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The environmental microbiology of melioidosis

Inglis, Timothy J. J.*,†; Mee, Brian J.; Chang, Barbara J.

Reviews in Medical Microbiology: January 2001 - Volume 12 - Issue 1 - p 13-20
Bacterial Infections
Free

Melioidosis is an unusual bacterial infection. While on the one hand melioidosis can present as an acute, rapidly fatal septicaemia, the causative agent, Burkholderia pseudomallei, can also cause localised soft tissue infection or seroconversion without clinically evident infection. Distinctive epidemiological features of melioidosis include a high prevalence in southeast Asia and northern Australia, a predilection for those with prior co-morbidities such as diabetes mellitus, and an association with soil or surface water exposure. Melioidosis is also notable for primary infection or secondary recurrence after an interval of many years. The ability of B. pseudomallei to survive in soil or water for prolonged periods may explain the relevance of soil or water exposure to melioidosis. However, the means of transmission, definitive reservoir, principal means of exposure and mechanisms of pathogenesis have yet to be fully understood. Careful attention to the environmental microbiology of B. pseudomallei will provide important insights into the normal behaviour of this species and help to explain the environmental origins of melioidosis.

*Division of Microbiology and Infectious Diseases, Western Australian Centre for Pathology and Medical Research, †Department of Microbiology, University of Western Australia, Nedlands, Western Australia, Australia

Address for correspondence: Dr T. J. J. Inglis, Division of Microbiology, PathCentre, Locked Bag 2009, Nedlands, WA 6009, Australia. Fax: +618 9381 7139. e-mail: tim.inglis@health.wa.gov.au

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INTRODUCTION

Melioidosis is an infection that challenges conventional infectious disease paradigms. In its most acute form, a rapidly fatal septicaemia, Burkholderia pseudomallei behaves like a Gram-negative bacillus belonging to the closely related genus Pseudomonas [1]. However, the ability of B. pseudomallei to survive inside phagocytic cells for prolonged periods implies that the species can adapt to a facultative intracellular lifestyle [2]. Despite affecting the lungs in a large proportion of acute cases, melioidosis does not appear to be transmitted via the respiratory route and direct transmission has been documented only in rare circumstances [3]. Many domestic and wild mammalian species contract melioidosis but no animal has been convincingly proven to act as a reservoir for human infection [4–6]. The principal source of human infection is therefore considered to be B. pseudomallei- contaminated soil or water [7–9]. The details of how melioidosis is transmitted are still under debate. The majority of documented melioidosis cases occur in southeast Asia and northern Australia, with the highest prevalence in northeast Thailand and the highest incidence of septicaemic pneumonia in northwestern Australia [10,11]. Endemic melioidosis has been recorded in isolated locations elsewhere in the tropical belt, possibly representing the tip of an unrecognised disease iceberg [12]. It is not known whether intervening gaps in recorded melioidosis distribution are due to a genuine absence of disease or a lack of disease awareness and laboratory technology.

The different presentations of melioidosis require different therapeutic strategies. Optimal results dictate a multi-disciplinary approach, which is often under-resourced in locations where the disease is most common. The septicaemic form of melioidosis is best treated with intravenous fourth generation cephalosporins or carbapenems [13,14]. Convalescent septicaemic and subacute soft tissue infections require a combination of oral agents known to have good tissue penetration and activity against B. pseudomallei [15]. There is at present no licensed vaccine for melioidosis and B. pseudomallei is too widely distributed in the environment in endemic regions to make primary environmental control feasible.

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ENVIRONMENTAL EXPOSURE TO B. PSEUDOMALLEI

The link between contaminated soil and melioidosis was first recognized in 1955 when bacteriologists cultured B. pseudomallei from soil and muddy water samples in French Indo-China [7]. Later, during the Vietnam conflict, many servicemen contracted melioidosis. Infection was thought to be due to inhalation of the dust generated by helicopter blades [16]. Late-onset infections have been reported in a small proportion of US Vietnam veterans up to 26 years after leaving southeast Asia [17]. There is further evidence of the importance of soil or water exposure from studies on civilian cases in northern Australia, Thailand and Malaysia [9,18,19]. A significant association was found in these studies between occupational or recreational soil exposure and subsequent melioidosis. Whether rice farmers in Thailand or gardeners in the Australian Northern Territory, a history of soil exposure is now a recognised melioidosis risk factor. However, in Australia most acute cases are recorded during the wet season when it is thought the B. pseudomallei count in the soil increases. Unusually wet weather in northern Australia resulted in a cluster of cases in Darwin in 1991 [20]. This centre is aware of anecdotal reports of additional case clusters in Katherine (NT) during 1999 and in Townsville (Queensland) during 2000. A cyclone occurring in the Pilbara region of Western Australia in mid-2000 was followed by the first reported cases of acute melioidosis from this area centre and appears to have extended the boundary of the endemic area.

During the end of the 1997 dry season (October and December), a small cluster of acute melioidosis cases in northwestern Australian provided a unique opportunity to study environmental determinants of melioidosis [21]. The preliminary outbreak investigation excluded soil and suggested that the potable water supply might be a potential environmental source of B. pseudomallei [22]. Further investigation of the community water supply led to the recovery of the outbreak strain of B. pseudomallei from an aerating device in the water treatment plant [23]. Removal of the aerator, cleansing of the water storage tanks and improved chlorination eradicated B. pseudomallei from the water supply. Two cases of melioidosis occurred in the community later but both had a history of potential exposure during the 1997 outbreak and had clinical features of infection before the aerator was removed. Another potable water-related incident occurred in the Northern Territory around the same time, though the water supply was unchlorinated and therefore not investigated as thoroughly [11]. These incidents, particularly the Western Australia outbreak, led to the inclusion of water supply as a potential source of melioidosis in a consensus statement drawn up by an Australian working group in 1999 [24]. The same group noted that there was insufficient data on which to base bacteriological guidelines. Further studies are required to determine whether bacteriological standards or surrogate indicators can be used to determine acute melioidosis risk in northern Australia.

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B. PSEUDOMALLEI SURVIVAL IN THE ENVIRONMENT

Persistence of B. pseudomallei in the environment has attracted considerable attention in view of the probable role of exposure to environmental B. pseudomallei as a precursor to subsequent infection. The species has been recovered from a variety of soil types but appears to prefer moist clay soils [25]. Studies have concentrated on surface soil at locations most likely to result in human contact. Within the first 90 cm or so, bacterial count falls in the layers closer to the surface, particularly during the dry season. B. pseudomallei will survive in a viable form without any nutrients and can persist in distilled water for several years [26]. The species tolerates a wide pH spectrum, temperature range and ultraviolet radiation [27]. Recently it has been reported that viable B. pseudomallei can be detected using epifluorescent supravital stains at a pH too low to allow detection of bacteria by conventional viable count [28]. This and reports of soils which are positive by B. pseudomallei-specific PCR [29], but which do not contain culturable bacteria, raise the possibility of survival of environmental B. pseudomallei in a viable but non-culturable (VBNC) state.

The terminology used to describe persistence of bacteria under stressful environmental conditions is controversial. Authorities even disagree over whether a VBNC state exists despite accurate predictions of persistence and fluctuation of Vibrio cholerae in marine habitats based on a proposed VBNC state [30]. In the case of B. pseudomallei, a new selective medium has been designed that improves bacterial recovery when compared with previously used media such as Ashdown's selective agar [31], supporting the view that the culturability of some B. pseudomallei cells is medium-dependent. The relative virulence of these cells has yet to be compared with B. pseudomallei cells from any stage of a conventional growth cycle. Further data are needed before we can be sure that survival in a VBNC state occurs. Moreover, it is not clear whether this effect can be attributed to physicochemical stress, sublethal damage or entry into a sessile bacterial form more typical of a biofilm.

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THE PHYSIOLOGY OF ENVIRONMENTAL B. PSEUDOMALLEI

Like other members of the genus, B. pseudomallei has an unusually wide range of potential metabolic substrates. Carbon storage is achieved under conditions of substrate excess by formation of cytoplasmic inclusions composed of polyhydroxybutyrate [32]. These inclusions account for the ‘safety-pin’ appearance of Gram-stained bacilli. It has been noted that strains of B. pseudomallei can grow under anaerobic conditions and utilize nitrate in distinction to B. cepacia which cannot [33]. From this it can be inferred that B. pseudomallei can utilise nitrate under anaerobic conditions as an alternative terminal electron acceptor. The growth and survival of B. pseudomallei under substrate-limited conditions has not been studied in detail. Safety considerations pose a particular challenge to experimentation involving large volumes of liquid media whether by chemostat or bacteriostat. Nevertheless, delineation of a starvation–stress response for B. pseudomallei would help to explain how the species adapts to changes in the physico-chemical environment.

The flagellar apparatus renders B. pseudomallei motile with positive aerotaxis. A pellicle forms at the broth–air interface of liquid cultures, possibly because of aerotaxis. The majority of strains will form rugose colonies on solid media after incubation in air for several days. The appearance of these colonies is similar to that of the upper surface of the pellicle formed on liquid media. Rare variant strains that produce persistently smooth colonies have been reported [34]. Their appearance on solid media resembles mucoid Pseudomonas aeruginosa and they can be inhibited by the selective media currently used for recovery of B. pseudomallei [31]. On the other hand, mucoid variants of B. pseudomallei have been shown to have an inhibitory effect on strains of the classical phenotype through ammonia production, giving this type a survival advantage. Taking these considerations into account, selective media would be expected to cause under-representation of the mucoid colony type when used for isolation from environmental or clinical specimens. A small colony variant has been described recently which produces three distinct polysaccharide antigens with variable expression according to whether or not glycerol was present in the growth medium [35].

There are insufficient data on the normal habitat of B. pseudomallei to extrapolate from physiological behaviour in vitro. However, the physiological features reviewed above are likely to assist its known capacity to survive in a substrate-limited environment for prolonged periods despite a variety of physical or chemical stresses. Moreover, aerotaxis may assist B. pseudomallei in finding air–water interfaces between soil particles in moist soil and possibly promote a net movement of bacteria towards the surface when dry topsoil becomes moistened by rainfall. Ploughing, harrowing or digging topsoil should increase aeration of the uppermost layer and therefore promote the growth of bacteria such as B. pseudomallei with a predominantly aerobic metabolism.

A metabolic variant of B. pseudomallei has been described with a group of consistent substrate utilisation differences, most notably the ability to assimilate l-arabinose [36]. The precise taxonomic status of these strains is controversial as DNA hybridisation studies indicate insufficient dissimilarity to justify classification as a separate species. Nevertheless, the physiological differences and the lack of human virulence have been used to argue for a new species called Burkholderia thailandensis [37]. In one study from southeast Asia, B. thailandensis strains comprised approximately half of all environmental isolates but were absent from clinical specimens. B. thailandensis has recently been reported to cause occasional human infection [38]. There are several other distinctive features of B. thailandensis including differences in surface antigen expression and molecular genetics which provide researchers with a useful tool for work on the cellular and molecular pathogenesis of melioidosis [39,40].

The pellicle formed by B. pseudomallei at the air–fluid interface is a type of multi-cellular organisation that resembles a benthic mat or biofilm. Biofilms form when bacteria colonise solid–liquid interfaces, and are common to many environmental Gram-negative species. Biofilm formation is thought to be an important aspect of B. pseudomallei ecology and has been proposed as a reason for antibiotic resistance in vivo [41]. Multi-cellular organisation of this bacterial species is likely to be under the same autoregulation or density control mechanisms as that of other aquatic bacteria. Quorum sensing, as this process is called, has recently been described in B. pseudomallei, implying that B. pseudomallei growth control is sensitive to cell density and is achieved through phase-dependent intercellular signalling [42].

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INTERACTIONS WITH OTHER ORGANISMS

Interaction with other bacteria

Little is known about ecological interactions with bacteria other than the inhibitory effect of ammonia produced by some B. pseudomallei strains [34]. Other Burkholderia species, most commonly B. cepacia, are found in soil and water specimens and therefore have to be excluded when attempting isolation of B. pseudomallei [21,22,32]. Whether these species or particular strains within each species gain advantage by bacteriocin expression (as does Pseudomonas aeruginosa) has yet to be determined [43]. At the time of writing, a putative response by B. pseudomallei to intercellular signalling by other bacterial species is plausible but speculative.

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Interaction with protozoa

Intracellular parasitisation of non-pathogenic protozoa by bacteria, particularly between Legionella and Acanthamoeba species, is a well-documented phenomenon [44,45]. This interaction has been used to explore the adaptation of Legionella species to a facultative intracellular habitat and develop a cellular model for bacterial virulence [46]. In vitro experiments on B. pseudomallei and Acanthamoeba co-cultures indicate that B. pseudomallei is capable of intra-amoebic survival [47]. These observations provide a possible explanation for prolonged bacterial survival during periods of drought and other hostile environmental conditions. It is notable that Acanthamoeba, Hartmanella and Vahlkampfia species were identified in specimens obtained during the investigation of the 1997 melioidosis outbreak in Western Australia (M. Henderson, personal communication). It is unclear whether the presence of these protozoa can be used as an indicator of melioidosis risk in the potable water supply.

Co-culture investigations in vitro indicated that the outcome of bacteria–protozoa interaction depended on bacterial inoculum density (multiplicity of infection) and stage of bacterial growth cycle [47]. Synchronisation of the bacterial cell cycle through sudden change in external environmental factors might thus be worth investigating as an environmental determinant of infectivity. If bacterial density and growth cycle are both prerequisites for cellular invasion, a role for bacterial intercellular signalling or quorum sensing is also implied. B. pseudomallei has still to be demonstrated in amoebae recovered from environmental samples in a melioidosis-endemic setting. Moreover, human infection has yet to be attributed to an encounter with free-living amoebae infected by B. pseudomallei. However, the concept of a failed or prematurely curtailed symbiosis may be as useful to investigation of the cellular pathogenesis of melioidosis as it has been with legionellosis [48]. Such endosymbiotic relationships have received closer attention since the discovery of the link between mitochondria and the alpha-1 proteobacteria [49]. Careful study of the interaction between B. pseudomallei and free-living amoebae may thus have wider implications for biology, including the evolutionary significance of bacterial endosymbiosis.

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Interaction with plants

Many members of the genus Burkholderia have a close association with plants. Some are plant pathogens [50], but others are not known to cause plant disease and can be found as nitrogen-fixing bacteria in the plant soil around roots [51]. While B. pseudomallei has not yet been formally associated with root nodules from a specific plant species, a non-outbreak strain was isolated from root soil surrounding the roots of a wattle (Acacia colei) during investigation of the 1997 melioidosis outbreak in Western Australia [22]. Despite extensive sampling of soil in and around the affected community, the only other B. pseudomallei-positive soil specimen was obtained from the rhizosphere. Possible associations with other leguminous plant species are worth investigating to determine whether vegetation can be used as a marker of environmental contamination risk or used for bioremediation of contaminated soils.

There is particular interest in the bacteriology of the rice plant rhizosphere. Burkholderia species other than B. pseudomallei have been recovered from this location [51,52]. No analysis of preferential support for B. pseudomallei by rice plants has been published to date, but the effect of new rice strains on the epidemiology of melioidosis in southeast Asia could provide indirect evidence of a preference for specific varieties. Could the green revolution have enhanced the distribution or virulence of B. pseudomallei? As some aspects of land management increase B. cepacia in soil, it is possible that agriculture will inadvertently increase distribution or count of B. pseudomallei and thus increase disease risk [53]. Conversely, a better understanding of the inter-relationship between methods of land management and the presence of B. pseudomallei could be applied to reduce disease risk.

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Interaction with non-human animals

B. pseudomallei is known to cause infection in a variety of animal species. There was an epizootic in laboratory animals in a medical research centre in Malaya shortly after human melioidosis was first reported [4]. Over the ensuing decades, melioidosis was described in a variety of domestic animal species including pigs and goats. More recently, melioidosis has been recognised in native wild animals in northern Australia and even in captive marine mammals [5,6]. Despite the long history of B. pseudomallei infection in animal species, there has been surprisingly little evidence for a major zoonotic reservoir of human infection. Perhaps the nearest evidence is the demonstration of indistinguishable ribotypes of B. pseudomallei from infected goats and one human case in southwestern Australia [54]. An alternative interpretation of this data is that the human and animal cases are linked by exposure to the same environmental source. Epidemiological studies so far have failed to implicate occupational or recreational contact with animals as a major risk for human infection. Nevertheless, the recognition of melioidosis in livestock or wild native species may be a useful indicator of the environmental distribution of B. pseudomallei and an early warning of potential human disease risk.

There are significant variations in the extent of disease caused by B. pseudomallei in different animal species, with some species experiencing asymptomatic infection on exposure to doses fatal to other species via the same route. The differences in susceptibility to B. pseudomallei have recently been exploited by modelling disease in laboratory animals, including BALB/c and C57BL/6 mice [55]. There are obvious limitations to how closely these results can be extrapolated to human infection, but the interaction between B. pseudomallei and these animals will throw further light on the pathogenesis of melioidosis.

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THE NORMAL HABITAT OF B. PSEUDOMALLEI

It should now be possible to begin a reconstruction of the natural habitat of B. pseudomallei from what is known about the physico-chemical and biological environment of the species. Recent observations have shifted the previous emphasis on bacterial presence in soil to bacterial survival in water and moist soil [22,23]. Although soil conditions clearly have a major determinant effect on the growth and survival of B. pseudomallei, water movement due to natural flow, irrigation, potable water distribution and sewage disposal provide a more efficient means of disseminating the bacteria. That should not diminish the possible contribution of soil movement due to agriculture, construction work, erosion or geological events [22]. Climatic factors explain some aspects of the known distribution of melioidosis [9,22], but the documented spread of B. pseudomallei outside the tropical zone and the ability of some strains to survive low temperatures should lead to caution over the geographical distribution [54,56–58]. As this is a uniquely environmental disease, climate and the biogeochemical functions of B. pseudomallei in its normal inanimate habitat can be expected to have a determining effect on the geographical distribution of the disease.

Many aspects of the pathogenesis of melioidosis have yet to be explained. The demonstrable ability of B. pseudomallei to survive inside free-living amoebae provides an attractive analogy for persistence within mammalian phagocytic cells such as tissue macrophages [2,47]. Close association with the roots of nitrogen fixing plants highlights another specific aspect of the ecology of the burkholderias [23]. These observations suggest a means of improving isolation rates through selection of sites for environmental sampling, and indicate priorities for future environmental control studies. Furthermore, the identification of a potential natural intracellular habitat for B. pseudomallei provides a model for more detailed cell biology investigations including bacterial adhesion, penetration and intracellular trafficking. The recovery of B. pseudomallei from around leguminous plant roots underlines the importance of nitrogen metabolism in the interaction between B. pseudomallei and eukaryotic species. Could a limiting effect of some aspect of bacterial nitrogen metabolism provide a reason why chronic renal failure is a key risk factor for acute septicaemic melioidosis? What is the significance of the arabinogalactan present in rice anthers, given that arabinose-utilising environmental strains are largely non-pathogenic, and that galactose is a component of the B. pseudomallei capsular polysaccharide [36,59,60]?

Although we neither know the full extent and complexity of these inter-species relationships nor their environmental context, it is clear that B. pseudomallei lies at the centre of a complex ecological web in which melioidosis also belongs. At present, the critical environmental events that cause melioidosis remain obscure. We can expect detailed investigation of the interaction between B. pseudomallei and its immediate natural environment to throw more light on the environmental origins of this disease.

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Keywords:

Melioidosis; Burkholderia pseudomallei; bacterial ecology.

© 2001 Lippincott Williams & Wilkins, Inc.