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As one of the six species of the genus Listeria, L. monocytogenes belongs to a genus of the Corynebacteriaceae family that comprises of L. monocytogenes, L. grayi, L. innocua, L.Â ivanovii, L. seeligeri, and L. welshimeri. While L. monocytogenes is pathogenic to both humans and animals, L. ivanovii is only pathogenic to animals, mainly sheep and cattle. The rest of the Listeria species are known not to cause any disease (Cossart 2007).
L. monocytogenes shows resistance to extreme environmental conditions. For example, high concentrations of salt (up to 10% NaCl), a wide pH range (4.5 - 9) as well as low temperatures (Vázquez-Boland et al. 2001). From a clinical perspective, L. monocytogenes is also able to colonize and persist in the gallbladder, which has a high pH content (Begley et al. 2009). This finding suggests the occurrence of both long-term and chronic infections, as well as the ability of the bacterium to survive within the microenvironments of the gastrointestinal tract. Though the optimum growth temperature range for L.Â monocytogenes growth is between 30Â°C - 37Â°C, not only does growth has been shown to occur between 3Â°C - 45Â°C (Junttila et al. 1988), the bacteria has been demonstrated to grow at temperatures between 1Â°C - 4Â°C (Farber and Peterkin 1991). This adaptability of L. monocytogenes to a variety of temperatures suits it for survival and growth in either processed or refrigerated food items.
Though L. monocytogenes is ubiquitous in the environment, this bacterium is most commonly found in decaying vegetation and also soil (Weis and Seeliger 1975). Following ingestion of L.Â monocytogenes by a susceptible person, the bacteria can make the transition from being a saprophyte to a parasite that promotes the survival and replication of the bacterium within host cells (Freitag 2006). L. monocytogenes has been demonstrated to be capable colonizing a variety of inert surfaces, as well as able to form biofilms on food-processing surfaces (Roberts and Wiedmann 2003). The presence of L.Â monocytogenes in numerous environments such as farms, soil, water, silage produced from contaminated grasses and also food processing facilities, indicates that there L. monocytogenes has many opportunities to contaminate the food production process (Cossart and Bierne 2001).
Though human listeriosis outbreaks following ingestion of L. monocytogenes contaminated food items have been reported previously, only recently the bacterium has been recognized as a major cause of human infections (Lecuit 2007). This is due to increased numbers of susceptible, immuno-compromised individuals, the increase in large-scale agro-industrial plant development, as well as an increased reliance on refrigerated food items.
Fresh vegetables are an example of origins of contaminants; it can be contaminated from the soil or from manure that were originally used as fertilizers and food. Animals may also carry the bacterium asymptomatically and contaminate foods of animal origin. Other examples of food items that are most linked to listeriosis outbreaks include ready-to-eat meats, undercooked meats, cold cuts, pâté (McLauchlin et al. 1991), salads, dairy products, especially soft cheeses (Linnan et al. 1988) and milk that is either inadequately pasteurized or contaminated post-pasteurization (Fleming et al. 1985, Jackson et al. 2011).
Listeriosis in Humans
A previous study has demonstrated that asymptomatic carriage of L. monocytogenes does occur within the intestinal tract of 5% or more of healthy humans (Grif et al. 2003). However extremely rare infections also occur in healthy adults and young children, where there is increasing evidence in recent years to prove that listeriosis may also occur in healthy individuals within just 24 h post ingestion of highly contaminated food (Swaminathan and Gerner-Smidt 2007).
Those most at risk from listeriosis are generally immuno-compromised individuals such as diabetics, AIDS patients, those with renal failure, organ transplant patients, cancer patients and also elderly adults. Diarrhoea is usually an early symptom of the infection. Advanced symptoms in these susceptible individuals include septicemia or meningoencephalitis. The main clinical features of L.Â monocytogenes meningitis are abnormal movements, seizures, as well as alteration of consciousness. L. monocytogenes meningitis cases have been shown to have the highest mortality rate (22%) in comparison to all types of bacterial meningitis (Lecuit and Cossart 2001). Certain patients are known to experience rare and localized infections, for example due to direct inoculation of the bacterium (Schlech 2000, Lecuit and Cossart 2001).
Others at high risk of the disease also include pregnant women, foetus and also newborns (Vázquez-Boland et al. 2001). Due to the fact that pregnant women have a naturally depressed, cell-mediated immune system (Weinberg 1984), pregnant women is more likely to acquire listeriosis upon post consumption of contaminated food compared to other individuals. By gaining access to the maternal circulation, L. monocytogenes colonizes the placenta to induce placentitis, infecting the defenseless foetus. As opposed to maternal illness, the severity of infection within foetal and neonatal is much higher. The common fatal symptoms include pre-term labour, amnionitis, spontaneous abortion, stillbirth as well as early onset sepsis (Vázquez-Boland et al. 2001).
The occurrence of fatality due to listeriosis is continuing to decrease in industrialized countries in recent years. This has been attributed to the stricter implementation of food quality and safety (Allerberger and Wagner 2010).
Listeriosis in Animals
Various species of animals can be infected with L. monocytogenes, but clinical disease is rare. The bacterium can also live within the intestines of healthy animals without causing any infections. Though most cases of animal listeriosis are generally seen in ruminants, this disease can also occur in poultry and other birds, pigs, dogs, cats, domestic and wild rabbits, and many other small mammals. Infected ruminants have been shown to experience encephalitis, septicemia, and even abortions (Schoder et al. 2003). The course of disease in sheep and goats is more rapid and death may occur 24 - 48 h upon the onset of symptoms. In cattle however, the course of disease is less acute.
Many L. monocytogenes-infected animals excrete the bacterium in faeces and milk. This is a common source of animal to animal spread of infection. Grass silage is presumed to be the source of infection, as it can be contaminated with large numbers of L. monocytogenes. This is because the low pH of silage can enhance the growth of L monocytogenes cells (Pham 2006). Besides silage, the bacterium has also been isolated from other sources such as water troughs, manure, soil and animal feeds. L. monocytogenes infection may also cause mastitis in cattle and sheep (Wagner et al. 2005).
In ruminants such as sheep, infections that lead to lesions in the brain stem, result in characteristic clinical symptoms (Rebhun and deLahunta 1982). Typical symptoms of listeriosis in ruminants include turning or twisting of the head to one side and walking in circles, drooping of the eyelid and ear caused by paralysis of the unilateral facial nerve. The infected ruminant may also drool saliva as a result of partial pharyngeal paralysis (Rebhun and deLahunta 1982).
Animals that excrete L. monocytogenes cells in faeces have been suggested as the primary cause of entry of this pathogen into food-processing plants (Schonberg and Gerigk 1991). The growth and multiplication of L. monocytogenes cells is usually promoted by not only the high humidity, but also the nutrient rich waste present within certain food production plants. Hence, it is not surprising that animal listeriosis does pose a serious contamination risk for the food industry in general.
Pathophysiology of L. monocytogenes
L. monocytogenes is usually ingested with contaminated food (Error: Reference source not found). In immuno-compromised individuals, L. monocytogenes invades the epithelial cells of the intestines and spreads to other parts of the body by cell-to-cell spread. L. monocytogenes secretes invasins (InlA + InlB) to enable it to penetrate cells of the intestinal epithelial lining (Gaillard et al. 1987, Mengaud et al. 1996). L. monocytogenes cells that cross the intestinal epithelial barrier are then carried by the lymph or blood to the mesenteric lymph nodes, the spleen, and also the liver (Marco et al. 1992, Pron et al. 1998). Entry into the host's monocytes, macrophages, or polymorphonuclear leukocytes promotes growth of L. monocytogenes, and the infection becomes blood-borne (septicemic) dissemination.
L. monocytogenes then enters the liver after the intestinal translocation and carriage by the bloodstream (Marco et al. 1992, Dramsi et al. 1998). Hepatocytes are generally the main site of L. monocytogenes multiplication within the liver (Vázquez-Boland et al. 2001). When there is an inadequate immune response by the host, L. monocytogenes usually multiplies unlimitedly within the liver parenchyma, possibly releasing the bacteria into blood to cause bacteremia. Via the bloodstream, L. monocytogenes cells will reach the brain in order to cross the blood-brain barrier (Kirk 1993). High levels of L. monocytogenes cells in the brain accompanied by bacteremia will generally result in meningoencephalitis (Tunkel and Scheld 1993, Tuomanen 1996).
In pregnant women, L. monocytogenes usually gains access to the foetus by entering the endothelial layer of the placental barrier (Gray and Killinger 1966). The bacterial cells will reach the bloodstream of the foetus by firstly colonizing the trophoblast layer. The bacteria then will reach the bloodstream of the foetus by translocating across the endothelial barrier. This will usually result in infection and the possible subsequent death of the foetus within the uterus, or occasionally even the premature birth of a severely infected neonate (Vázquez-Boland et al. 2001).
Virulence Factors of L. monocytogenes
A wide array of virulence factors is wielded by L. monocytogenes to assist the bacterium to interact and manipulate the host cells. Virulence genes of L. monocytogenes are known to be optimally expressed at 37Â°C, but expressions almost does not occur at 30Â°C (Freitag et al. 2009). The key transcriptional activator of L. monocytogenes virulence factor genes, known as PrfA, is also known to be thermo-regulated. PrfA is usually activated upon the ingestion of L.Â monocytogenes contaminated foods (Error: Reference source not found). PrfA is also known to regulate a variety of the bacterium's virulence genes (Camejo et al. 2011, Stavru et al. 2011) as well as other core genome genes.
Internalins (InlA and InlB), which are L. monocytogenes surface proteins, have been previously shown to involve in the invading of the host cells (Seveau et al. 2007). InlA is known to bind E-cadherin, which is the host cell's adhesion molecule, whereas InlB binds to Met, which is the hepatocyte growth factor (HGF) receptor. By the binding of internalin proteins to E-cadherin and Met, L.Â monocytogenes cells are able to gain entry into the host cells; this is done by taking advantage of the endocytic machinery of the host cells (Pizarro-Cerda and Cossart 2006).
Once internalized within the host cell, L. monocytogenes mediates escape from membrane-bound vacuoles through the secretion of Listeriolysin O (a pore-forming haemolysin) (Gaillard et al. 1987), as well as two phospholipases: phosphatidylinositol (PI) phospholipase (PLC-A) (Camilli et al. 1993) and phosphatidylcholine (PC) phospholipase C (PLC-B) (Grundling et al. 2003). Together, these proteins assist in breaking down the host phagosome that contains L. monocytogenes cells. This is done to allow the bacterium to escape into the host cytosol (Kathariou et al. 1987, Camilli et al. 1991, Mengaud et al. 1991, Vázquez-Boland et al. 1992, Schnupf and Portnoy 2007, Scortti et al. 2007). Upon entering the host cell's cytosol, L. monocytogenes cells begin to replicate (O'Riordan et al. 2003, Joseph and Goebel 2007), and then with the assistance of actin polymerization mediated cell-cell spread , the bacterium moves through the host cell for the purpose of migrating into the neighbouring host cells. The actin polymerization mediated cell-cell spread process is directed by ActA. The ActA protein binds and activates Arp2/3, which is a seven-protein host complex. Arp2/3 has been shown to induce actin polymerization as well as generate actin filaments (Pizarro-Cerda and Cossart 2006). Upon entry into the adjacent host cell, L.Â monocytogenes cells secretes both Listeriolysin O and also PC-PLC to assist the bacteria in escaping from the double-membrane secondary vacuoles, known as listeriopods, which were formed as a result of cell-to-cell spread (Freitag et al. 2009).
Invasion of Mammalian Cells by L. monocytogenes
L. monocytogenes has evolved a number of strategic methods to evade or resist killing by the innate immune response of mammalian phagocytic cells that are usually known to phagocytose and degrade most pathogens that invade the host cells (Ryter and De Chastellier 1983). The bacterium is able to multiply in a variety of mammalian cell types such as professional phagocytic cells, for example, J774 macrophage-like cells (Portnoy et al. 1992), as well as non-professional phagocytes such as epithelial cells (Rácz et al. 1972), endothelial cells (Drevets et al. 1995) and hepatocytes (Conlan and North 1992). Marco et al. (1992) previously demonstrated that in mice that were infected with L. monocytogenes cells, the bacterium first infected the macrophage cells, followed by infection of the hepatocytes in the liver. L.Â monocytogenes has also been shown in a separate study to be able to efficiently invade hepatocytes in vitro (Wood et al. 1993).
Within mammalian host cells, L. monocytogenes is internalized within membrane-bound phagosomes upon adhering to host cells. The bacterium then escapes into the host cytosol from the phagosome by disrupting the phagosomal membrane. Within the host cytosol, L.Â monocytogenes grows and multiplies, and then proceeds to infect neighbouring host cells (Freitag et al. 2009). Gaillard et al. (1987) showed L.Â monocytogenes was able to initiate entry into human colon carcinoma cell line Caco-2, and multiply within the host cytosol. That same study also provided evidence to show that L. monocytogenes was able to induce phagocytosis by Caco-2 cells. Francis and Thomas (1996) demonstrated recovery of a higher numbers of L.Â monocytogenes cells of hemolytic strains from both HeLa and Caco-2 cell lines, in comparison to non-hemolytic strains. Furthermore, the extensive morphological changes that the host cells exhibited not only included loss of confluence and host cell lysis, but also the presence of very high counts of L.Â monocytogenes cells within the host cells were detected (Francis and Thomas 1996).
Is there an Environmental Reservoir for L. monocytogenes?
Although L. monocytogenes causes severe disease in human and animal hosts, unlike other Gram-negative intracellular pathogens, this pathogen has no recognized animal reservoir. Several studies have suggested that as a result of interaction with soil-borne organisms such as protozoa, a number of intracellular pathogens are able to maintain its virulence genes (Barker and Brown 1994, Adiba et al. 2010, Lamrabet et al. 2012). For example, Salmonella spp. interacts with protozoans such as amoebae and the ciliate Tetrahymena pyriformis (Tezcan-Merdol et al. 2004, Brandl et al. 2005). Miltner and Bermudez (2000) have suggested the possible role of Acanthamoeba castellanii as an environmental host for the pathogenic Mycobacterium avium. Furthermore, protozoan cells that were harbouring Legionella pneumophila were identified as the cause of a fatal outbreak of Legionnaires' disease outbreak during a convention in Philadelphia in 1976.
While Acanthamoeba spp. is known to harbour a number of bacterial pathogens, L.Â monocytogenes has been recently demonstrated to be phagocytosed and rapidly degraded by the host amoeba within just 2 h of ingestion (Akya et al. 2010). In view of this result, the question arises whether protozoa could act as a potential reservoir for L. monocytogenes.
Interactions between Bacteria and Protozoa
Protozoa are unicellular eukaryotic microorganisms that are ubiquitously present in diverse habitats. They feed heterotrophically and are generally recognised as the major consumers of bacteria in the environment. Protozoan cells can be present either singly or as colonies of cells (eg. Volvox spp.), may swim freely (Paramecium spp.), or are parasitic for other animals (eg. Trypanosoma spp.).
Briefly, there are three main groups of bacterivorous protozoa: amoebae, ciliates and flagellates. Amoebae feed on algae, bacteria, plant cells, and smaller protozoans. Amoebae move by forming pseudopods (temporary foot-like structures) with diverse morphologies. Ciliates can be found almost everywhere there is water, such as lakes, rivers, oceans and also soil. They are characterized by large numbers of hair-like organelles (called cilia) that are involved in movement of the cells, chemotaxis, as well as predation of bacteria (Fenchel 1987). Flagellates have whip-like appendages (called flagella) for the main purposes of locomotion as well as to direct food particles or cells into its mouth-like opening.
The major cause of bacterial mortality in the environment is suggested to be mainly as a result of feeding of bacteria by protozoans (Pernthaler 2005). A majority of protozoa feed by phagocytosis, a process by which they engulf bacteria and digest them within a food vacuole. Briefly, once the bacterial prey is captured, it is packaged into a food vacuole. Once inside the protozoan food vacuole, the process of digestion commences (Fenchel 1987). This is carried out through the release of host proteases and lysozymes into the food vacuole in order to break down the bacteria within the food vacuole. This will supply the protozoa with energy and nutrients for its growth. The acidic environment within the food vacuoles assists the protozoa in disabling the bacterial prey for digestion. The products of the digestion process are then released into the cytoplasm. However, previous studies have clearly demonstrated that not all bacteria are digested as food source. Some types of bacteria survive within the protozoa in order to persist and utilize those protozoan cells as a host. The major outcomes in a bacteria-protozoa interaction (Error: Reference source not found) include:
Upon phagocytosis, the bacteria multiplies to high numbers within the vacuoles, resulting in massive enlargement of these vacuoles that will eventually cause lysis of the host, releasing free bacteria into the extracellular environment, e.g. Legionella pneumophila (Rowbotham 1983).
The same process as (1), except that following lysis of host, free bacteria is released alongside intact vacuoles containing infectious bacteria, e.g. L. pneumophila (Rowbotham 1983).
Ingested bacteria multiply within the host but not able to cause lysis of the host, e.g. Coxiella burnetti (La Scola and Raoult 2001).
Ingested bacteria survive within encysted protozoa, e.g. L. pneumophila (Rowbotham 1983).
A number of important studies have previously shown that pathogenic bacteria that are able to survive within protozoans can be protected from external stresses such as chemical disinfectants and antibiotics (King et al. 1988, Berk et al. 1998, Brandl et al. 2005, Bichai et al. 2008). It is likely that the ability of a number of intracellular bacterial pathogens to resist killing by its host protozoan cells may have resulted in their evolution as pathogens of the mammalian kingdom. Indeed it is possible that protozoan cells are the link between bacteria that inhabits the environment and the bacteria that cause diseases in mammals such as humans.
Protozoa as Model Organisms for Study of Pathogenesis
Protozoan cells have been previously utilized as model organisms for studies in various fields such as evolution and ecology (Friman et al. 2008), population and community biology (Holyoak and Lawler 2005), the role of organelles (Smith et al. 2007) as well as toxicity studies (Stefanidou et al. 2008). The use of protozoa in the study of host-pathogen interactions has its advantages and has increasingly become more common in recent years, most importantly in infectious diseases studies. Infection studies generally utilized mammalian species such as mice and sometimes even humans as the host systems. By using the mammals as the host system, the analysis is not only expensive, prolonged and subject to extensive ethical review, it is also technically challenging and complex. In contrast, similar studies in protozoans are more convenient, quicker and also cost-effective (Montagnes et al. 2012).
As model systems, protozoan cells can also help in understanding better the mechanisms of infectious diseases within mammalian cells (Montagnes et al. 2012). Intracellular bacterial pathogens have been previously shown to escape the phagolysosomes of protozoa and mammalian phagocytic cells by utilizing similar mechanisms. Hence, protozoa are useful models for studying the pathogenesis of opportunistic, human pathogens. In terms of evolution, single-celled organisms such as protozoans are older than multi-celled organisms such as mammals. Hence the possibility that several mammalian pathogenic bacteria would have evolved from intracellular pathogens within protozoan cells, cannot be ruled out (Montagnes et al. 2012). For example, the interactions of L. pneumophila-mammalian cells and L. pneumophila-protozoa sharing a number of phenotypical and molecular similarities between them demonstrate this fact (Barker and Brown 1994, Fields 1996).
It is now clear that protozoan-bacterial pathogen interactions play an important role in transmission of human disease. This was especially evident when protozoa harbouring L.Â pneumophila were identified as the cause of a Legionnaires' disease outbreak during an American Legion convention in Philadelphia in 1976. A total of 34 fatalities out of 221 cases were reported during that outbreak. Therefore, it is inevitable that studies on bacteria-protozoa interactions can provide crucial steps into possible prevention of infectious diseases.
The amoeba Dictyostelium discoideum is an example of a useful model in the study of human pathogens, including L. pneumophila (Solomon et al. 2000), Neisseria meningitidis (Colucci et al. 2008) as well as Salmonella enterica serovar Typhimurium (Annesley and Fisher 2009). Other protozoan models commonly studied include the ciliate Tetrahymena spp. (Friman et al. 2008), the marine flagellate Oxyrrhis spp. (Montagnes et al. 2011), and also the choanoflagellate Monosiga spp. (Behringer et al. 2009). There are a number of other advantages with using protozoa as a model system, including the ease with the protozoans can grown in large amounts as well as the simple storage and maintenance techniques. In general, protozoa cultures can be maintained on simple, inexpensive media, such as bacterial suspensions. Protozoa cultures may be easily isolated from a variety of natural and artificial environments. Protozoan cells can also be stored as stock cultures over a long period of time, for example by suspending concentrated suspensions of protozoan cells in DMSO with storage at -20°C. These stock cultures can be revived with only a little effort.
Interactions between Pathogenic Bacteria and Protozoa
Intracellular bacterial pathogens of humans that parasitize protozoans exist within a privileged environment, protected from external stresses. Thus bacteria-protozoa interactions are likely to have important ecological as well as public health consequences.
The association of pathogens and protozoa may have contributed to the survival and persistence of bacterial pathogens in various natural and artificial environments. Encapsulation of bacterial pathogens within protozoan cells and provides a protective effect against environmental stress, such as predation, starvation, disinfectants and high temperatures. A number of pathogens can survive for extended periods of time within cysts of protozoan cells, and cannot be detected by methodologies based in culture and cannot be killed by the normal anti-bacterial methods and other adverse environmental conditions (Greub and Raoult 2004). A number of studies have shown that following internalization within protozoans, the pathogens have increased virulence and demonstrate increase pathogenicity following infection of mammalian cells (Rasmussen et al. 2005, Steinberg and Levin 2007, Adiba et al. 2010). The definite reasons for this occurrence remain to be explained. The role of protozoans as possible reservoirs of pathogenic bacteria has been widely studied, yet little is understood about the true nature of the interaction at cellular level (Barker and Brown 1994). Besides acting as a potential reservoir for the maintenance of pathogenic bacteria in the environment, these eukaryotic organisms are also vectors for the transmission of human and animal disease. How this occurs and the role of that process in evolution of these pathogens is not fully known. Following uptake by protozoa, several well-known intracellular bacterial pathogens of humans have been shown to evade digestion by the host and multiply within vacuoles (Rowbotham 1983, Barker and Brown, 1994, Tezcan-Merdol et al. 2004). Maintenance of virulence genes in environmental pathogens has been increasingly understood to be a result of interactions between pathogenic bacteria and soil-borne microorganisms, such as protozoans (Brown and Barker 1999, Adiba et al. 2010, Lamrabet et al. 2012).
L. monocytogenes is a facultative intracellular pathogen that is reported to be isolated from a wide range of environments, including processed foods (Vázquez-Boland et al. 2001). Thus consumption of L. monocytogenes-contaminated food results in human listeriosis, a disease associated with a high mortality rate, particularly in immune-compromised individuals (Schlech 2000). For this reason, food products contaminated by L.Â monocytogenes are a major concern for the food industry and public health authorities alike. However, while it is accepted that L. monocytogenes is ubiquitous, little is known of the ecology of this organism in natural environments. In particular, the role of protozoans as a natural reservoir for L. monocytogenes is not well studied, even though this bacterium has evolved an array of virulence factors that are critical for uptake and establishment of an intracellular lifestyle within mammalian cells (Cossart et al. 2003).
Protozoans are known to feed on bacteria through ingestion by phagocytosis (Barker and Brown 1994). This interaction yields different outcomes depending on the type of bacteria involved. For example, Rowbotham's (1980) landmark study demonstrated that Acanthamoeba polyphaga could harbour replicating Legionella pneumophila. Since that study, a number of published reports have examined the relationship between L.Â pneumophila and a wide number of free-living protozoans. The virulence of L.Â pneumophila was enhanced by intracellular growth in A. castellanii (Cirillo et al. 1999). Furthermore, resuscitation of viable but non-culturable L. pneumophila cells was triggered following ingestion by A.Â castellanii (Steinert et al. 1997). The ability of L. pneumophila to multiply intracellularly within mammalian cells has also been well-documented (Horwitz and Silverstein 1980).
In a separate study, co-culture of Helicobacter pylori with A. castellanii not only resulted in a 100 fold increase of bacterial counts, intact and metabolically active H. pylori were also found located within amoebic vacuoles (Winiecka-Krusnell et al. 2002). Brandl et al. (2005) later showed survival of Green Fluorescent Protein (GFP) labelled S. enterica serovar Thompson cells located in vesicle-like structures secreted by both starved and fed Tetrahymena cells. Similarly Mycobacterium avium was shown to be able to survive within the outer walls of Acanthamoeba polyphaga cysts (Steinert et al. 1998). Interestingly, S.Â enterica and E. coli O157:H7 cells encapsulated within expelled protozoan vesicles are able to multiply and exit from these structures (Gourabathini et al. 2008). These observations suggested that the vesicle-like structures may provide a protected environmental reservoir for pathogenic bacteria.
Zhou et al. (2007) showed that L. monocytogenes had no predatory effect on A.Â castellanii, and the presence of amoebae can actually enhance the growth of the bacteria. However, a study by Akya et al. (2009) provided data that showed co-cultures of L.Â monocytogenes with Acanthamoeba spp. resulted in active phagocytosis and killing of the bacteria by these amoebae within 5 h of feeding.
In both natural and artificial habitats such as rivers, drainage systems and water distribution systems, bacteria are typically present in planktonic and biofilm communities (Barker and Brown 1994). Biofilms represent a mode of growth for bacteria including mammalian bacterial pathogens. This environment comprises extracellular products, and inorganic and organic debris that promote biofilm bacteria survival during exposure to stressors, such as UV exposure (Espeland and Wetzel 2001), dehydration and salinity (LeMagrex-Debar et al. 2000), antibiotics (Mah and O'Toole 2001) as well as grazing by protozoa (Snelling et al. 2006). Natural bacterial biofilms are typically colonized by amoebae, flagellates and ciliates (Weitere et al. 2003, Parry 2004). However, it is not known whether different strains of bacteria respond differently at the physiological level to grazing by protozoa. Huws et al. (2005) showed that A. castellanii and the ciliate Colpoda maupasi, were able to graze on biofilm material. Grazing protozoa can have a significant impact on the integrity and composition of biofilm communities. Grazing, for example, results in reduction of biofilm biomass and may cause rapid changes in morphological and taxonomical community composition in areas where planktonic bacteria are present (Hahn and Hofle 2001, Jurgens and Matz 2002). In this context, Jackson and Jones (1991) showed that amoebae were able to graze biofilm bacteria to a point where the biofilm disruption was accompanied by sloughing of the biofilm structure.
When protozoa are not present, L. pneumophila has been shown to persist and remain viable for up to 15 d within artificial biofilms that were made using filter-sterilized tap water or distilled water (Lau and Ashbolt 2009). However, other studies have provided evidence that 30% - 40% of biofilm samples isolated from sources such as hospital water supplies, dental units and taps showed presence of Acanthamoeba spp. (Barbeau and Buhler 2001, Carlesso et al. 2007). Though biofilms have generally been shown to provide protection against predation by protozoans, one study has demonstrated that the effect of protozoan grazing on the biofilm community is a crucial factor that controls the biofilm composition in aquatic ecosystems (Pedersen 1990). Furthermore, biofilms may also be able to restrict predation by protozoans as biofilm cells have been shown to secrete defensive factors that inhibit predation by either protozoan or bacterial competitors (Matz and Kjelleberg 2005).
A number of studies have demonstrated that mitochondria are recruited to vacuoles containing intracellular pathogens (Horwitz 1983, Sinai et al. 1997, Chong et al. 2009). Recruitment of mitochondria to macrophage phagosomes resulting in an upregulation of mitochondrial reactive oxygen species (mROS) for bacterial killing by the mitochondrial pathway was shown in a previous study (West et al. 2011). Factors that might promote survival include traits of the bacteria as well as of the host. Other factors may be serotype specific. It is known that different serotypes of L. monocytogenes exhibit different levels of pathogenicity. Of the 13 serotypes of L. monocytogenes that can cause disease, more than 90% of human isolates belong to only three serotypes: 1/2a, 1/2b, and 4b. Serotype 4b isolates are responsible for 33% - 50% of sporadic human cases worldwide (Ward et al. 2004). Sandgren et al. (2005) has shown that following inoculation of mice models with six serotypes of Streptococcus pneumoniae known to cause invasive disease in humans, differing levels of host immune response was observed, suggesting that invasive disease caused by different serotypes may result in different degrees of host response. Different serotypes of S.Â pneumoniae even exhibit variations in resistance to phagocytosis (Guckian et al. 1980) as well as vary in the activation of the alternative complement pathway (Fine 1975). The nature of the disease caused by S. Typhimurium is suggested to be largely dependent on the specific serotype-host combination (Watson et al. 1999). Another study has shown that even among strains of S. pneumoniae of the same multilocus sequence type (ST) and serotype, differences in the virulence of these strains in a mouse infection model were observed; and these differences were attributed to the genetic differences between the bacterial strains of the same serotype (Silva et al. 2006). Listeria monocytogenes is a facultative intracellular pathogen capable of survival and proliferation within mammalian cells such as macrophages and epithelial cells (Vázquez-Boland et al. 2001). As a food-borne pathogen, L. monocytogenes is the causative agent of human listeriosis that occurs following consumption of contaminated foods such as dairy and processed food. Upon entering a mammalian host cell via a passive or host-induced phagocytosis, L. monocytogenes is enclosed within phagosomes. At this stage, the host acidifies the phagosome to eliminate invading bacteria. In this acidic environment, Listeriolysin O secreted by the bacterial cells is activated, interacts with the phagosomal membrane. This interaction leads to destabilization of the membrane and eventual escape into the nutrient-rich cytosol of the host cell. Upon entering the host cytosol, L. monocytogenes begins to divide and express ActA (Tilney and Portnoy 1989), which in turn leads to recruitment of host cell proteins that are essential in the formation of actin tails and actin-based motility (Vázquez-Boland et al. 2001). Actin-based motility within the cytoplasm results in migration of bacterial cells to adjacent uninfected host cells via listeriopods (double- membraned phagosome). An infectious cycle is completed upon escape of L. monocytogenes from the listeriopods within the new host cell (Vázquez-Boland et al. 2001).
Mammalian hosts employ a wide array of anti-microbial mechanisms to kill intracellular pathogens. Examples of these mechanisms include phagosome-lysosome fusion and vacuole acidification (Flannagan et al. 2009), proteases (Pham 2006) and production of nitric oxide (Vouldoukis et al. 1995, Nathan and Shiloh 2000, Bekker et al. 2001). Although most types of bacterial cells are successfully internalized and eliminated by host mammalian cells, several intra-cellular pathogens have evolved an array of methods to escape host defence mechanisms in order for intracellular survival within host mammalian cells. For example, Legionella pneumophila is known to survive and replicate within professional phagocytes by redirecting the maturation of phagosomes to create an intracellular environment that is conducive for intracellular bacterial replication (Hart et al. 1987), whereas several species of mycobacterium are capable of preventing phagosome-lysosome fusion within macrophages (Brüggemann et al. 2006). Furthermore there is evidence that L. monocytogenes inhibits host-induced apoptosis in macrophages (Barsig and Kaufmann 1997), induces a delay in host phagosome maturation (Henry et al. 2006), as well as inhibiting phagosome-lysosome fusion within macrophages (Bouvier et al. 1994). These reports indicate that L. monocytogenes engages several protective mechanisms to avoid, or delay, killing by host cells.
Phagosome-lysosome Fusion mediates killing of L. monocytogenes
The ability of L. monocytogenes to survive within intracellular compartments within mammalian cells has been clearly demonstrated (Dramsi et al. 1993, Wood et al. 1993, Francis and Thomas 1996). However, this study has demonstrated that Colpoda RR efficiently inactivates L. monocytogenes within hours of feeding on this bacterial pathogen. Indeed, Colpoda RR are able to feed on planktonic DRDC8 cells as well as graze efficiently on biofilms of DRDC8 (Chapter 2). Furthermore, the fact that DRDC8 cells are unable to invade ColpodaÂ RR cells when phagocytosis is inhibited by the K+ efflux inhibitor tetraethylammonium chloride (see Chapter 2) indicated that internalization of these bacteria within vacuoles was mediated by Colpoda RR. This conclusion contrasts with the well established InlA and InlB mediated invasion of mammalian cells by DRDC8.
Phagosome-lysosome fusion and vacuole acidification are important in degradation of phagocytosed bacterial cells by mammalian cells (Styrt and Klempner 1988, Huynh and Grinstein 2007). Bacterial cells are phagocytosed into an intracellular vacuole where microbicidal agents are deployed by the maturation process. During maturation, acidification of the vacuoles occurs due to presence of V-ATPases, following phagosome-lysosome fusion (Huynh and Grinstein 2007). Acidification of vacuoles results in the lytic activity of a variety of degradative enzymes such as proteases, as well as promoting the generation of hydrogen peroxide (Huynh and Grinstein 2007). In this study, acridine orange as well as the fluid phase probe Lysosensorâ„¢ Blue DND-167 were used to characterize the DRDC8-containing vacuoles within Colpoda RR. Acridine orange  has been shown to concentrate within lysosomes, becoming protonated and sequestered, causing them to appear as bright orange granules in fluorescence microscopy, whereas Lysosensorâ„¢ Blue DND-167 is a non-fluorescent probe compound that freely diffuses through the membrane and becomes highly fluorescent at an acidic pH (ca. < 5.0). Consequently this probe is useful for identifying trafficking of acidic vacuoles. In the context of this study, this probe was used to show that DRDC8-containing vacuoles within Colpoda RR fused with small acidic vesicles (presumably lysosomes), an outcome mirrored in Colpoda RR stained with acridine orange. As expected, the content of the larger vesicles became acidic shortly after phagosome-lysosome fusion. These results support the idea that for Colpoda at least, lysosome mediated acidification of vacuoles containing bacteria is a likely precursor to killing of bacteria and digestion. In this respect, the process mimics well-described cellular processes used by mammalian phagocytic cells. The fact that killing of intra-ciliate DRDC8 cells, was impaired by inhibitors that target phagosome-lysosome fusion (NH4Cl) and vacuole acidification (bafilomycin A1 and monensin), lends strong support for the role of these cellular processes in Colpoda.
Ammonium chloride is a weak base that interferes with the maturation of the phagosome and as a consequence leads to inhibition of phagosome-lysosome fusion (Hart and Young 1991). However there are instances of contradictory outcomes reported that concern survival of bacteria within NH4Cl-treated host cells. For example, Harley and Drasar (1989) reported that L.Â pneumophila within NH4Cl-treated HeLa cells were able to survive and replicate within intracellular compartments, but Byrd and Horwitz (1991) later showed that L.Â pneumophila survived without replication within treated human monocytes. Nevertheless, in the current study, this weak base clearly blocked maturation of the phagolysosome to the point where DRDC8 was able to survive within Colpoda vacuoles, although no evidence for growth and cell division was detected.
The involvement of acidification of the phagosome following fusion with lysosomes, in killing of intra-ciliate DRDC8 was confirmed by use of neutralizing inhibitors such as bafilomycin A1 and monensin. Bafilomycin A has a membrane-permeant activity and specifically inhibits vacuolar-type proton translocating ATPases (V-ATPases) that are involved in vacuole acidification within treated cells (Conte et al. 1996), whereas monensin intercalates into vacuole membranes and mediates the exchange of monovalent cations through the membrane resulting in the increase of the vacuolar pH (Nakazato and Hatano 1991). The effect of these inhibitors on survival of DRDC8 within Colpoda cells is directly comparable with studies that report impaired killing of bacteria within macrophage cell lines. For example, killing of intra-amoebic L. monocytogenes by A. polyphaga was impaired following treatments with both bafilomycin A1 and monensin (Akya et al. 2009). Furthermore, bafilomycin A1 treatment of murine RAW 264.7 macrophages inhibited killing and degradation of phagocytosed E. coli (Frankenberg et al. 2008). Similarly, inhibition of acidification of Staphylococcus aureus-containing vacuoles also resulted in impaired bacterial killing by alveolar macrophages (Bidani et al. 2000). Di et al. (2006) has demonstrated the use of alveolar macrophages that carried cystic fibrosis transmembrane conductance regulator chloride channel mutations to directly implicate the role of vacuole acidification in the killing of bacteria.
Exactly why DRDC8 is able to survive within inhibitor-treated Colpoda RR, but is unable to replicate, is unclear. Several reports have suggested that survival without replication of M.Â avium or L. pneumophila within activated macrophages is due to replication restriction mediated by enhanced phagosome-lysosome fusion (Schaible et al. 1998, Santic et al. 2005). However this is an unlikely explanation for Colpoda cells treated with inhibitors designed to prevent fusion of lysosomes with the phagosome. An alternative, and more likely reason to explain this observation is that inhibition by NH4Cl, bafilomycin and monensin was sufficient to allow survival, but insufficiently complete to allow growth of survivor L.Â monocytogenes cells within the phagosome. Other intact antibacterial mechanisms may also have been sufficient to repress growth without affecting survival. Furthermore, as Listeriolysin O expression is reduced at temperatures used for co-culture (Datta and Kothary 1993), L.Â monocytogenes would not be able to mediate destruction of the ciliate vacuole membrane to escape and replicate within the cytosol of Colpoda RR.
Proteases assist killing of L. monocytogenes
In addition to the role of lysosome fusion with phagocytic vacuoles and vacuolar acidification, a variety of endopeptidases (cysteine and aspartate proteases), exopeptidases (cysteine and serine proteases) and hydrolases located within phagosomes are also reported to degrade bacterial components within the phagosomes (Pillay et al. 2002). Substrates for the exopeptidases are generated by the C1 family of cysteine proteases of endopeptidases (Pillay et al. 2002).
Since the experimental data presented confirmed that impairment of proteases of Colpoda RR by protease inhibitors allowed the survival of DRDC8 within the ciliates, this outcome indicates that endo- and exo-peptidase of Colpoda RR are likely to be a factor involved in the killing and degradation of internalized DRDC8. Since the serine and cysteine protease inhibitors in the cocktail used in this study would have likely impaired the endopeptidases, the activity of exopeptidases would have also been limited as the endopeptidase activity is required to generate substrates for the exopeptidases. Although the effect of protease inhibitors on survival of DRDC8 within Colpoda cells is novel for work with bacteriophagous protozoans, previous studies have highlighted the antibacterial role of serine proteases within mammalian cells. For example, Rosenberger et al. (2004) showed that protease regulation of macrophage cathelicidin-related antimicrobial peptide (CRAMP) expression and activity impaired the replication of intracellular S.Â Typhimurium. Similarly, Standish and Weiser (2009) demonstrated that inhibition of neutrophil granule proteases by serine protease inhibitors resulted in survival of Streptococcus pneumoniae within neutrophils. Whether serine proteases (or other proteases) in particular are involved in limiting the bacteriocidal response to L. monocytogenes within Colpoda cells is unknown. This question could easily be resolved by treatment of Colpoda cells used in co-cultures with specific protease inhibitors.
L. monocytogenes is insensitive to Nitric Oxide
Nitric oxide is a toxic radical synthesised from L-arginine by inducible nitric oxide synthase (iNOS). It is a central component of the mammalian innate immunity and an important bactericidal agent (Nathan and Shiloh 2000) central to killing ingested pathogens by activated macrophages (Nathan 1997). Intracellular pathogens such as Mycobacterium tuberculosis and Leishmania major are killed by a nitric oxide-dependent mechanism (Vouldoukis et al. 1995, Bekker et al. 2001). Many other published studies implicate nitric oxide produced by mammalian host cells in the killing of internalized bacteria. One of these studies has for example, showed that L-NMMA inhibition of nitric oxide synthesis within activated macrophages correlates with loss of control of intracellular replication of Rhodococcus equi (Darrah et al. 2000). This type of study implicates nitric oxide as a mediator of the killing of intracellular R. equi by activated macrophages. Furthermore, an increased S.Â Typhimurium burden was detected within reticuloendothelial organs of iNOS mutant mice (Mastroeni et al. 2000). Increased levels of nitric oxide production were detected within peritoneal macrophages in response to interferon gamma in a separate study (Vazquez-Torres et al. 2000). In that study, the elevated levels of NO appeared to increase antibacterial activity against S.Â Typhimurium.
A recent report by von Bargen et al. (2011) indicated that following infection with R. equi, macrophages produced ca. 10 ÂµM NO at 24 h post infection. Summersgill et al. (1992) showed L.Â pneumophila were killed by nitric oxide released by murine macrophages, and the level of nitrite measured at 24 h post infection was ca. 58.6 ÂµM. However, the data presented in this present study mirrors the outcome of a separate study by Beckerman et al. (1993) that showed killing of L. monocytogenes occurred even after treatment of activated macrophages with L-NMMA. The outcome of this present study could be an indication that though Colpoda RR does produce low levels of NO, the ciliate does not employ it as a mechanism of defence against phagocytosed pathogens.
NADPH oxidase within hosts is activated upon phagocytosis, resulting in the generation of intracellular superoxide (Takeya and Sumimoto 2003). The superoxide plays an important role in the oxygen-dependent antibacterial mechanisms of phagocytic cells (Fang 2004). Phagocytic cells are known to use a combination of oxidative mechanisms, including superoxide in addition to non-oxidative mechanisms such as pH, to defend against the wide range of phagocytosed pathogenic bacteria (Roos and Winterbourn 2002). It has been previously established that the NADPH oxidase is important for eliminating L.Â monocytogenes within host cells (Shiloh et al. 1999). The outcomes of the present study that showed less superoxide production by Colpoda RR following feeding with DRDC8 in comparison to control co-cultures with E. coli DH5Î± and heat-killed DRDC8 controls mirrored the outcome of a separate study that demonstrated macrophages produced less superoxide following Burkholderia cenocepacia infection as compared to heat-inactivated B.Â cenocepacia and E. coli controls (Keith et al. 2009). Interestingly, a study provided evidence of Listeriolysin O inhibiting host NADPH oxidase by preventing its localization to phagosomes hence allowing the bacterium to escape the phagosome while avoiding the microbicidal respiratory burst (Lam et al. 2011). Inhibitor mediated uptake of L. monocytogenes by Colpoda
Phagocytosis is the primary mechanism used by ciliates for the uptake of bacteria. An invagination pinches off to form a large endocytic vesicle (phagosome) containing the ingested material (Alberts et al. 1994). This process is called endocytosis. The phagosome travels within the cell and ultimately fuses with a lysosome. The ingested substance is then digested (Alberts et al. 1994). A significantly reduced uptake of bacteria by all inhibitor-treated ciliates (except L-NMMA) compared to untreated ciliates seemed to indicate that phagocytosis of DRDC8 by treated Colpoda RR was affected by the inhibitory effects of the chemicals. This phenomenon has been a feature of a number of previous studies. One of the studies showed that besides inhibiting vacuole acidification, bafilomycin A1 also conferred inhibitory effects on the uptake of E. coli by alveolar macrophages (Bidani and Heming 1995). Treatment of A. polyphaga with monensin also resulted in a reduced uptake of L.Â monocytogenes by the amoeba compared to untreated control amoeba cells (Akya et al. 2009). One reason for this effect may be related to reduced Ca2+ levels within Colpoda cells. Calcium (Ca2+) is regarded as a requirement for phagocytosis (Peck and Duborgel 1985) and vacuole acidification (Downey et al. 1999), although a few other studies have reported conflicting outcomes concerning the importance of Ca2+ for phagosome-lysosome fusion (Jaconi et al. 1990, Zimmerli et al. 1996, Dewitt and Hallett 2002). Importantly, increases in vacuole pH within macrophages, resulting from treatment by either NH4Cl or bafilomycin A1 result in several fold decreases in Ca2+ concentrations within the macrophage cells (Christensen et al. 2002). Thus, it seems likely that reduced levels of endogenous Ca2+ brought about by NH4Cl, bafilomycin and monensin treatment, are responsible for reduced uptake of L. monocytogenes by treated Colpoda cells. The AS buffer used to suspend Colpoda cells has sufficient concentrations of Ca2+ to promote active phagocytosis and phagosome-lysosome fusion within Colpoda RR in co-cultures with DRDC8. This suggests that the effects of the inhibitors used is localized within cells and is likely to be transitory and dependent on continued presence of those inhibitors. However, it is not known why treatment of the ciliates with protease inhibitor cocktail also resulted in a reduced bacteria uptake, although it is interesting to speculate that this effect may be caused by the effect of proteases on signalling cascades necessary for phagocytosis.
When phagocytic cells "feed" on bacterial cells, the host phagosome undergoes a maturation process in which actin undergoes depolymerisation (Bengtsson et al. 1993), enabling fusion of the phagosomes with lysosomes (Flannagan et al. 2009). The phagosome-lysosome structure formed is an acidic, hydrolytic compartment in which the bacterial cell may be killed and digested. Defensins, cathelicidins, lysozymes, and a variety of proteases are recruited by the phagosome to kill the pathogen (Flannagan et al. 2009). It was interesting to note that inhibition of a single mechanism within Colpoda RR resulted survival of DRDC8 within the ciliate. This observation indicated that killing of L.Â monocytogenes by Colpoda RR cannot be attributed to any one single mechanism. Phagocytosis and killing of bacteria by mammalian cells is a pathway that consists of a cascade of defined processes. Survival of bacteria within mammalian cells following inhibition of either one of these processes has been well established for mammalian cells. Interference with the uptake of bacteria such as Pseudomonas aeruginosa and Yersinia enterocolitica by macrophages and neutrophils by direct impairment of the phagocytic machinery of the host has been previously demonstrated (Garrity-Ryan et al. 2000, Grosdent et al. 2002). Other types of bacteria such as Mycobacterium tuberculosis and S. Typhimurium have strategies to either counteract the acid accumulation within phagolysosomes, or have acquired genes that encode proteins that assist the bacteria to withstand the low pH within phagolysosomes (Park et al. 1996, Vandal et al. 2008). P. aeruginosa, Enterococcus faecalis, Proteus mirabilis and Streptococcus pyogenes have been shown to protect themselves by actively degrading antimicrobial peptides such as proteases that are recruited by the phagosome (Schmidtchen et al. 2002), whereas Staphylococcus aureus are able to resist the effects of phagosome-recruited antimicrobial peptides (Peschel et al. 2001). Hence it is not unusual to note the intra-ciliate survival of DRDC8 when one of the ciliate defence mechanisms was inhibited in this present study. Furthermore, it also shows that the Colpoda RR defence mechanism machinery is similar to that seen in mammalian cells.
As a pathogen of both humans and animals, Listeria monocytogenes is one of the most virulent and ubiquitous pathogens as this facultative intracellular bacterium is well adapted to survive in the environment. As the etiological agent for listeriosis, L. monocytogenes causes infection when contaminated food is ingested (Cossart and Bierne 2001). By comparison with the large number of published reports concerning L.Â monocytogenes, there have been few studies dedicated to the ecology of L. monocytogenes in natural environments. In particular, the role of protozoans as a potential reservoir for L.Â monocytogenes has not been well studied, even though this bacterium has evolved an array of virulence factors that are important for invasion of cells and establishment of an intracellular lifestyle within mammalian cells (Cossart et al. 2003). L. monocytogenes has shown to have parasitizing effects on mammalian cells (e.g. HeLa and Caco-2 cell lines) and is pathogenic in mice (Francis and Thomas 1996). By contrast, previously demonstrated outcomes of co-cultures of L.Â monocytogenes with Acanthamoeba spp. have shown otherwise. Akya et al. (2010) showed that amoebae are able to actively phagocytose and kill bacteria located in phagolysosomal compartments within 2 h post ingestion. Interestingly, several groups have reported secretion of pathogenic prey bacteria from protozoan cells within membrane bound faecal pellets. For example, Rowbotham (1980 and 1986) described release of vesicle-like structures (faecal pellets) that contained an infectious dose of L. pneumophila. Following predation and phagocytosis by amoebae, the bacteria were released within pellets. These pellet associated bacterial cells were in addition to those released from lysed amoeba cells. Encapsulation of viable L. pneumophila cells within faecal pellets has also been confirmed for other protozoan hosts, such as Tetrahymena spp. (McNealy 2001) as well as A. polyphaga and A. castellanii (Berk et al. 1998). A separate study went further to show that L. pneumophila located within faecal pellets of A. castellanii were able to survive to up to 6 months within medium that had a poor nutrient availability (Bouyer et al. 2007). Furthermore, co-cultures of T. pyriformis with strains of Escherichia coli resulted in secretion of faecal pellet-like structures that contained viable, culturable bacteria (Schlimme et al. 1997). Brandl et al. (2005) also showed that up to 50 viable S. enterica cells are present per faecal pellet secreted by Tetrahymena sp. However, Gourabathini et al. (2008) demonstrated that while Tetrahymena sp. released faecal pellets containing E. coli O157:H7 and S. enterica following grazing on these bacteria, this process did not occur when the ciliates grazed on L. monocytogenes.
Encapsulation of bacterial cells within membrane bound pellet-like structures presents these cells with a significant advantage in terms of survival in the presence of antibacterial agents. Berk et al. (1998) showed that faecal pellet encapsulated L.Â pneumophila cells retained viability following exposure to biocides (e.g. free chlorine, and which others) as well as cycles of freeze-thaw treatment. Similarly, S. enterica cells contained within Tetrahymena sp. faecal pellets exhibit higher resistance to low concentrations of chlorine in comparison to free-swimming bacteria (Brandl et al. 2005). However, to date there is no unequivocal evidence to support the contention that L. monocytogenes is either secreted within faecal pellet structures by predatory protozoans, or that faecal pellet-associated cells are more resistant to bactericidal agents than individual cells.
Rowbotham (1980) was the first to provide evidence of amoebae as a vector of Legionella spp. More recently, Berk et al. (1998) demonstrated Acanthamoeba spp. expelled faecal pellets that contained bacterial cells into the extracellular environment. Since then, only a few published studies have demonstrated faecal pellet production by other protozoans following co-culture with different species of bacterial pathogens.
The genus Colpoda belongs to the class Colpodea, and is usually 20 - 50 Î¼m in length with a reniform shape. Colpoda ciliates feed on bacteria as the major food source. Like L.Â monocytogenes, Colpoda can be found in a variety of environments, especially in soil and vegetation. For this reason, Colpoda may be a good model organism to analyze interactions between L. monocytogenes and protozoans. In this study, we have demonstrated that Colpoda ciliates secrete faecal pellets containing live L.Â monocytogenes DRDC8 cells following co-culture. This outcome is unique, as previous studies have shown the inability of other protozoa species to secrete faecal pellets containing bacteria following co-cultures with L.Â monocytogenes. The amoeba A.Â palestinensis and the ciliate T. pyriformis did not produce any faecal pellets following feeding with L. monocytogenes (Brandl et al. 2005, Gourabathini et al. 2008). The ciliate Glaucoma sp. produced only a few faecal pellets following co-culture with L. monocytogenes (Gourabathini et al. 2008). Brandl et al. (2005) showed that Tetrahymena sp. strain MB125 expelled only few faecal pellets following co-culture with L.Â monocytogenes, and these faecal pellets contained only few cells of L. monocytogenes. It is interesting that although the present study provided clear evidence to show Colpoda RR and MLS-5 secreted faecal pellets containing bacteria following co-cultures with L.Â monocytogenes and S. Typhimurium, Gourabathini et al. 2008 reported that Colpoda steinii did not secrete any faecal pellets containing bacteria following co-culture with
these bacteria-containing faecal pellets should still be considered as a possible clinically important vector of disease transmission.
It has been well documented that host protozoans may enhance survival of bacterial pathogens within protozoans by protecting bacteria from exposure to disinfectants and sanitizers. Pathogens such as S. Typhimurium and Campylobacter jejuni have demonstrated resistance to free chlorine following ingestion by T. pyriformis (King et al. 1988). C. jejuni present within T.Â pyriformis and A. castellanii also exhibit increased resistance to Virudine, a disinfectant commonly used in the poultry industry (Snelling et al. 2005). Furthermore, Adekambi et al. (2006) reported that as many as 26 species of water-associated mycobacteria cells that were encapsulated within cysts of A.Â polyphaga were able to survive up to 24 h exposure to free chlorine. Bacteria encapsulated within faecal pellets secreted by protozoans have also been demonstrated to be resistant to biocidal agents. Brandl et al. (2005) for example, reported that S. Thompson cells located within faecal pellets of Tetrahymena sp. were protected during treatment with calcium hypochlorite (Ca(ClO)2) concentrations of 0.13 and 0.42%. Similarly, Berk et al. (1998) showed that L.Â pneumophila cells within faecal pellets of Acanthamoeba spp. are resistant to external stresses such as cooling tower biocides, freeze-thawing and sonication processes. Consequently it is not surprising that DRDC8 cells encapsulated within faecal pellets of Colpoda RR and MLS-5 are resistant to concentrations of gentamycin (0 - 100 Âµg mL-1) and NaOCl (0 - 10%) that are normally lethal to washed cell suspensions of this pathogen. The membranes that make up the faecal pellets may either act as a barrier to prevent the permeation of the biocides into the pellets, and/or organic materials present within the pellets may inactivate or prevent the biocide molecules damaging the bacterial cells. The remarkable resistance of these pellet-located DRDC8 cells to these otherwise biocidal concentrations of disinfectants indicates that the faecal pellets are likely to be an excellent protective shield for the bacteria in the presence of disinfectants and cleaning agents used in food processing environments.