly lost viability and couldnot be recovered after 10 days incubation (data notshown). Co-cultures were also incubated at 4³C. Atthis temperature, E. coli 0157 survived and multi-plied within Acanthamoeba trophozoites and after3 days incubation the count had increased by 1 logcycle (data not shown). Bacteria suspended in amoe-ba-saline without trophozoites decreased by 1 logcycle over the same period.

Phase contrast microscopy of co-cultures incu-bated at 25³C showed that some trophozoites con-tained ten or more E. coli cells, within membranebound vacuoles (Fig. 2). Staining with the BacLightkit revealed internalised, green £uorescent, live bac-teria (data not shown). As the cells multiplied thegreen £uorescence was lost because of dilution ofthe stain, as was observed in numerous cases. Insome vacuoles both green and red £uorescent bacte-rial cells were observed, although bacteria initiallyinternalised within the amoebae had demonstratedgreen £uorescence. Red £uorescence indicates cellswith damaged membranes which may be the resultof the stress induced by the intracellular environ-ment. On several occasions the trophozoites weredirectly observed expelling internalised live bacteriafrom food vacuoles into the environment. Digestionof internalised E. coli occurred in some of the acan-thamoebae at both 4 and 25³C. When the bacteriawere hydrolysed, the STYO 9 component of the Bac-Light stain was released, staining the trophozoitesbright green and no intact bacteria were observed.The trophozoites which permitted growth of E. colidid not absorb the BacLight stain and remained col-ourless. The BacLight stain also revealed E. coli lo-cated in the outer wall of A. polyphaga cysts, verysimilar to the report for Mycobacterium avium,which has shown to be associated with the outercyst wall in A. polyphaga [6].

Our results show that there is a dynamic and mu-tually bene¢cial interaction between E. coli 0157 andA. polyphaga trophozoites in laboratory microcosms.In some instances the trophozoites digested the in-ternalised bacteria as a food source but in others thebacteria multiplied within membrane bound va-cuoles. Growth of E. coli was enhanced when lysedamoebae were used as the suspending medium. It hasbeen noted that Acanthamoeba lysate can have aninhibitory e¡ect on bacterial growth through thepresence of free radicals [20], but E. coli grew rapidlyin our amoeba-lysate. Acanthamoebae are an abun-dant source of amino acids, enzymes, fatty acids andlipids [21,22] and clearly provided E. coli with nu-trients for growth.

The mechanisms that determine the internalisationand digestion of bacteria by amoebae are complexand not fully understood [23]. However, these mech-anisms are likely to be in£uenced by the physiolog-ical characteristics of both the protozoa and the bac-terial prey. In E. coli, the rpoS-encoded sigma factorcs is the master regulator in a complex network ofstationary-phase-responsive genes involved in thegeneral response (GSR) [24]. The GSR has a majorrole in resistance to environmental stress conditions

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Fig. 2. Phase-contrast micrograph of A. polyphaga after 3 daysat 25³C in co-culture with E. coli 0157. The trophozooite con-tains two vacuoles containing ten or more E. coli cells (indicatedby arrows). Barr 10 Wm.

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and we speculate that this may include the ability ofE. coli to evade digestion when internalised withinprotozoa. Recently, Steinert et al. [6] have shownthat E. coli can survive in A. polyphaga at 33³C,although they did not study an 0157 vero-cytotoxi-genic strain. We also found that a non-pathogenicstrain of E. coli (K12 W3110) could survive withinA. polyphaga (unpublished data). Thus the ability ofvero-cytotoxin-producing E. coli to survive in amoe-bae is not a unique phenomenon. Vero-cytotoxin-producing E. coli can also survive and replicate inbovine mammary cells indicating that the organismis able to resist lysosomal attack [25]. It has beensuggested that this intracellular location may providea reservoir of bacteria for the contamination ofworkers, equipment and carcasses at the time ofslaughter. Further, it has been noted by King et al.[7] that internalised coliform bacteria can resist di-gestion by protozoa and survive the e¡ects of chlori-nation. Our results con¢rm bacterial survival withinprotozoa and indicate that protozoa have importantpotential to act as vehicles for the dissemination ofE. coli (or other pathogens) in the environment.Amoeba trophozoites could provide a protectiveniche for E. coli against adverse conditions, espe-cially so if the organism is able to survive in amoebacysts as has been shown for Legionella pneumophila[26] and Vibrio cholerae [27]. Bacteria trapped withinamoeba cysts could be blown through the air andenhance distribution. Grazing cattle almost certainlyingest protozoa in silage and grass and if the ingestedprotozoa contain pathogens, this may be a signi¢-cant route of transmission between and within cattleherds.

A possible link between bacterial evolution in thenatural environment and animal pathogenicity is thegeneral stress response, involving RpoS, and its dualin£uence on environmental survival and bacterialvirulence in animals [24,28,29]. It is possible thatthe co-evolution of bacteria and protozoa hasequipped some species of bacteria both for environ-mental survival and for invasion of and survival inanimal cells/tissues [2^4]. We suggest that this area ofmedical ecology requires further investigation to en-hance our understanding of environmental patho-gens such as E. coli, where protozoa have a potentialrole in transmission.

Acknowledgments

This work was supported in part by grants fromBBSRC (GR/J96086) and The Wellcome Trust(041505/Z/94/Z).

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