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Processes controlling the transmission of bacterial pathogens in the environment
Research in Microbiology 158 (2007) 195e202 www.elsevier.com/locate/resmic
Review
Processes controlling the transmission of bacterial pathogens in the environment Fitnat H. Yildiz* Department of Environmental Toxicology, University of California Santa Cruz, Santa Cruz, CA 95064, USA Received 3 October 2006; accepted 1 December 2006 Available online 13 January 2007
Abstract Many pathogens in the environment can be transmitted to human populations and cause outbreaks and epidemics. Transmission is a multifactorial process influenced by the physiology of the pathogen as it exits its initial host, the mechanisms it uses for surviving outside the host, the physiology of the pathogen as it enters the next susceptible host and its ability to establish a successful infection. Few studies so far have focused on the processes responsible for modulating microbial survival in non-host environments and the transmission dynamics between infected and susceptible hosts, as well as the interplay between hosts. A better understanding of these mechanisms is thus necessary for predicting and preventing future outbreaks. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Pathogen; Environmental survival; Biofilm formation; Phase variation; Protozoan grazing
1. Introduction Infectious diseases remain one of the leading causes of mortality worldwide. As new diseases continue to be discovered, some of the old scourges re-emerge to threaten human populations who have not previously been exposed to them. Although we have a relatively good understanding of the mechanisms by which pathogenic microorganisms cause disease, the biology of human pathogens in non-host environments is only beginning to be elucidated. This review discusses recent developments in ‘‘environmental pathogenesis,’’ highlighting those processes shown to be critical for the transmission of pathogenic bacteria. It has become clear that transmission of a pathogen from one host to another is a multi-factorial process influenced by the quantity, growth form (e.g., planktonic or biofilm) and physiology of the pathogen exiting its initial host; by the mechanisms the pathogen
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uses to survive in a non-host environment, which can control the abundance of the pathogen and thus exposure to it; and by the growth form and physiology of the pathogen entering the next susceptible host, which determine its ability to establish a successful infection. Each of these processes is discussed in detail below, drawing from examples of pathogenic bacteria with documented environmental prevalence. These include Vibrio cholerae, the causative agent of cholera, which is a natural inhabitant of aquatic ecosystems around the world and is transmitted via contaminated food or water [31]; Legionella pneumophila, the causative agent of Legionnaires’ disease, which is transmitted via aerosols generated from either natural or manmade environmental sources [2]; non-tuberculous Mycobacteria (NTM), such as M. avium, M. marinum, M. ulcerans, which are opportunistic environmental pathogens that cause infections of the respiratory and gastrointestinal tracts and skin, and can be acquired from surface water, potable water and groundwater by inhalation, ingestion or skin abrasion [47]; Campylobacter jejuni, the leading cause of bacterial gastroenteritis in the world, which is adapted to grow in the avian and
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mammalian intestinal tracts and is transmitted to humans by consumption of contaminated poultry, milk or water [44]. Studies of these pathogens has revealed some of the mechanisms important for enhanced survival outside their hosts and increased virulence among human populations. As our understanding of these mechanisms increases, we should be able to find ways to target them and thereby reduce the transmission of pathogenic organisms. 2. Exit from the host The physiological state of a microorganism shed from the infected host is one of the critical factors determining its fate in the non-host environment and its transmission to the next host. Information in this area is, however, very limited. It was recently reported that V. cholerae present in the stools of cholera patients are in a hyperinfectious state [40]. These studies showed that V. cholera shed in stools outcompete laboratory grown V. cholerae by 10-100 fold in an infant mouse competition assay [40] and that the dose of V. cholerae needed for oral infection is 10-fold lower for the pathogen shed in stools than its in vitro grown counterpart [9]. Although this hyperinfectious state is maintained for only a short time after exiting the host, it is thought to facilitate the transmission of V. cholera during epidemics [9,40]. It is therefore important to elucidate its molecular underpinnings. Whole genome expression profiling revealed that the expression levels of genes required for nutrient acquisition and motility were increased, while those required for bacterial chemotaxis decreased in V. cholerae cells shed in stools from cholera patients, as compared to cultures grown in vitro to a stationary growth phase [40]. Using genomic DNA as a reference, Bina et al. determined that highly expressed genes in the V. cholerae from stools encoded hypothetical proteins, conserved hypothetical proteins, and proteins of unknown functions, whereas genes with intermediate levels of expression encoded virulence factors cholera toxin and toxin coregulated pilus [7]. This study also revealed that the gene expression pattern of V. cholerae from cholera stools was more similar to that of mid-exponential phase bacteria harvested from rabbit ileal loops than that of cultures grown in vitro to stationary phase. A recent study suggested that a state of altered chemotaxis of V. cholerae present in stools may be responsible for enhanced virulence [9]. Although these studies begin to provide insights into the hyperinfectious phenotype of the V. cholerae present in stool samples, the molecular mechanisms involved remain largely unknown. In addition to looking at the expression of genes that may be responsible for hyperinfectivity, a number of studies have focused on the morphological properties of pathogens. For example, studies have shown that V. cholerae shed in human stools is heterogeneous, containing both planktonic and biofilm-like aggregate forms [20]. A competition assay using a reference strain revealed that the average infectivity of the aggregate form is significantly higher than that of the corresponding planktonic cells [20]. It was, therefore, suggested that the hyperinfectivity of V. cholerae shed in human stools
might be due to the presence of the aggregate form of V. cholerae, which delivers a high infectious dose of pathogen to the human host due to better capacity to survive in vivo stresses. The phenotype of V. cholerae dispersed into an aquatic environment is also of epidemiological significance, as different forms of the pathogen are likely to exhibit different survival properties which in turn determine the likelihood of transmission to the next human host. However, more functional studies need to be conducted to evaluate the significance of these different morphological forms of V. cholerae. 3. Environmental growth and survival Upon exiting their host, many pathogens persist in dynamic ecological conditions before infecting another host. Understanding those processes critical for the pathogens’ environmental survival may provide clues for how best to reduce their ability to interact with human populations. The processes described below have been shown to facilitate environmental survival of different pathogens. 3.1. Environmental stress response Bacterial pathogens have developed ways to respond to the physical and chemical factors they encounter in their natural habitats. Laboratory and field studies have shown that in the free-living state, V. cholerae can adaptively respond to fluctuations in environmental parameters, such as salinity, temperature, pH and nutrient availability. For example, upon nutrient deprivation, comma-shaped V. cholerae cells become coccoid-shaped as a result of reductive division, significantly decrease their volume, and reduce their lipid, carbohydrate, RNA and protein contents [28]. In addition to these metabolic and structural changes, nutrient starvation induces resistance to oxidative stress and osmotic shock [66]. These responses are critical to surviving environmental stress. Indeed, mutants lacking the alternative sigma factor RpoS, which controls starvation and stress response in V. cholerae, are impaired in their ability to survive diverse environmental stresses [66]. C. jejuni has multiple environmental survival strategies, which differ from those used by other enteric bacteria, as recently reviewed by Murphy et al [44]. For instance, stationary phase/stress responses are not present in C. jejuni [32], and RpoS, a protein that governs the expression of many of the genes that function in stationary phase/stress response, is not present in C. jejuni [46]. Despite this deficiency, a recent study showed that C. jejuni can initiate a stringent response to nutrient deprivation which is critical for its survival and transmission [23]. In aquatic ecosystems L. pneumophila replicates within amoebae. Upon exhaustion of nutrients in the host cell, L. pneumophila stops replication and activates transmission traits that include production of cytotoxicity factors to escape the host cell, activation of motility for dissemination, increasing extracellular stress resistance and avoidance of phagosomee lysosome fusion in the new host cell [57]. It has been reported that expression of many of these transmission traits is initiated
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by production of the stringent response signal (p)ppGpp and controlled by the alternative sigma factors RpoS and FliA and the regulators of the two component regulatory system [5,6,26,27,41,57]. It has also been shown that upon extended exposure to unfavorable environmental conditions, such as low growth temperature and nutrient starvation, V. cholerae enters into a state of dormancy, a viable-but-non-culturable (VBNC) state during which DNA replication and cell division are incapacitated but metabolic activity is retained [12]. In the VBNC state V. cholerae cannot be grown by standard culturing techniques, but when VBNC cells are inoculated into ligated rabbit ileal loops or given to human volunteers the V. cholerae cells isolated from these hosts can then be cultured [12]. Many bacterial pathogens, including L. pneumophila, C. jejuni, and Mycobacterium spp. can apparently enter into a VBNC state (as recently reviewed by Oliver) [45]. Although it is not clear what this state actually represents, entry into a VBNC state, or any form of dormancy from which pathogens can exit when environmental conditions become more favorable, could represent a step critical in the transmission of many human pathogens. Indeed, such a process could ensure the survival of bacterial pathogens in the environment, even under harsh conditions, and thus increase their abundance. 3.2. Biofilm formation Biofilms are microbial communities composed of microorganisms and the extra polymeric substances they produce, attached to abiotic and biotic surfaces [14]. Biofilm formation by pathogenic microorganisms is a well documented process thought to enhance the survival of pathogenic organisms in certain environments [15,22]. Growth on surfaces may be advantageous as surfaces adsorb and thus concentrate nutrients that are otherwise scarce in the surrounding liquid. In addition, as biofilms contain different species of bacteria, pathogens can engage in favorable metabolic interactions with other members of the consortium. Lastly, the formation of a biofilm provides some protection from grazing predators and from toxic compounds such as antimicrobial agents [14,15]. V. cholerae form biofilms by attaching to surfaces of phytoplankton and zooplankton [16,29,58]. Phytoplanktons excrete a variety of organic compounds that can support the growth of associated (biofilm) or free-living (planktonic) V. cholerae [29,58]. Similarly, attachment to chitinous surfaces of zooplankton and the subsequent degradation of chitin to Nacetyl glucosamine, which serves both as a carbon and nitrogen source, by several chitinases produced by V. cholerae, can support growth of the pathogen in an aquatic environment. Indeed, laboratory microcosm studies have shown that during co-culture, V. cholerae effectively attaches to surfaces of zooplankton and phytoplankton and this association increases the survival period of the organism [29]. Thus, attachment of V. cholerae to plankton and subsequent biofilm growth facilitates the environmental survival and growth of this pathogen. This property of the pathogen may, in turn, aid its transmission. In cholera-epidemic areas, there is a correlation between
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phytoplankton and zooplankton blooms and the timing of epidemics. This suggests that the plankton in V. cholerae’s aquatic habitat acts as a reservoir for the organism, and that the plankton’s life cycle controls the abundance of V. cholerae populations and epidemic cycles. Recent studies have shown that V. cholerae found in the surface waters of cholera endemic areas exists in biofilmlike aggregates [20] in which cells are in a so-called ‘‘conditionally viable’’ state. ‘‘Conditionally viable environmental cells’’ (CVECs) are viable but ‘‘metabolically impeded’’ V. cholera cells. CVECs can regain metabolic activity under specific in vitro conditions, as wells by inoculation into ligated rabbit ileal loops [20], suggesting that these forms of V. cholerae might play a critical role in the transmission of the pathogen. The source of CVECs is thought to be biofilm-like aggregate populations of V. cholerae present in human stools [20]. Similarly, the active and passive detachment of V. cholerae from various surfaces it colonizes in aquatic habitats could also lead to formation of bacterial aggregates. We know very little about how the physical and chemical factors likely to be encountered by V. cholerae in its natural aquatic habitats affect the biofilm formation and detachment from biofilms. Fig. 1 depicts the developmental cycle of biofilm formation by V. cholerae under laboratory conditions. Studying the conditions and mechanics of transitioning from planktonic (cells exist in free-living, unattached state) to biofilm states and vice versa, as well as the conditions promoting biofilm detachment, could provide further information on transmission of V. cholerae. C. jejuni can also form biofilms. Similarly to V. cholerae, the organism’s growth in biofilms is thought to be important for environmental persistence and transmission to the next host [8,30,44]. It was recently shown that C. jejuni form monoculture biofilms at solid-liquid and air-water interfaces and can also form biofilm-like bacterial aggregates in liquid cultures [30]. Furthermore, the biofilm growth mode has been shown to confer resistance to the pathogen to environmental stresses [30]. C. jejuni is a microaerophilic organism and therefore sensitive to atmospheric oxygen levels, a property that greatly decreases its ability to survive in the environment. It is thought that the microaerobic conditions created within biofilms are conducive to the growth of C. jejuni [8,30]. A recent study revealed that attachment of C. jejuni to pre-existing biofilms is enhanced in mixed-species biofilms compared to monospecies biofilms, and that belonging to a biofilm enhances C. jejuni’s resistance to biocides [60]. Furthermore, C. jejuni strains isolated from different sources exhibit variable propensities to form biofilms and abilities to survive in non-host environments [44]. Although limited in number, such studies suggest that the formation of C. jejuni biofilms on food and food processing environments increases pathogen survival. In addition, the increased resistance to biocides when the pathogen is in a biofilm growth mode increases its transmission risk. The presence of Legionella spp. in biofilms, and in particular, in the pipes of water distribution systems, is well established [2]. Studies have shown that L. pneumophila can persist (meaning that the organism does not increase in
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Fig. 1. Biofilm developmental cycle in V. cholerae. Biofilm formation begins with attachment of single or aggregated cells to surfaces, followed by formation of microcolonies and mature biofilm structures that are characterized by pillars and channels. Biofilm developmental cycle is completed by detachment of bacteria from biofilms. Three-dimensional images of V. cholerae biofilms at different stage of biofilm development are shown.
biomass) in a biofilm environment, but growth of the pathogen requires the presence of protozoa in which L. pneumophilia can reproduce [43]. The presence of heterotrophic bacteria that support the growth of protozoa further enhances growth of L. pneumophila in biofilm [33,34,43]. A recent study analyzed biofilm formation of L. pneumophila in a rich growth medium that supports replication of L. pneumophila in the absence of protozoa [34]. It showed that L. pneumophila can attach to surfaces under static growth conditions in rich medium, but they are unable to grow on the surfaces [34]. However, incubation of L. pneumophila biofilms with protozoa (Acanthamoeba castellanii) enhances biofilm formation, as it is required for the replication of L. pneumophilia in biofilms. Under dynamic flow conditions, L. pneumophila was able to attach to surfaces but could not grow and form mature biofilms [34]. A capture assay, performed to test the ability of L. pneumophila to enter and grow in single or mixed cultures of defined heterotrophic bacteria, revealed that L. pneumophila simply persists and does not proliferate in these biofilms in the absence of protozoa [34]. Taken together, these studies show that biofilm growth of L. pneumophila requires the presence of protozoa, which are prevalent in aquatic and engineered water systems. This association is, in turn, critical for transmission of the pathogen via the biofilm route. Mycobacteria have been identified as efficient colonizers of water surfaces. M. avium has been isolated from biofilm samples of drinking water distribution systems [61] and recent laboratory studies have shown that M. avium can form and grow in biofilms. One study also showed that M. avium biofilms or aggregates of M. avium detached from biofilms exhibit increased chlorine resistance relative to cells grown in suspension [56],
suggesting once again that the biofilm growth mode increases survival and, in turn, transmission of M. avium. Other environmental Mycobacteria also form biofilms and can be dispersed into bulk water or in aerosols as clumps (formed by detachment from biofilms) [25]. Such behavior could enhance transmission by increasing the chances of exposure to high infectious doses of pathogens by human populations [25]. M. ulcerans forms biofilms on the surfaces of algae and the addition of algal extracts increases the growth rate of M. ulcerans in biofilms [36]. Because genotype analysis revealed that these environmental isolates have the same genotypic profile as clinical isolates [36], the association between M. ulcerans and algae might have implications for increasing transmission to human populations. Furthermore, it has been speculated that the capacity of M. ulcerans to form biofilms in the setae of aquatic insect predators facilitates its transmission [35,37]. For transmission to occur, the new host has to encounter the pathogen and to either ingest, inhale or be otherwise exposed to a sufficient infectious dose of the pathogen from the environment. Biofilms are not only important for the pathogen’s survival in the environment, but can also further facilitate its transmission if the biofilm is along a pathway of infection, such as ingestion of contaminated water or inhalation of pathogen-containing aerosols. Particles that have detached from biofilms frequently exceed the minimum infectious dose required by many pathogenic biofilm-forming bacteria. In addition, the increased resistance of biofilm-dwelling bacteria to physical stressors (e.g. acidity) could potentially reduce the infectious dose by increasing the number of viable organisms that survive in vivo stresses. Field studies have shown that removing particles >20 mm from drinking water by filtration
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reduces cholera incidence by 48% [13], suggesting that transmission of V. cholerae to new hosts is, in part, taking place when the organism is in a biofilm state. 3.3. Generation of new genetic variants Changes in the pathogens’ genetic make-up through the accumulation of mutations and acquisition of new genes could enhance their environmental survival, their transmission potential and pathogenicity, as well as leading to the emergence of new pathogens. Exposure to various stress conditions, such as nutrient limitation, or exposure to biocides and antimicrobial agents, has led to the generation of phenotypic variants in several important and common pathogens. A well studied example is a phase variation event in V. cholerae which results in the generation of two morphologically different variants termed smooth and rugose [42] (Fig. 2). The phenotypic properties of these two variants differ greatly. The rugose variant has an increased capacity to form biofilms and exhibits increased resistance to osmotic and oxidative stress compared to the smooth variant [62,67]. The switch between the two colonial variants is bidirectional [48,67], but the rugose to smooth transition occurs more frequently, as demonstrated by the smooth sectoring observed on the edges of rugose colonies [67] (Fig. 2). Conditions that result in conversion from the smooth to the rugose form include carbon limitation and treatment with bactericidal agents [67]. Epidemic strains have an increased capacity to switch between variants, suggesting that this ability may increase the transmission potential of V. cholerae [48]. The switching is also of critical importance from a public heath perspective as the conversion to a rugose colonial form is associated with increased survival in chlorinated water [49]. Increased production of Vibrio polysaccharide by the rugose variant, as depicted in Fig. 2, was shown to be responsible for chlorine resistance [67]. Because of the association between morphology and phenotypic properties, it has been proposed that the generation of V. cholerae cells with different phenotypes enhances the likelihood of transmission for the organism. Gene capture systems such as an integron island are also critical for enhancing genetic diversity, plasticity and versatility [50e52]. V. cholerae has an integron island [39] and transcription of many genes in the integron island are positively regulated by HapR, master regulator of the quorum sensing system of V. cholerae and RpoS [68]. HapR production is induced at high cell densities and RpoS accumulates in response to stress and during stationary phase growth. Consequently, integron island genes might be expected to be activated in crowded, stressed communities containing non-replicating organisms [68]. When viewed from an environmental and evolutionary survival perspective, this behavior could confer a survival advantage because it would integrate a gene acquisition program with a survival-enhancing stress response. Viruses are particularly relevant for generation of genetic diversity and for the emergence of new pathogenic strains of V. cholerae. The genes coding cholera toxin are contained in the genome of the filamentous bacteriophage CTXF [63]
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and environmental non-pathogenic V. cholerae strains can be infected by CTXF, a process that leads to the generation of new pathogenic variants [17]. These studies demonstrate that the life cycles of V. cholerae and Vibrio phages are interwoven, and this relationship may be particularly relevant to the transmission of the pathogen. While it is clear that biofilms can enhance the survival of pathogens by providing increased resistance to environmental stresses, it is also possible that biofilms enhance the emergence of new genetic variants. The close spatial arrangement of bacteria in biofilms may permit increased rates of lateral gene transfer, thus allowing the transfer of virulence and resistance genes. The transfer of virulence genes could both enhance the pathogenicity of existing pathogens and lead to the emergence of new pathogens, especially by transforming organisms that currently infect other vertebrates but not humans. 3.4. Interaction with protozoa and viruses Bacterial pathogens in the environment become part of the microbial food web and their abundance can be modulated by protozoa, which are ubiquitous in natural environments. Protozoa graze on bacteria. Whereas some pathogens are readily killed by protozoan grazing, others survive and grow in protozoa. These pathogens exit protozoa either in vesicles or following protozoal lysis, which releases large numbers of bacteria into the environment [24]. The best studied example of this phenomenon is that of Legionella-protozoa interaction [2]. L. pneumophila can replicate intracellularly in various species of protozoa, where it is protected from environmental stresses [2]. Since growth inside protozoa increases the chances of survival of L. pneumophila in the environment, it increases the transmission potential of the pathogen. M. avium remains viable in protozoan vacuoles and can survive encystment of protozoa, a property thought to facilitate its transmission [53]. The pathogenicity of L. pneumophila and M. avium, both intracellular pathogens that replicate in human alveolar macrophages, has been shown to be enhanced by prior growth in protozoa [10,11], suggesting that interaction with protozoa increases the transmission chances of these pathogens by enhancing their virulence properties and priming them for human infection. Protozoan grazing reduces the planktonic V. cholerae population in coastal marine environments [65]. Matz et al. have shown that biofilms formed by V. cholerae are resistant to protozoan grazing relative to planktonic V. cholerae, and that grazing selects for phenotypic variants of V. cholerae with enhanced biofilm-forming capacity [38]. It has also been reported that V. cholerae can multiply within protozoa and survive encystment [1]. Taken together, these studies indicate that V. cholerae-protozoan interactions control V. cholerae population numbers and transmission dynamics. C. jejuni populations in natural aquatic environments can be negatively impacted by protozoan grazing activity [54]. But interaction of C. jejuni with protozoa may increase the colonization chances of broilers and, in turn, increases the likelihood of
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Fig. 2. (A) A scanning electron micrograph of a rugose colony variant switching to a smooth variant. (B,C) Transmission electron micrograph of thin sections stained with ruthenium red of rugose (B) and smooth (C) cells grown on cellulose membrane atop nutrient agar.
transmission. A recent study showed that in an in vitro coculture assay, incubation of C. jejuni with protozoa, initially decreases the number of viable C. jejuni cells; however, C. jejuni remains viable longer after internalization by protozoa relative to C. jejuni in suspension [55]. Furthermore, ingestion by protozoa increases C. jejuni resistance to disinfection [55]. Thus, there is growing evidence that protozoan grazing is one of the critical ecological factors controlling abundance and transmission of various bacterial pathogens in the environment. Viruses are highly abundant in natural environments and can control the abundance and diversity of microbial populations [64]. It has been demonstrated that environmental phage populations infect V. cholerae strains and that the numbers of these phages inversely correlates with those of V. cholerae and of cholera cases [18,19]. It has been further shown that epidemic and non-epidemic V. cholerae strains harbor lysogenic phages that can influence the epidemiology of V. cholerae [19]. Upon exposure to environmental stresses, lysogenic Vibrio-phages can enter into a lytic cycle and generate phage particles, which reduce the population of susceptible V. cholerae strains in aquatic ecosystems. There is also a correlation between the prevalence of environmental phages and a reduction in the C. jejuni population in commercial broiler chickens [4]. Furthermore, host-specific bacteriophages have been used to control populations of C. jejuni cells on artificially contaminated chicken [3]. Hence, phages can also play a role in transmission of C. jejuni. 4. Conclusions The survival of microbes in the environment and their transmission to new hosts are influenced by multiple factors, some of which were discussed above. The pathogen load in the environment needs to be better estimated for improving risk assessment. A number of newly developed molecular biology methods for detecting pathogenic species facilitate the study of the distribution of these microorganisms in their native environment. For example, PCR and RTePCR methods have been used to study pathogens, and natural communities
of microbes in aquatic environments. These methods are relatively rapid, extremely sensitive, and can be applied to determine the abundance of specific species or broadly-related phylogenetic groups. Culture- and molecular-based detection methods have been evolving to incorporate our growing knowledge of pathogen ecology and evolution. Elegant examples of such studies include the use of protozoan coculture [59] to enrich for pathogens, and the application of antibiotics to remove rapidly growing, interfering bacteria (but to which pathogenic bacteria are resistant), allowing the isolation of culturable pathogenic bacteria from the environment [21]. Many questions remain regarding the physiological state of pathogens when they exit the host and how they survive in and adjust to the non-host environment. For example, in any given environment, are the pathogens surface-attached or free-floating, and what factors alter this distribution? How do physical/ chemical factors govern attachment and detachment to surfaces? Is one form of a pathogen more infectious than another? How do surface-attached bacteria respond to fluctuating environmental parameters, and what genes/processes are required for this response? What is the importance of these responses for long-term survival of a given pathogen? Do the physical and chemical factors likely to be encountered by a given pathogen in natural aquatic habitats favor the evolution of populations with enhanced transmission capabilities, and thus pathogens with new abilities? Answers to each of these questions would lead to a better understanding of processes controlling environmental fitness and transmission of pathogenic bacteria. Such knowledge would, in turn, allow us to better predict and control of infectious disease outbreaks. Acknowledgments Work in my laboratory is supported by grants from The Ellison Medical Foundation and NIH (AI055987). References [1] H. Abd, A. Weintraub, G. Sandstrom, Intracellular survival and replication of Vibrio cholerae O139 in aquatic free-living amoebae, Environ. Microbiol. 7 (2005) 1003e1008.
F.H. Yildiz / Research in Microbiology 158 (2007) 195e202 [2] R.M. Atlas, Legionella: from environmental habitats to disease pathology, detection and control, Environ. Microbiol. 1 (1999) 283e293. [3] R.J. Atterbury, P.L. Connerton, C.E. Dodd, C.E. Rees, I.F. Connerton, Application of host-specific bacteriophages to the surface of chicken skin leads to a reduction in recovery of Campylobacter jejuni, Appl. Environ. Microbiol. 69 (2003) 6302e6306. [4] R.J. Atterbury, E. Dillon, C. Swift, P.L. Connerton, J.A. Frost, C.E. Dodd, C.E. Rees, I.F. Connerton, Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca, Appl. Environ. Microbiol. 71 (2005) 4885e4887. [5] M.A. Bachman, M.S. Swanson, The LetE protein enhances expression of multiple LetA/LetS-dependent transmission traits by Legionella pneumophila, Infect. Immun. 72 (2004) 3284e3293. [6] M.A. Bachman, M.S. Swanson, Genetic evidence that Legionella pneumophila RpoS modulates expression of the transmission phenotype in both the exponential phase and the stationary phase, Infect. Immun. 72 (2004) 2468e2476. [7] J. Bina, J. Zhu, M. Dziejman, S. Faruque, S. Calderwood, J. Mekalanos, ToxR regulon of Vibrio cholerae and its expression in vibrios shed by cholera patients, Proc. Natl. Acad. Sci. USA. 100 (2003) 2801e2806. [8] C.M. Buswell, Y.M. Herlihy, L.M. Lawrence, J.T. McGuiggan, P.D. Marsh, C.W. Keevil, S.A. Leach, Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescent-antibody and -rRNA staining, Appl. Environ. Microbiol. 64 (1998) 733e741. [9] S.M. Butler, E.J. Nelson, N. Chowdhury, S.M. Faruque, S.B. Calderwood, A. Camilli, Cholera stool bacteria repress chemotaxis to increase infectivity, Mol. Microbiol. 60 (2006) 417e426. [10] J.D. Cirillo, S. Falkow, L.S. Tompkins, L.E. Bermudez, Interaction of Mycobacterium avium with environmental amoebae enhances virulence, Infect. Immun. 65 (1997) 3759e3767. [11] J.D. Cirillo, S.L. Cirillo, L. Yan, L.E. Bermudez, S. Falkow, L.S. Tompkins, Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila, Infect. Immun. 67 (1999) 4427e4434. [12] R.R. Colwell, Viable but nonculturable bacteria: a survival strategy, J. Infect. Chemother. 6 (2000) 121e125. [13] R.R. Colwell, A. Huq, M.S. Islam, K.M. Aziz, M. Yunus, N.H. Khan, A. Mahmud, R.B. Sack, G.B. Nair, J. Chakraborty, D.A. Sack, E. RussekCohen, Reduction of cholera in Bangladeshi villages by simple filtration, Proc. Natl. Acad. Sci. USA. 100 (2003) 1051e1055. [14] J.W. Costerton, Z. Lewandowski, D.E. Caldwell, D.R. Korber, H.M. Lappin-Scott, Microbial biofilms, Annu. Rev. Microbiol. 49 (1995) 711e745. [15] R.M. Donlan, J.W. Costerton, Biofilms: survival mechanisms of clinically relevant microorganisms, Clin. Microbiol. Rev. 15 (2002) 167e193. [16] S.M. Faruque, M.J. Albert, J.J. Mekalanos, Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae, Microbiol. Mol. Biol. Rev. 62 (1998) 1301e1314. [17] S.M. Faruque, J.J. Mekalanos, Pathogenicity islands and phages in Vibrio cholerae evolution, Trends Microbiol. 11 (2003) 505e510. [18] S.M. Faruque, M.J. Islam, Q.S. Ahmad, A.S. Faruque, D.A. Sack, G.B. Nair, J.J. Mekalanos, Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage, Proc. Natl. Acad. Sci. USA. 102 (2005) 6119e6124. [19] S.M. Faruque, I.B. Naser, M.J. Islam, A.S. Faruque, A.N. Ghosh, G.B. Nair, D.A. Sack, J.J. Mekalanos, Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages, Proc. Natl. Acad. Sci. USA. 102 (2005) 1702e1707. [20] S.M. Faruque, K. Biswas, S.M. Udden, Q.S. Ahmad, D.A. Sack, G.B. Nair, J.J. Mekalanos, Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment, Proc. Natl. Acad. Sci. USA. 103 (2006) 6350e6355. [21] S.M. Faruque, M.J. Islam, Q.S. Ahmad, K. Biswas, A.S. Faruque, G.B. Nair, R.B. Sack, D.A. Sack, J.J. Mekalanos, An improved technique for isolation of environmental Vibrio cholerae with epidemic potential: monitoring the emergence of a multiple-antibiotic-resistant epidemic strain in Bangladesh, J. Infect. Dis. 193 (2006) 1029e1036.
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[22] C.A. Fux, J.W. Costerton, P.S. Stewart, P. Stoodley, Survival strategies of infectious biofilms, Trends Microbiol. 13 (2005) 34e40. [23] E.C. Gaynor, D.H. Wells, J.K. MacKichan, S. Falkow, The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes, Mol. Microbiol. 56 (2005) 8e27. [24] G. Greub, D. Raoult, Microorganisms resistant to free-living amoebae, Clin. Microbiol. Rev. 17 (2004) 413e433. [25] L. Hall-Stoodley, P. Stoodley, Biofilm formation and dispersal and the transmission of human pathogens, Trends Microbiol. 13 (2005) 7e10. [26] B.K. Hammer, M.S. Swanson, Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp, Mol. Microbiol. 33 (1999) 721e731. [27] B.K. Hammer, E.S. Tateda, M.S. Swanson, A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila, Mol. Microbiol. 44 (2002) 107e118. [28] M.A. Hood, J.B. Guckert, D.C. White, F. Deck, Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae, Appl. Environ. Microbiol. 52 (1986) 788e793. [29] A. Huq, E.B. Small, P.A. West, M.I. Huq, R. Rahman, R.R. Colwell, Ecological relationships between Vibrio cholerae and planktonic crustacean copepods, Appl. Environ. Microbiol. 45 (1983) 275e283. [30] G.W. Joshua, C. Guthrie-Irons, A.V. Karlyshev, B.W. Wren, Biofilm formation in Campylobacter jejuni, Microbiology 152 (2006) 387e396. [31] J.B. Kaper, J.G. Morris Jr., M.M. Levine, Cholera, Clin. Microbiol. Rev. 8 (1995) 48e86. [32] A.F. Kelly, S.F. Park, R. Bovill, B.M. Mackey, Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response, Appl. Environ. Microbiol. 67 (2001) 2248e2254. [33] M.W. Kuiper, B.A. Wullings, A.D. Akkermans, R.R. Beumer, D. van der Kooij, Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride, Appl. Environ. Microbiol. 70 (2004) 6826e6833. [34] J. Mampel, T. Spirig, S.S. Weber, J.A. Haagensen, S. Molin, H. Hilbi, Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions, Appl. Environ. Microbiol. 72 (2006) 2885e2895. [35] L. Marsollier, R. Robert, J. Aubry, J.P. Saint Andre, H. Kouakou, P. Legras, A.L. Manceau, C. Mahaza, B. Carbonnelle, Aquatic insects as a vector for Mycobacterium ulcerans, Appl. Environ. Microbiol. 68 (2002) 4623e4628. [36] L. Marsollier, T. Stinear, J. Aubry, J.P. Saint Andre, R. Robert, P. Legras, A.L. Manceau, C. Audrain, S. Bourdon, H. Kouakou, B. Carbonnelle, Aquatic plants stimulate the growth of and biofilm formation by Mycobacterium ulcerans in axenic culture and harbor these bacteria in the environment, Appl. Environ. Microbiol. 70 (2004) 1097e1103. [37] L. Marsollier, J. Aubry, E. Coutanceau, J.P. Andre, P.L. Small, G. Milon, P. Legras, S. Guadagnini, B. Carbonnelle, S.T. Cole, Colonization of the salivary glands of Naucoris cimicoides by Mycobacterium ulcerans requires host plasmatocytes and a macrolide toxin, mycolactone, Cell Microbiol. 7 (2005) 935e943. [38] C. Matz, D. McDougald, A.M. Moreno, P.Y. Yung, F.H. Yildiz, S. Kjelleberg, Biofilm formation and phenotypic variation enhance predation-driven persistence of Vibrio cholerae, Proc. Natl. Acad. Sci. USA. 102 (2005) 16819e16824. [39] D. Mazel, B. Dychinco, V.A. Webb, J. Davies, A distinctive class of integron in the Vibrio cholerae genome, Science 280 (1998) 605e608. [40] D.S. Merrell, S.M. Butler, F. Qadri, N.A. Dolganov, A. Alam, M.B. Cohen, S.B. Calderwood, G.K. Schoolnik, A. Camilli, Host-induced epidemic spread of the cholera bacterium, Nature 417 (2002) 642e645. [41] A.B. Molofsky, M.S. Swanson, Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication, Mol. Microbiol. 50 (2003) 445e461. [42] J.G. Morris Jr., M.B. Sztein, E.W. Rice, J.P. Nataro, G.A. Losonsky, P. Panigrahi, C.O. Tacket, J.A. Johnson, Vibrio cholerae O1 can assume a chlorine-resistant rugose survival form that is virulent for humans, J. Infect. Dis. 174 (1996) 1364e1368. [43] R. Murga, T.S. Forster, E. Brown, J.M. Pruckler, B.S. Fields, R.M. Donlan, Role of biofilms in the survival of Legionella pneumophila in a model potable-water system, Microbiology 147 (2001) 3121e3126.
202
F.H. Yildiz / Research in Microbiology 158 (2007) 195e202
[44] C. Murphy, C. Carroll, K.N. Jordan, Environmental survival mechanisms of the foodborne pathogen Campylobacter jejuni, J. Appl. Microbiol. 100 (2006) 623e632. [45] J.D. Oliver, The viable but nonculturable state in bacteria, J. Microbiol. Spec No 43 (2005) 93e100. [46] J. Parkhill, B.W. Wren, K. Mungall, J.M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R.M. Davies, T. Feltwell, S. Holroyd, K. Jagels, A.V. Karlyshev, S. Moule, M.J. Pallen, C.W. Penn, M.A. Quail, M.A. Rajandream, K.M. Rutherford, A.H. van Vliet, S. Whitehead, B.G. Barrell, The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences, Nature 403 (2000) 665e668. [47] T.P. Primm, C.A. Lucero, J.O. Falkinham 3rd, Health impacts of environmental mycobacteria, Clin. Microbiol. Rev. 17 (2004) 98e106. [48] M.H. Rashid, C. Rajanna, A. Ali, D.K. Karaolis, Identification of genes involved in the switch between the smooth and rugose phenotypes of Vibrio cholerae, FEMS Microbiol. Lett. 227 (2003) 113e119. [49] E.W. Rice, C.J. Johnson, R.M. Clark, K.R. Fox, D.J. Reasoner, M.E. Dunnigan, P. Panigrahi, J.A. Johnson, J.G. Morris Jr., Chlorine and survival of ‘‘rugose’’ Vibrio cholerae, Lancet 340 (1992) 740. [50] D.A. Rowe-Magnus, A.M. Guerout, D. Mazel, Super-integrons, Res. Microbiol. 150 (1999) 641e651. [51] D.A. Rowe-Magnus, A.M. Guerout, P. Ploncard, B. Dychinco, J. Davies, D. Mazel, The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons, Proc. Natl. Acad. Sci. USA. 98 (2001) 652e657. [52] D.A. Rowe-Magnus, A.M. Guerout, D. Mazel, Bacterial resistance evolution by recruitment of super-integron gene cassettes, Mol. Microbiol. 43 (2002) 1657e1669. [53] M.T. Rowe, I.R. Grant, Mycobacterium avium ssp. paratuberculosis and its potential survival tactics, Lett. Appl. Microbiol. 42 (2006) 305e311. [54] M. Schallenberg, P.J. Bremer, S. Henkel, A. Launhardt, C.W. Burns, Survival of Campylobacter jejuni in water: effect of grazing by the freshwater crustacean Daphnia carinata (Cladocera), Appl. Environ. Microbiol. 71 (2005) 5085e5088. [55] W.J. Snelling, J.P. McKenna, D.M. Lecky, J.S. Dooley, Survival of Campylobacter jejuni in waterborne protozoa, Appl. Environ. Microbiol. 71 (2005) 5560e5571.
[56] K.A. Steed, J.O. Falkinham 3rd, Effect of growth in biofilms on chlorine susceptibility of Mycobacterium avium and Mycobacterium intracellulare, Appl. Environ. Microbiol. 72 (2006) 4007e4011. [57] M.S. Swanson, B.K. Hammer, Legionella pneumophila pathogesesis: a fateful journey from amoebae to macrophages, Annu. Rev. Microbiol. 54 (2000) 567e613. [58] M.L. Tamplin, A.L. Gauzens, A. Huq, D.A. Sack, R.R. Colwell, Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters, Appl. Environ. Microbiol. 56 (1990) 1977e1980. [59] V. Thomas, K. Herrera-Rimann, D.S. Blanc, G. Greub, Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network, Appl. Environ. Microbiol. 72 (2006) 2428e2438. [60] N. Trachoo, J.F. Frank, Effectiveness of chemical sanitizers against Campylobacter jejuni-containing biofilms, J. Food Prot. 65 (2002) 1117e 1121. [61] M.J. Vaerewijck, G. Huys, J.C. Palomino, J. Swings, F. Portaels, Mycobacteria in drinking water distribution systems: ecology and significance for human health, FEMS Microbiol. Rev. 29 (2005) 911e934. [62] S.N. Wai, Y. Mizunoe, A. Takade, S.I. Kawabata, S.I. Yoshida, Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation, Appl. Environ. Microbiol. 64 (1998) 3648e3655. [63] M.K. Waldor, J.J. Mekalanos, Lysogenic conversion by a filamentous phage encoding cholera toxin, Science 272 (1996) 1910e1914. [64] K.E. Wommack, R.R. Colwell, Virioplankton: viruses in aquatic ecosystems, Microbiol. Mol. Biol. Rev. 64 (2000) 69e114. [65] A.Z. Worden, M. Seidel, S. Smriga, A. Wick, F. Malfatti, D. Bartlett, F. Azam, Trophic regulation of Vibrio cholerae in coastal marine waters, Environ. Microbiol. 8 (2006) 21e29. [66] F.H. Yildiz, G.K. Schoolnik, Role of rpoS in stress survival and virulence of Vibrio cholerae, J. Bacteriol. 180 (1998) 773e784. [67] F.H. Yildiz, G.K. Schoolnik, Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation, Proc. Natl. Acad. Sci. USA. 96 (1999) 4028e4033. [68] F.H. Yildiz, X.S. Liu, A. Heydorn, G.K. Schoolnik, Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant, Mol. Microbiol. 53 (2004) 497e515.
Review
Processes controlling the transmission of bacterial pathogens in the environment Fitnat H. Yildiz* Department of Environmental Toxicology, University of California Santa Cruz, Santa Cruz, CA 95064, USA Received 3 October 2006; accepted 1 December 2006 Available online 13 January 2007
Abstract Many pathogens in the environment can be transmitted to human populations and cause outbreaks and epidemics. Transmission is a multifactorial process influenced by the physiology of the pathogen as it exits its initial host, the mechanisms it uses for surviving outside the host, the physiology of the pathogen as it enters the next susceptible host and its ability to establish a successful infection. Few studies so far have focused on the processes responsible for modulating microbial survival in non-host environments and the transmission dynamics between infected and susceptible hosts, as well as the interplay between hosts. A better understanding of these mechanisms is thus necessary for predicting and preventing future outbreaks. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Pathogen; Environmental survival; Biofilm formation; Phase variation; Protozoan grazing
1. Introduction Infectious diseases remain one of the leading causes of mortality worldwide. As new diseases continue to be discovered, some of the old scourges re-emerge to threaten human populations who have not previously been exposed to them. Although we have a relatively good understanding of the mechanisms by which pathogenic microorganisms cause disease, the biology of human pathogens in non-host environments is only beginning to be elucidated. This review discusses recent developments in ‘‘environmental pathogenesis,’’ highlighting those processes shown to be critical for the transmission of pathogenic bacteria. It has become clear that transmission of a pathogen from one host to another is a multi-factorial process influenced by the quantity, growth form (e.g., planktonic or biofilm) and physiology of the pathogen exiting its initial host; by the mechanisms the pathogen
* Tel.: þ1 831 459 1588; fax: þ1 831 459 3524. E-mail address: [email protected] 0923-2508/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2006.12.005
uses to survive in a non-host environment, which can control the abundance of the pathogen and thus exposure to it; and by the growth form and physiology of the pathogen entering the next susceptible host, which determine its ability to establish a successful infection. Each of these processes is discussed in detail below, drawing from examples of pathogenic bacteria with documented environmental prevalence. These include Vibrio cholerae, the causative agent of cholera, which is a natural inhabitant of aquatic ecosystems around the world and is transmitted via contaminated food or water [31]; Legionella pneumophila, the causative agent of Legionnaires’ disease, which is transmitted via aerosols generated from either natural or manmade environmental sources [2]; non-tuberculous Mycobacteria (NTM), such as M. avium, M. marinum, M. ulcerans, which are opportunistic environmental pathogens that cause infections of the respiratory and gastrointestinal tracts and skin, and can be acquired from surface water, potable water and groundwater by inhalation, ingestion or skin abrasion [47]; Campylobacter jejuni, the leading cause of bacterial gastroenteritis in the world, which is adapted to grow in the avian and
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mammalian intestinal tracts and is transmitted to humans by consumption of contaminated poultry, milk or water [44]. Studies of these pathogens has revealed some of the mechanisms important for enhanced survival outside their hosts and increased virulence among human populations. As our understanding of these mechanisms increases, we should be able to find ways to target them and thereby reduce the transmission of pathogenic organisms. 2. Exit from the host The physiological state of a microorganism shed from the infected host is one of the critical factors determining its fate in the non-host environment and its transmission to the next host. Information in this area is, however, very limited. It was recently reported that V. cholerae present in the stools of cholera patients are in a hyperinfectious state [40]. These studies showed that V. cholera shed in stools outcompete laboratory grown V. cholerae by 10-100 fold in an infant mouse competition assay [40] and that the dose of V. cholerae needed for oral infection is 10-fold lower for the pathogen shed in stools than its in vitro grown counterpart [9]. Although this hyperinfectious state is maintained for only a short time after exiting the host, it is thought to facilitate the transmission of V. cholera during epidemics [9,40]. It is therefore important to elucidate its molecular underpinnings. Whole genome expression profiling revealed that the expression levels of genes required for nutrient acquisition and motility were increased, while those required for bacterial chemotaxis decreased in V. cholerae cells shed in stools from cholera patients, as compared to cultures grown in vitro to a stationary growth phase [40]. Using genomic DNA as a reference, Bina et al. determined that highly expressed genes in the V. cholerae from stools encoded hypothetical proteins, conserved hypothetical proteins, and proteins of unknown functions, whereas genes with intermediate levels of expression encoded virulence factors cholera toxin and toxin coregulated pilus [7]. This study also revealed that the gene expression pattern of V. cholerae from cholera stools was more similar to that of mid-exponential phase bacteria harvested from rabbit ileal loops than that of cultures grown in vitro to stationary phase. A recent study suggested that a state of altered chemotaxis of V. cholerae present in stools may be responsible for enhanced virulence [9]. Although these studies begin to provide insights into the hyperinfectious phenotype of the V. cholerae present in stool samples, the molecular mechanisms involved remain largely unknown. In addition to looking at the expression of genes that may be responsible for hyperinfectivity, a number of studies have focused on the morphological properties of pathogens. For example, studies have shown that V. cholerae shed in human stools is heterogeneous, containing both planktonic and biofilm-like aggregate forms [20]. A competition assay using a reference strain revealed that the average infectivity of the aggregate form is significantly higher than that of the corresponding planktonic cells [20]. It was, therefore, suggested that the hyperinfectivity of V. cholerae shed in human stools
might be due to the presence of the aggregate form of V. cholerae, which delivers a high infectious dose of pathogen to the human host due to better capacity to survive in vivo stresses. The phenotype of V. cholerae dispersed into an aquatic environment is also of epidemiological significance, as different forms of the pathogen are likely to exhibit different survival properties which in turn determine the likelihood of transmission to the next human host. However, more functional studies need to be conducted to evaluate the significance of these different morphological forms of V. cholerae. 3. Environmental growth and survival Upon exiting their host, many pathogens persist in dynamic ecological conditions before infecting another host. Understanding those processes critical for the pathogens’ environmental survival may provide clues for how best to reduce their ability to interact with human populations. The processes described below have been shown to facilitate environmental survival of different pathogens. 3.1. Environmental stress response Bacterial pathogens have developed ways to respond to the physical and chemical factors they encounter in their natural habitats. Laboratory and field studies have shown that in the free-living state, V. cholerae can adaptively respond to fluctuations in environmental parameters, such as salinity, temperature, pH and nutrient availability. For example, upon nutrient deprivation, comma-shaped V. cholerae cells become coccoid-shaped as a result of reductive division, significantly decrease their volume, and reduce their lipid, carbohydrate, RNA and protein contents [28]. In addition to these metabolic and structural changes, nutrient starvation induces resistance to oxidative stress and osmotic shock [66]. These responses are critical to surviving environmental stress. Indeed, mutants lacking the alternative sigma factor RpoS, which controls starvation and stress response in V. cholerae, are impaired in their ability to survive diverse environmental stresses [66]. C. jejuni has multiple environmental survival strategies, which differ from those used by other enteric bacteria, as recently reviewed by Murphy et al [44]. For instance, stationary phase/stress responses are not present in C. jejuni [32], and RpoS, a protein that governs the expression of many of the genes that function in stationary phase/stress response, is not present in C. jejuni [46]. Despite this deficiency, a recent study showed that C. jejuni can initiate a stringent response to nutrient deprivation which is critical for its survival and transmission [23]. In aquatic ecosystems L. pneumophila replicates within amoebae. Upon exhaustion of nutrients in the host cell, L. pneumophila stops replication and activates transmission traits that include production of cytotoxicity factors to escape the host cell, activation of motility for dissemination, increasing extracellular stress resistance and avoidance of phagosomee lysosome fusion in the new host cell [57]. It has been reported that expression of many of these transmission traits is initiated
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by production of the stringent response signal (p)ppGpp and controlled by the alternative sigma factors RpoS and FliA and the regulators of the two component regulatory system [5,6,26,27,41,57]. It has also been shown that upon extended exposure to unfavorable environmental conditions, such as low growth temperature and nutrient starvation, V. cholerae enters into a state of dormancy, a viable-but-non-culturable (VBNC) state during which DNA replication and cell division are incapacitated but metabolic activity is retained [12]. In the VBNC state V. cholerae cannot be grown by standard culturing techniques, but when VBNC cells are inoculated into ligated rabbit ileal loops or given to human volunteers the V. cholerae cells isolated from these hosts can then be cultured [12]. Many bacterial pathogens, including L. pneumophila, C. jejuni, and Mycobacterium spp. can apparently enter into a VBNC state (as recently reviewed by Oliver) [45]. Although it is not clear what this state actually represents, entry into a VBNC state, or any form of dormancy from which pathogens can exit when environmental conditions become more favorable, could represent a step critical in the transmission of many human pathogens. Indeed, such a process could ensure the survival of bacterial pathogens in the environment, even under harsh conditions, and thus increase their abundance. 3.2. Biofilm formation Biofilms are microbial communities composed of microorganisms and the extra polymeric substances they produce, attached to abiotic and biotic surfaces [14]. Biofilm formation by pathogenic microorganisms is a well documented process thought to enhance the survival of pathogenic organisms in certain environments [15,22]. Growth on surfaces may be advantageous as surfaces adsorb and thus concentrate nutrients that are otherwise scarce in the surrounding liquid. In addition, as biofilms contain different species of bacteria, pathogens can engage in favorable metabolic interactions with other members of the consortium. Lastly, the formation of a biofilm provides some protection from grazing predators and from toxic compounds such as antimicrobial agents [14,15]. V. cholerae form biofilms by attaching to surfaces of phytoplankton and zooplankton [16,29,58]. Phytoplanktons excrete a variety of organic compounds that can support the growth of associated (biofilm) or free-living (planktonic) V. cholerae [29,58]. Similarly, attachment to chitinous surfaces of zooplankton and the subsequent degradation of chitin to Nacetyl glucosamine, which serves both as a carbon and nitrogen source, by several chitinases produced by V. cholerae, can support growth of the pathogen in an aquatic environment. Indeed, laboratory microcosm studies have shown that during co-culture, V. cholerae effectively attaches to surfaces of zooplankton and phytoplankton and this association increases the survival period of the organism [29]. Thus, attachment of V. cholerae to plankton and subsequent biofilm growth facilitates the environmental survival and growth of this pathogen. This property of the pathogen may, in turn, aid its transmission. In cholera-epidemic areas, there is a correlation between
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phytoplankton and zooplankton blooms and the timing of epidemics. This suggests that the plankton in V. cholerae’s aquatic habitat acts as a reservoir for the organism, and that the plankton’s life cycle controls the abundance of V. cholerae populations and epidemic cycles. Recent studies have shown that V. cholerae found in the surface waters of cholera endemic areas exists in biofilmlike aggregates [20] in which cells are in a so-called ‘‘conditionally viable’’ state. ‘‘Conditionally viable environmental cells’’ (CVECs) are viable but ‘‘metabolically impeded’’ V. cholera cells. CVECs can regain metabolic activity under specific in vitro conditions, as wells by inoculation into ligated rabbit ileal loops [20], suggesting that these forms of V. cholerae might play a critical role in the transmission of the pathogen. The source of CVECs is thought to be biofilm-like aggregate populations of V. cholerae present in human stools [20]. Similarly, the active and passive detachment of V. cholerae from various surfaces it colonizes in aquatic habitats could also lead to formation of bacterial aggregates. We know very little about how the physical and chemical factors likely to be encountered by V. cholerae in its natural aquatic habitats affect the biofilm formation and detachment from biofilms. Fig. 1 depicts the developmental cycle of biofilm formation by V. cholerae under laboratory conditions. Studying the conditions and mechanics of transitioning from planktonic (cells exist in free-living, unattached state) to biofilm states and vice versa, as well as the conditions promoting biofilm detachment, could provide further information on transmission of V. cholerae. C. jejuni can also form biofilms. Similarly to V. cholerae, the organism’s growth in biofilms is thought to be important for environmental persistence and transmission to the next host [8,30,44]. It was recently shown that C. jejuni form monoculture biofilms at solid-liquid and air-water interfaces and can also form biofilm-like bacterial aggregates in liquid cultures [30]. Furthermore, the biofilm growth mode has been shown to confer resistance to the pathogen to environmental stresses [30]. C. jejuni is a microaerophilic organism and therefore sensitive to atmospheric oxygen levels, a property that greatly decreases its ability to survive in the environment. It is thought that the microaerobic conditions created within biofilms are conducive to the growth of C. jejuni [8,30]. A recent study revealed that attachment of C. jejuni to pre-existing biofilms is enhanced in mixed-species biofilms compared to monospecies biofilms, and that belonging to a biofilm enhances C. jejuni’s resistance to biocides [60]. Furthermore, C. jejuni strains isolated from different sources exhibit variable propensities to form biofilms and abilities to survive in non-host environments [44]. Although limited in number, such studies suggest that the formation of C. jejuni biofilms on food and food processing environments increases pathogen survival. In addition, the increased resistance to biocides when the pathogen is in a biofilm growth mode increases its transmission risk. The presence of Legionella spp. in biofilms, and in particular, in the pipes of water distribution systems, is well established [2]. Studies have shown that L. pneumophila can persist (meaning that the organism does not increase in
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Fig. 1. Biofilm developmental cycle in V. cholerae. Biofilm formation begins with attachment of single or aggregated cells to surfaces, followed by formation of microcolonies and mature biofilm structures that are characterized by pillars and channels. Biofilm developmental cycle is completed by detachment of bacteria from biofilms. Three-dimensional images of V. cholerae biofilms at different stage of biofilm development are shown.
biomass) in a biofilm environment, but growth of the pathogen requires the presence of protozoa in which L. pneumophilia can reproduce [43]. The presence of heterotrophic bacteria that support the growth of protozoa further enhances growth of L. pneumophila in biofilm [33,34,43]. A recent study analyzed biofilm formation of L. pneumophila in a rich growth medium that supports replication of L. pneumophila in the absence of protozoa [34]. It showed that L. pneumophila can attach to surfaces under static growth conditions in rich medium, but they are unable to grow on the surfaces [34]. However, incubation of L. pneumophila biofilms with protozoa (Acanthamoeba castellanii) enhances biofilm formation, as it is required for the replication of L. pneumophilia in biofilms. Under dynamic flow conditions, L. pneumophila was able to attach to surfaces but could not grow and form mature biofilms [34]. A capture assay, performed to test the ability of L. pneumophila to enter and grow in single or mixed cultures of defined heterotrophic bacteria, revealed that L. pneumophila simply persists and does not proliferate in these biofilms in the absence of protozoa [34]. Taken together, these studies show that biofilm growth of L. pneumophila requires the presence of protozoa, which are prevalent in aquatic and engineered water systems. This association is, in turn, critical for transmission of the pathogen via the biofilm route. Mycobacteria have been identified as efficient colonizers of water surfaces. M. avium has been isolated from biofilm samples of drinking water distribution systems [61] and recent laboratory studies have shown that M. avium can form and grow in biofilms. One study also showed that M. avium biofilms or aggregates of M. avium detached from biofilms exhibit increased chlorine resistance relative to cells grown in suspension [56],
suggesting once again that the biofilm growth mode increases survival and, in turn, transmission of M. avium. Other environmental Mycobacteria also form biofilms and can be dispersed into bulk water or in aerosols as clumps (formed by detachment from biofilms) [25]. Such behavior could enhance transmission by increasing the chances of exposure to high infectious doses of pathogens by human populations [25]. M. ulcerans forms biofilms on the surfaces of algae and the addition of algal extracts increases the growth rate of M. ulcerans in biofilms [36]. Because genotype analysis revealed that these environmental isolates have the same genotypic profile as clinical isolates [36], the association between M. ulcerans and algae might have implications for increasing transmission to human populations. Furthermore, it has been speculated that the capacity of M. ulcerans to form biofilms in the setae of aquatic insect predators facilitates its transmission [35,37]. For transmission to occur, the new host has to encounter the pathogen and to either ingest, inhale or be otherwise exposed to a sufficient infectious dose of the pathogen from the environment. Biofilms are not only important for the pathogen’s survival in the environment, but can also further facilitate its transmission if the biofilm is along a pathway of infection, such as ingestion of contaminated water or inhalation of pathogen-containing aerosols. Particles that have detached from biofilms frequently exceed the minimum infectious dose required by many pathogenic biofilm-forming bacteria. In addition, the increased resistance of biofilm-dwelling bacteria to physical stressors (e.g. acidity) could potentially reduce the infectious dose by increasing the number of viable organisms that survive in vivo stresses. Field studies have shown that removing particles >20 mm from drinking water by filtration
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reduces cholera incidence by 48% [13], suggesting that transmission of V. cholerae to new hosts is, in part, taking place when the organism is in a biofilm state. 3.3. Generation of new genetic variants Changes in the pathogens’ genetic make-up through the accumulation of mutations and acquisition of new genes could enhance their environmental survival, their transmission potential and pathogenicity, as well as leading to the emergence of new pathogens. Exposure to various stress conditions, such as nutrient limitation, or exposure to biocides and antimicrobial agents, has led to the generation of phenotypic variants in several important and common pathogens. A well studied example is a phase variation event in V. cholerae which results in the generation of two morphologically different variants termed smooth and rugose [42] (Fig. 2). The phenotypic properties of these two variants differ greatly. The rugose variant has an increased capacity to form biofilms and exhibits increased resistance to osmotic and oxidative stress compared to the smooth variant [62,67]. The switch between the two colonial variants is bidirectional [48,67], but the rugose to smooth transition occurs more frequently, as demonstrated by the smooth sectoring observed on the edges of rugose colonies [67] (Fig. 2). Conditions that result in conversion from the smooth to the rugose form include carbon limitation and treatment with bactericidal agents [67]. Epidemic strains have an increased capacity to switch between variants, suggesting that this ability may increase the transmission potential of V. cholerae [48]. The switching is also of critical importance from a public heath perspective as the conversion to a rugose colonial form is associated with increased survival in chlorinated water [49]. Increased production of Vibrio polysaccharide by the rugose variant, as depicted in Fig. 2, was shown to be responsible for chlorine resistance [67]. Because of the association between morphology and phenotypic properties, it has been proposed that the generation of V. cholerae cells with different phenotypes enhances the likelihood of transmission for the organism. Gene capture systems such as an integron island are also critical for enhancing genetic diversity, plasticity and versatility [50e52]. V. cholerae has an integron island [39] and transcription of many genes in the integron island are positively regulated by HapR, master regulator of the quorum sensing system of V. cholerae and RpoS [68]. HapR production is induced at high cell densities and RpoS accumulates in response to stress and during stationary phase growth. Consequently, integron island genes might be expected to be activated in crowded, stressed communities containing non-replicating organisms [68]. When viewed from an environmental and evolutionary survival perspective, this behavior could confer a survival advantage because it would integrate a gene acquisition program with a survival-enhancing stress response. Viruses are particularly relevant for generation of genetic diversity and for the emergence of new pathogenic strains of V. cholerae. The genes coding cholera toxin are contained in the genome of the filamentous bacteriophage CTXF [63]
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and environmental non-pathogenic V. cholerae strains can be infected by CTXF, a process that leads to the generation of new pathogenic variants [17]. These studies demonstrate that the life cycles of V. cholerae and Vibrio phages are interwoven, and this relationship may be particularly relevant to the transmission of the pathogen. While it is clear that biofilms can enhance the survival of pathogens by providing increased resistance to environmental stresses, it is also possible that biofilms enhance the emergence of new genetic variants. The close spatial arrangement of bacteria in biofilms may permit increased rates of lateral gene transfer, thus allowing the transfer of virulence and resistance genes. The transfer of virulence genes could both enhance the pathogenicity of existing pathogens and lead to the emergence of new pathogens, especially by transforming organisms that currently infect other vertebrates but not humans. 3.4. Interaction with protozoa and viruses Bacterial pathogens in the environment become part of the microbial food web and their abundance can be modulated by protozoa, which are ubiquitous in natural environments. Protozoa graze on bacteria. Whereas some pathogens are readily killed by protozoan grazing, others survive and grow in protozoa. These pathogens exit protozoa either in vesicles or following protozoal lysis, which releases large numbers of bacteria into the environment [24]. The best studied example of this phenomenon is that of Legionella-protozoa interaction [2]. L. pneumophila can replicate intracellularly in various species of protozoa, where it is protected from environmental stresses [2]. Since growth inside protozoa increases the chances of survival of L. pneumophila in the environment, it increases the transmission potential of the pathogen. M. avium remains viable in protozoan vacuoles and can survive encystment of protozoa, a property thought to facilitate its transmission [53]. The pathogenicity of L. pneumophila and M. avium, both intracellular pathogens that replicate in human alveolar macrophages, has been shown to be enhanced by prior growth in protozoa [10,11], suggesting that interaction with protozoa increases the transmission chances of these pathogens by enhancing their virulence properties and priming them for human infection. Protozoan grazing reduces the planktonic V. cholerae population in coastal marine environments [65]. Matz et al. have shown that biofilms formed by V. cholerae are resistant to protozoan grazing relative to planktonic V. cholerae, and that grazing selects for phenotypic variants of V. cholerae with enhanced biofilm-forming capacity [38]. It has also been reported that V. cholerae can multiply within protozoa and survive encystment [1]. Taken together, these studies indicate that V. cholerae-protozoan interactions control V. cholerae population numbers and transmission dynamics. C. jejuni populations in natural aquatic environments can be negatively impacted by protozoan grazing activity [54]. But interaction of C. jejuni with protozoa may increase the colonization chances of broilers and, in turn, increases the likelihood of
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Fig. 2. (A) A scanning electron micrograph of a rugose colony variant switching to a smooth variant. (B,C) Transmission electron micrograph of thin sections stained with ruthenium red of rugose (B) and smooth (C) cells grown on cellulose membrane atop nutrient agar.
transmission. A recent study showed that in an in vitro coculture assay, incubation of C. jejuni with protozoa, initially decreases the number of viable C. jejuni cells; however, C. jejuni remains viable longer after internalization by protozoa relative to C. jejuni in suspension [55]. Furthermore, ingestion by protozoa increases C. jejuni resistance to disinfection [55]. Thus, there is growing evidence that protozoan grazing is one of the critical ecological factors controlling abundance and transmission of various bacterial pathogens in the environment. Viruses are highly abundant in natural environments and can control the abundance and diversity of microbial populations [64]. It has been demonstrated that environmental phage populations infect V. cholerae strains and that the numbers of these phages inversely correlates with those of V. cholerae and of cholera cases [18,19]. It has been further shown that epidemic and non-epidemic V. cholerae strains harbor lysogenic phages that can influence the epidemiology of V. cholerae [19]. Upon exposure to environmental stresses, lysogenic Vibrio-phages can enter into a lytic cycle and generate phage particles, which reduce the population of susceptible V. cholerae strains in aquatic ecosystems. There is also a correlation between the prevalence of environmental phages and a reduction in the C. jejuni population in commercial broiler chickens [4]. Furthermore, host-specific bacteriophages have been used to control populations of C. jejuni cells on artificially contaminated chicken [3]. Hence, phages can also play a role in transmission of C. jejuni. 4. Conclusions The survival of microbes in the environment and their transmission to new hosts are influenced by multiple factors, some of which were discussed above. The pathogen load in the environment needs to be better estimated for improving risk assessment. A number of newly developed molecular biology methods for detecting pathogenic species facilitate the study of the distribution of these microorganisms in their native environment. For example, PCR and RTePCR methods have been used to study pathogens, and natural communities
of microbes in aquatic environments. These methods are relatively rapid, extremely sensitive, and can be applied to determine the abundance of specific species or broadly-related phylogenetic groups. Culture- and molecular-based detection methods have been evolving to incorporate our growing knowledge of pathogen ecology and evolution. Elegant examples of such studies include the use of protozoan coculture [59] to enrich for pathogens, and the application of antibiotics to remove rapidly growing, interfering bacteria (but to which pathogenic bacteria are resistant), allowing the isolation of culturable pathogenic bacteria from the environment [21]. Many questions remain regarding the physiological state of pathogens when they exit the host and how they survive in and adjust to the non-host environment. For example, in any given environment, are the pathogens surface-attached or free-floating, and what factors alter this distribution? How do physical/ chemical factors govern attachment and detachment to surfaces? Is one form of a pathogen more infectious than another? How do surface-attached bacteria respond to fluctuating environmental parameters, and what genes/processes are required for this response? What is the importance of these responses for long-term survival of a given pathogen? Do the physical and chemical factors likely to be encountered by a given pathogen in natural aquatic habitats favor the evolution of populations with enhanced transmission capabilities, and thus pathogens with new abilities? Answers to each of these questions would lead to a better understanding of processes controlling environmental fitness and transmission of pathogenic bacteria. Such knowledge would, in turn, allow us to better predict and control of infectious disease outbreaks. Acknowledgments Work in my laboratory is supported by grants from The Ellison Medical Foundation and NIH (AI055987). References [1] H. Abd, A. Weintraub, G. Sandstrom, Intracellular survival and replication of Vibrio cholerae O139 in aquatic free-living amoebae, Environ. Microbiol. 7 (2005) 1003e1008.
F.H. Yildiz / Research in Microbiology 158 (2007) 195e202 [2] R.M. Atlas, Legionella: from environmental habitats to disease pathology, detection and control, Environ. Microbiol. 1 (1999) 283e293. [3] R.J. Atterbury, P.L. Connerton, C.E. Dodd, C.E. Rees, I.F. Connerton, Application of host-specific bacteriophages to the surface of chicken skin leads to a reduction in recovery of Campylobacter jejuni, Appl. Environ. Microbiol. 69 (2003) 6302e6306. [4] R.J. Atterbury, E. Dillon, C. Swift, P.L. Connerton, J.A. Frost, C.E. Dodd, C.E. Rees, I.F. Connerton, Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca, Appl. Environ. Microbiol. 71 (2005) 4885e4887. [5] M.A. Bachman, M.S. Swanson, The LetE protein enhances expression of multiple LetA/LetS-dependent transmission traits by Legionella pneumophila, Infect. Immun. 72 (2004) 3284e3293. [6] M.A. Bachman, M.S. Swanson, Genetic evidence that Legionella pneumophila RpoS modulates expression of the transmission phenotype in both the exponential phase and the stationary phase, Infect. Immun. 72 (2004) 2468e2476. [7] J. Bina, J. Zhu, M. Dziejman, S. Faruque, S. Calderwood, J. Mekalanos, ToxR regulon of Vibrio cholerae and its expression in vibrios shed by cholera patients, Proc. Natl. Acad. Sci. USA. 100 (2003) 2801e2806. [8] C.M. Buswell, Y.M. Herlihy, L.M. Lawrence, J.T. McGuiggan, P.D. Marsh, C.W. Keevil, S.A. Leach, Extended survival and persistence of Campylobacter spp. in water and aquatic biofilms and their detection by immunofluorescent-antibody and -rRNA staining, Appl. Environ. Microbiol. 64 (1998) 733e741. [9] S.M. Butler, E.J. Nelson, N. Chowdhury, S.M. Faruque, S.B. Calderwood, A. Camilli, Cholera stool bacteria repress chemotaxis to increase infectivity, Mol. Microbiol. 60 (2006) 417e426. [10] J.D. Cirillo, S. Falkow, L.S. Tompkins, L.E. Bermudez, Interaction of Mycobacterium avium with environmental amoebae enhances virulence, Infect. Immun. 65 (1997) 3759e3767. [11] J.D. Cirillo, S.L. Cirillo, L. Yan, L.E. Bermudez, S. Falkow, L.S. Tompkins, Intracellular growth in Acanthamoeba castellanii affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila, Infect. Immun. 67 (1999) 4427e4434. [12] R.R. Colwell, Viable but nonculturable bacteria: a survival strategy, J. Infect. Chemother. 6 (2000) 121e125. [13] R.R. Colwell, A. Huq, M.S. Islam, K.M. Aziz, M. Yunus, N.H. Khan, A. Mahmud, R.B. Sack, G.B. Nair, J. Chakraborty, D.A. Sack, E. RussekCohen, Reduction of cholera in Bangladeshi villages by simple filtration, Proc. Natl. Acad. Sci. USA. 100 (2003) 1051e1055. [14] J.W. Costerton, Z. Lewandowski, D.E. Caldwell, D.R. Korber, H.M. Lappin-Scott, Microbial biofilms, Annu. Rev. Microbiol. 49 (1995) 711e745. [15] R.M. Donlan, J.W. Costerton, Biofilms: survival mechanisms of clinically relevant microorganisms, Clin. Microbiol. Rev. 15 (2002) 167e193. [16] S.M. Faruque, M.J. Albert, J.J. Mekalanos, Epidemiology, genetics, and ecology of toxigenic Vibrio cholerae, Microbiol. Mol. Biol. Rev. 62 (1998) 1301e1314. [17] S.M. Faruque, J.J. Mekalanos, Pathogenicity islands and phages in Vibrio cholerae evolution, Trends Microbiol. 11 (2003) 505e510. [18] S.M. Faruque, M.J. Islam, Q.S. Ahmad, A.S. Faruque, D.A. Sack, G.B. Nair, J.J. Mekalanos, Self-limiting nature of seasonal cholera epidemics: Role of host-mediated amplification of phage, Proc. Natl. Acad. Sci. USA. 102 (2005) 6119e6124. [19] S.M. Faruque, I.B. Naser, M.J. Islam, A.S. Faruque, A.N. Ghosh, G.B. Nair, D.A. Sack, J.J. Mekalanos, Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages, Proc. Natl. Acad. Sci. USA. 102 (2005) 1702e1707. [20] S.M. Faruque, K. Biswas, S.M. Udden, Q.S. Ahmad, D.A. Sack, G.B. Nair, J.J. Mekalanos, Transmissibility of cholera: in vivo-formed biofilms and their relationship to infectivity and persistence in the environment, Proc. Natl. Acad. Sci. USA. 103 (2006) 6350e6355. [21] S.M. Faruque, M.J. Islam, Q.S. Ahmad, K. Biswas, A.S. Faruque, G.B. Nair, R.B. Sack, D.A. Sack, J.J. Mekalanos, An improved technique for isolation of environmental Vibrio cholerae with epidemic potential: monitoring the emergence of a multiple-antibiotic-resistant epidemic strain in Bangladesh, J. Infect. Dis. 193 (2006) 1029e1036.
201
[22] C.A. Fux, J.W. Costerton, P.S. Stewart, P. Stoodley, Survival strategies of infectious biofilms, Trends Microbiol. 13 (2005) 34e40. [23] E.C. Gaynor, D.H. Wells, J.K. MacKichan, S. Falkow, The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes, Mol. Microbiol. 56 (2005) 8e27. [24] G. Greub, D. Raoult, Microorganisms resistant to free-living amoebae, Clin. Microbiol. Rev. 17 (2004) 413e433. [25] L. Hall-Stoodley, P. Stoodley, Biofilm formation and dispersal and the transmission of human pathogens, Trends Microbiol. 13 (2005) 7e10. [26] B.K. Hammer, M.S. Swanson, Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp, Mol. Microbiol. 33 (1999) 721e731. [27] B.K. Hammer, E.S. Tateda, M.S. Swanson, A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila, Mol. Microbiol. 44 (2002) 107e118. [28] M.A. Hood, J.B. Guckert, D.C. White, F. Deck, Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibrio cholerae, Appl. Environ. Microbiol. 52 (1986) 788e793. [29] A. Huq, E.B. Small, P.A. West, M.I. Huq, R. Rahman, R.R. Colwell, Ecological relationships between Vibrio cholerae and planktonic crustacean copepods, Appl. Environ. Microbiol. 45 (1983) 275e283. [30] G.W. Joshua, C. Guthrie-Irons, A.V. Karlyshev, B.W. Wren, Biofilm formation in Campylobacter jejuni, Microbiology 152 (2006) 387e396. [31] J.B. Kaper, J.G. Morris Jr., M.M. Levine, Cholera, Clin. Microbiol. Rev. 8 (1995) 48e86. [32] A.F. Kelly, S.F. Park, R. Bovill, B.M. Mackey, Survival of Campylobacter jejuni during stationary phase: evidence for the absence of a phenotypic stationary-phase response, Appl. Environ. Microbiol. 67 (2001) 2248e2254. [33] M.W. Kuiper, B.A. Wullings, A.D. Akkermans, R.R. Beumer, D. van der Kooij, Intracellular proliferation of Legionella pneumophila in Hartmannella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride, Appl. Environ. Microbiol. 70 (2004) 6826e6833. [34] J. Mampel, T. Spirig, S.S. Weber, J.A. Haagensen, S. Molin, H. Hilbi, Planktonic replication is essential for biofilm formation by Legionella pneumophila in a complex medium under static and dynamic flow conditions, Appl. Environ. Microbiol. 72 (2006) 2885e2895. [35] L. Marsollier, R. Robert, J. Aubry, J.P. Saint Andre, H. Kouakou, P. Legras, A.L. Manceau, C. Mahaza, B. Carbonnelle, Aquatic insects as a vector for Mycobacterium ulcerans, Appl. Environ. Microbiol. 68 (2002) 4623e4628. [36] L. Marsollier, T. Stinear, J. Aubry, J.P. Saint Andre, R. Robert, P. Legras, A.L. Manceau, C. Audrain, S. Bourdon, H. Kouakou, B. Carbonnelle, Aquatic plants stimulate the growth of and biofilm formation by Mycobacterium ulcerans in axenic culture and harbor these bacteria in the environment, Appl. Environ. Microbiol. 70 (2004) 1097e1103. [37] L. Marsollier, J. Aubry, E. Coutanceau, J.P. Andre, P.L. Small, G. Milon, P. Legras, S. Guadagnini, B. Carbonnelle, S.T. Cole, Colonization of the salivary glands of Naucoris cimicoides by Mycobacterium ulcerans requires host plasmatocytes and a macrolide toxin, mycolactone, Cell Microbiol. 7 (2005) 935e943. [38] C. Matz, D. McDougald, A.M. Moreno, P.Y. Yung, F.H. Yildiz, S. Kjelleberg, Biofilm formation and phenotypic variation enhance predation-driven persistence of Vibrio cholerae, Proc. Natl. Acad. Sci. USA. 102 (2005) 16819e16824. [39] D. Mazel, B. Dychinco, V.A. Webb, J. Davies, A distinctive class of integron in the Vibrio cholerae genome, Science 280 (1998) 605e608. [40] D.S. Merrell, S.M. Butler, F. Qadri, N.A. Dolganov, A. Alam, M.B. Cohen, S.B. Calderwood, G.K. Schoolnik, A. Camilli, Host-induced epidemic spread of the cholera bacterium, Nature 417 (2002) 642e645. [41] A.B. Molofsky, M.S. Swanson, Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication, Mol. Microbiol. 50 (2003) 445e461. [42] J.G. Morris Jr., M.B. Sztein, E.W. Rice, J.P. Nataro, G.A. Losonsky, P. Panigrahi, C.O. Tacket, J.A. Johnson, Vibrio cholerae O1 can assume a chlorine-resistant rugose survival form that is virulent for humans, J. Infect. Dis. 174 (1996) 1364e1368. [43] R. Murga, T.S. Forster, E. Brown, J.M. Pruckler, B.S. Fields, R.M. Donlan, Role of biofilms in the survival of Legionella pneumophila in a model potable-water system, Microbiology 147 (2001) 3121e3126.
202
F.H. Yildiz / Research in Microbiology 158 (2007) 195e202
[44] C. Murphy, C. Carroll, K.N. Jordan, Environmental survival mechanisms of the foodborne pathogen Campylobacter jejuni, J. Appl. Microbiol. 100 (2006) 623e632. [45] J.D. Oliver, The viable but nonculturable state in bacteria, J. Microbiol. Spec No 43 (2005) 93e100. [46] J. Parkhill, B.W. Wren, K. Mungall, J.M. Ketley, C. Churcher, D. Basham, T. Chillingworth, R.M. Davies, T. Feltwell, S. Holroyd, K. Jagels, A.V. Karlyshev, S. Moule, M.J. Pallen, C.W. Penn, M.A. Quail, M.A. Rajandream, K.M. Rutherford, A.H. van Vliet, S. Whitehead, B.G. Barrell, The genome sequence of the food-borne pathogen Campylobacter jejuni reveals hypervariable sequences, Nature 403 (2000) 665e668. [47] T.P. Primm, C.A. Lucero, J.O. Falkinham 3rd, Health impacts of environmental mycobacteria, Clin. Microbiol. Rev. 17 (2004) 98e106. [48] M.H. Rashid, C. Rajanna, A. Ali, D.K. Karaolis, Identification of genes involved in the switch between the smooth and rugose phenotypes of Vibrio cholerae, FEMS Microbiol. Lett. 227 (2003) 113e119. [49] E.W. Rice, C.J. Johnson, R.M. Clark, K.R. Fox, D.J. Reasoner, M.E. Dunnigan, P. Panigrahi, J.A. Johnson, J.G. Morris Jr., Chlorine and survival of ‘‘rugose’’ Vibrio cholerae, Lancet 340 (1992) 740. [50] D.A. Rowe-Magnus, A.M. Guerout, D. Mazel, Super-integrons, Res. Microbiol. 150 (1999) 641e651. [51] D.A. Rowe-Magnus, A.M. Guerout, P. Ploncard, B. Dychinco, J. Davies, D. Mazel, The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons, Proc. Natl. Acad. Sci. USA. 98 (2001) 652e657. [52] D.A. Rowe-Magnus, A.M. Guerout, D. Mazel, Bacterial resistance evolution by recruitment of super-integron gene cassettes, Mol. Microbiol. 43 (2002) 1657e1669. [53] M.T. Rowe, I.R. Grant, Mycobacterium avium ssp. paratuberculosis and its potential survival tactics, Lett. Appl. Microbiol. 42 (2006) 305e311. [54] M. Schallenberg, P.J. Bremer, S. Henkel, A. Launhardt, C.W. Burns, Survival of Campylobacter jejuni in water: effect of grazing by the freshwater crustacean Daphnia carinata (Cladocera), Appl. Environ. Microbiol. 71 (2005) 5085e5088. [55] W.J. Snelling, J.P. McKenna, D.M. Lecky, J.S. Dooley, Survival of Campylobacter jejuni in waterborne protozoa, Appl. Environ. Microbiol. 71 (2005) 5560e5571.
[56] K.A. Steed, J.O. Falkinham 3rd, Effect of growth in biofilms on chlorine susceptibility of Mycobacterium avium and Mycobacterium intracellulare, Appl. Environ. Microbiol. 72 (2006) 4007e4011. [57] M.S. Swanson, B.K. Hammer, Legionella pneumophila pathogesesis: a fateful journey from amoebae to macrophages, Annu. Rev. Microbiol. 54 (2000) 567e613. [58] M.L. Tamplin, A.L. Gauzens, A. Huq, D.A. Sack, R.R. Colwell, Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters, Appl. Environ. Microbiol. 56 (1990) 1977e1980. [59] V. Thomas, K. Herrera-Rimann, D.S. Blanc, G. Greub, Biodiversity of amoebae and amoeba-resisting bacteria in a hospital water network, Appl. Environ. Microbiol. 72 (2006) 2428e2438. [60] N. Trachoo, J.F. Frank, Effectiveness of chemical sanitizers against Campylobacter jejuni-containing biofilms, J. Food Prot. 65 (2002) 1117e 1121. [61] M.J. Vaerewijck, G. Huys, J.C. Palomino, J. Swings, F. Portaels, Mycobacteria in drinking water distribution systems: ecology and significance for human health, FEMS Microbiol. Rev. 29 (2005) 911e934. [62] S.N. Wai, Y. Mizunoe, A. Takade, S.I. Kawabata, S.I. Yoshida, Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation, Appl. Environ. Microbiol. 64 (1998) 3648e3655. [63] M.K. Waldor, J.J. Mekalanos, Lysogenic conversion by a filamentous phage encoding cholera toxin, Science 272 (1996) 1910e1914. [64] K.E. Wommack, R.R. Colwell, Virioplankton: viruses in aquatic ecosystems, Microbiol. Mol. Biol. Rev. 64 (2000) 69e114. [65] A.Z. Worden, M. Seidel, S. Smriga, A. Wick, F. Malfatti, D. Bartlett, F. Azam, Trophic regulation of Vibrio cholerae in coastal marine waters, Environ. Microbiol. 8 (2006) 21e29. [66] F.H. Yildiz, G.K. Schoolnik, Role of rpoS in stress survival and virulence of Vibrio cholerae, J. Bacteriol. 180 (1998) 773e784. [67] F.H. Yildiz, G.K. Schoolnik, Vibrio cholerae O1 El Tor: identification of a gene cluster required for the rugose colony type, exopolysaccharide production, chlorine resistance, and biofilm formation, Proc. Natl. Acad. Sci. USA. 96 (1999) 4028e4033. [68] F.H. Yildiz, X.S. Liu, A. Heydorn, G.K. Schoolnik, Molecular analysis of rugosity in a Vibrio cholerae O1 El Tor phase variant, Mol. Microbiol. 53 (2004) 497e515.