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As one of the six species of the genus Listeria, L. monocytogenes belongs to a genus of the Corynebacteriaceae household that comprises of L. monocytogenes, L. grayi, L. innocua, L.A ivanovii, L. seeligeri, and L. welshimeri. While L. monocytogenes is infective to both worlds and animate beings, L. ivanovii is merely infective to animate beings, chiefly sheep and cowss. The remainder of the Listeria species are known non to do any disease ( Cossart 2007 ) .

L. monocytogenes shows opposition to extreme environmental conditions. For illustration, high concentrations of salt ( up to 10 % NaCl ) , a broad pH scope ( 4.5 – 9 ) every bit good as low temperatures ( Vazquez-Boland et al. 2001 ) . From a clinical position, L. monocytogenes is besides able to colonise and prevail in the gall bladder, which has a high pH content ( Begley et al. 2009 ) . This determination suggests the happening of both long-run and chronic infections, every bit good as the ability of the bacteria to last within the microenvironments of the GI piece of land. Though the optimal growing temperature scope for L.A monocytogenes growing is between 30A°C – 37A°C, non merely does growing has been shown to happen between 3A°C – 45A°C ( Junttila et al. 1988 ) , the bacterium has been demonstrated to turn at temperatures between 1A°C – 4A°C ( Farber and Peterkin 1991 ) . This adaptability of L. monocytogenes to a assortment of temperatures suits it for endurance and growing in either processed or refrigerated nutrient points.

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Though L. monocytogenes is omnipresent in the environment, this bacteria is most normally found in disintegrating flora and besides dirt ( Weis and Seeliger 1975 ) . Following consumption of L.A monocytogenes by a susceptible individual, the bacteriums can do the passage from being a saprophytic organism to a parasite that promotes the endurance and reproduction of the bacteria within host cells ( Freitag 2006 ) . L. monocytogenes has been demonstrated to be capable colonising a assortment of inert surfaces, every bit good as able to organize biofilms on food-processing surfaces ( Roberts and Wiedmann 2003 ) . The presence of L.A monocytogenes in legion environments such as farms, dirt, H2O, silage produced from contaminated grasses and besides nutrient processing installations, indicates that there L. monocytogenes has many chances to pollute the nutrient production procedure ( Cossart and Bierne 2001 ) .

Listeriosis

Though human listeria meningitis eruptions following consumption of L. monocytogenes contaminated nutrient points have been reported antecedently, merely late the bacteria has been recognized as a major cause of human infections ( Lecuit 2007 ) . This is due to increased Numberss of susceptible, immuno-compromised persons, the addition in large-scale agro-industrial works development, every bit good as an increased trust on refrigerated nutrient points.

Fresh veggies are an illustration of beginnings of contaminations ; it can be contaminated from the dirt or from manure that were originally used as fertilisers and nutrient. Animals may besides transport the bacteria asymptomatically and pollute nutrients of carnal beginning. Other illustrations of nutrient points that are most linked to listeriosis outbreaks include ready-to-eat meats, undercooked meats, cold cuts, pate ( McLauchlin et al. 1991 ) , salads, dairy merchandises, particularly 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 Worlds

A old survey has demonstrated that symptomless passenger car of L. monocytogenes does happen within the enteric piece of land of 5 % or more of healthy worlds ( Grif et al. 2003 ) . However highly rare infections besides occur in healthy grownups and immature kids, where there is increasing grounds in recent old ages to turn out that listeria meningitis may besides happen in healthy persons within merely 24 h station consumption of extremely contaminated nutrient ( Swaminathan and Gerner-Smidt 2007 ) .

Those most at hazard from listeria meningitis are by and large immuno-compromised persons such as diabetics, AIDS patients, those with nephritic failure, organ graft patients, malignant neoplastic disease patients and besides aged grownups. Diarrhoea is normally an early symptom of the infection. Advanced symptoms in these susceptible persons include blood poisoning or cerebromeningitis. The chief clinical characteristics of L.A monocytogenes meningitis are unnatural motions, ictuss, every bit good as change of consciousness. L. monocytogenes meningitis instances have been shown to hold the highest mortality rate ( 22 % ) in comparing to all types of bacterial meningitis ( Lecuit and Cossart 2001 ) . Certain patients are known to see rare and localised infections, for illustration due to direct vaccination of the bacteria ( Schlech 2000, Lecuit and Cossart 2001 ) .

Others at high hazard of the disease besides include pregnant adult females, fetus and besides neonates ( Vazquez-Boland et al. 2001 ) . Due to the fact that pregnant adult females have a of course depressed, cell-mediated immune system ( Weinberg 1984 ) , pregnant adult females is more likely to get listeria meningitis upon station ingestion of contaminated nutrient compared to other persons. By deriving entree to the maternal circulation, L. monocytogenes colonizes the placenta to bring on placentitis, infecting the defenseless fetus. As opposed to maternal unwellness, the badness of infection within fetal and neonatal is much higher. The common fatal symptoms include pre-term labor, amnionitis, self-generated abortion, spontaneous abortion every bit good as early oncoming sepsis ( Vazquez-Boland et al. 2001 ) .

The happening of human death due to listeria meningitis is go oning to diminish in industrialised states in recent old ages. This has been attributed to the stricter execution of nutrient quality and safety ( Allerberger and Wagner 2010 ) .

Listeriosis in Animals

Assorted species of animate beings can be infected with L. monocytogenes, but clinical disease is rare. The bacteria can besides populate within the bowels of healthy animate beings without doing any infections. Though most instances of carnal listeria meningitis are by and large seen in ruminants, this disease can besides happen in domestic fowl and other birds, hogs, Canis familiariss, cats, domestic and wild coneies, and many other little mammals. Infected ruminants have been shown to see phrenitis, blood poisoning, and even abortions ( Schoder et al. 2003 ) . The class of disease in sheep and caprine animals is more rapid and decease may happen 24 – 48 H upon the oncoming of symptoms. In cattle nevertheless, the class of disease is less acute.

Many L. monocytogenes-infected animate beings excrete the bacteria in fecal matters and milk. This is a common beginning of animate being to carnal spread of infection. Grass silage is presumed to be the beginning of infection, as it can be contaminated with big Numberss of L. monocytogenes. This is because the low pH of silage can heighten the growing of L monocytogenes cells ( Pham 2006 ) . Besides silage, the bacteria has besides been isolated from other beginnings such as H2O troughs, manure, dirt and animate being provenders. L. monocytogenes infection may besides do mastitis in cowss and sheep ( Wagner et al. 2005 ) .

In ruminants such as sheep, infections that lead to lesions in the encephalon root, consequence in characteristic clinical symptoms ( Rebhun and deLahunta 1982 ) . Typical symptoms of listeria meningitis in ruminants include turning or distortion of the caput to one side and walking in circles, drooping of the eyelid and ear caused by palsy of the one-sided facial nervus. The septic ruminant may besides salivate saliva as a consequence of partial guttural palsy ( Rebhun and deLahunta 1982 ) .

Animals that excrete L. monocytogenes cells in fecal matters have been suggested as the primary cause of entry of this pathogen into food-processing workss ( Schonberg and Gerigk 1991 ) . The growing and generation of L. monocytogenes cells is normally promoted by non merely the high humidness, but besides the food rich waste nowadays within certain nutrient production workss. Hence, it is non surprising that carnal listeria meningitis does present a serious taint hazard for the nutrient industry in general.

Pathophysiology of L. monocytogenes

L. monocytogenes is normally ingested with contaminated nutrient ( Mistake: Reference beginning non found ) . In immuno-compromised persons, L. monocytogenes invades the epithelial cells of the bowels and spreads to other parts of the organic structure by cell-to-cell spread. L. monocytogenes secretes invasins ( InlA + InlB ) to enable it to perforate cells of the enteric epithelial liner ( Gaillard et al. 1987, Mengaud et Al. 1996 ) . L. monocytogenes cells that cross the enteric epithelial barrier are so carried by the lymph or blood to the mesenteric lymph nodes, the lien, and besides the liver ( Marco et al. 1992, Pron et Al. 1998 ) . Entry into the host ‘s monocytes, macrophages, or polymorphonuclear leucocytes promotes growing of L. monocytogenes, and the infection becomes blood-borne ( septicemic ) airing.

L. monocytogenes so enters the liver after the enteric translocation and passenger car by the blood stream ( Marco et al. 1992, Dramsi et Al. 1998 ) . Hepatocytes are by and large the chief site of L. monocytogenes generation within the liver ( Vazquez-Boland et al. 2001 ) . When there is an unequal immune response by the host, L. monocytogenes normally multiplies unlimitedly within the liver parenchyma, perchance let go ofing the bacterium into blood to do bacteriemia. Via the blood stream, L. monocytogenes cells will make the encephalon in order to traverse the blood-brain barrier ( Kirk 1993 ) . High degrees of L. monocytogenes cells in the encephalon accompanied by bacteriemia will by and large ensue in cerebromeningitis ( Tunkel and Scheld 1993, Tuomanen 1996 ) .

In pregnant adult females, L. monocytogenes normally additions entree to the fetus by come ining the endothelial bed of the placental barrier ( Gray and Killinger 1966 ) . The bacterial cells will make the blood stream of the fetus by foremost colonising the trophoblast bed. The bacterium so will make the blood stream of the fetus by translocating across the endothelial barrier. This will normally ensue in infection and the possible subsequent decease of the fetus within the womb, or on occasion even the premature birth of a badly septic newborn ( Vazquez-Boland et al. 2001 ) .

Virulence Factors of L. monocytogenes

A broad array of virulency factors is wielded by L. monocytogenes to help the bacteria to interact and pull strings the host cells. Virulence cistrons of L. monocytogenes are known to be optimally expressed at 37A°C, but looks about does non happen at 30A°C ( Freitag et al. 2009 ) . The cardinal transcriptional activator of L. monocytogenes virulency factor cistrons, known as PrfA, is besides known to be thermo-regulated. PrfA is normally activated upon the consumption of L.A monocytogenes contaminated nutrients ( Mistake: Reference beginning non found ) . PrfA is besides known to modulate a assortment of the bacteria ‘s virulency cistrons ( Camejo et al. 2011, Stavru et Al. 2011 ) every bit good as other nucleus genome cistrons.

Internalins ( InlA and InlB ) , which are L. monocytogenes surface proteins, have been antecedently shown to affect in the invading of the host cells ( Seveau et al. 2007 ) . InlA is known to adhere E-cadherin, which is the host cell ‘s adhesion molecule, whereas InlB binds to Met, which is the hepatocyte growing factor ( HGF ) receptor. By the binding of internalin proteins to E-cadherin and Met, L.A monocytogenes cells are able to derive 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 secernment of Listeriolysin O ( a pore-forming hemolysin ) ( Gaillard et al. 1987 ) , every bit good as two phospholipases: phosphatidylinositol ( PI ) phospholipase ( PLC-A ) ( Camilli et al. 1993 ) and phosphatidylcholine ( Personal computer ) phospholipase C ( PLC-B ) ( Grundling et al. 2003 ) . Together, these proteins assist in interrupting down the host phagosome that contains L. monocytogenes cells. This is done to let the bacteria to get away into the host cytosol ( Kathariou et al. 1987, Camilli et Al. 1991, Mengaud et Al. 1991, Vazquez-Boland et Al. 1992, Schnupf and Portnoy 2007, Scortti et Al. 2007 ) . Upon come ining the host cell ‘s cytosol, L. monocytogenes cells begin to retroflex ( O’Riordan et al. 2003, Joseph and Goebel 2007 ) , and so with the aid of actin polymerisation mediated cell-cell spread, the bacteria moves through the host cell for the intent of migrating into the neighboring host cells. The actin polymerisation mediated cell-cell spread procedure is directed by ActA. The ActA protein binds and activates Arp2/3, which is a seven-protein host composite. Arp2/3 has been shown to bring on actin polymerisation every bit good as generate actin fibrils ( Pizarro-Cerda and Cossart 2006 ) . Upon entry into the next host cell, L.A monocytogenes cells secretes both Listeriolysin O and besides PC-PLC to help the bacteriums in get awaying from the double-membrane secondary vacuoles, known as listeriopods, which were formed as a consequence of cell-to-cell spread ( Freitag et al. 2009 ) .

Invasion of Mammalian Cells by L. monocytogenes

L. monocytogenes has evolved a figure of strategic methods to hedge or defy violent death by the innate immune response of mammalian phagocytic cells that are normally known to phagocytose and degrade most pathogens that invade the host cells ( Ryter and De Chastellier 1983 ) . The bacteria is able to multiply in a assortment of mammalian cell types such as professional phagocytic cells, for illustration, J774 macrophage-like cells ( Portnoy et al. 1992 ) , every bit good as non-professional scavenger cells such as epithelial cells ( Racz et al. 1972 ) , endothelial cells ( Drevets et al. 1995 ) and hepatocytes ( Conlan and North 1992 ) . Marco et Al. ( 1992 ) antecedently demonstrated that in mice that were infected with L. monocytogenes cells, the bacteria foremost infected the macrophage cells, followed by infection of the hepatocytes in the liver. L.A monocytogenes has besides been shown in a separate survey to be able to expeditiously occupy 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 bacteria so escapes into the host cytosol from the phagosome by interrupting the phagosomal membrane. Within the host cytosol, L.A monocytogenes grows and multiplies, and so returns to infect neighboring host cells ( Freitag et al. 2009 ) . Gaillard et Al. ( 1987 ) showed L.A monocytogenes was able to originate entry into human colon carcinoma cell line Caco-2, and multiply within the host cytosol. That same survey besides provided grounds to demo that L. monocytogenes was able to bring on phagocytosis by Caco-2 cells. Francis and Thomas ( 1996 ) demonstrated recovery of a higher Numberss of L.A monocytogenes cells of haemolytic strains from both HeLa and Caco-2 cell lines, in comparing to non-hemolytic strains. Furthermore, the extended morphological alterations that the host cells exhibited non merely included loss of meeting and host cell lysis, but besides the presence of really high counts of L.A monocytogenes cells within the host cells were detected ( Francis and Thomas 1996 ) .

Is there an Environmental Reservoir for L. monocytogenes?

Although L. monocytogenes causes terrible disease in human and carnal hosts, unlike other Gram-negative intracellular pathogens, this pathogen has no recognized animate being reservoir. Several surveies have suggested that as a consequence of interaction with soil-borne beings such as Protozoa, a figure of intracellular pathogens are able to keep its virulency cistrons ( Barker and Brown 1994, Adiba et Al. 2010, Lamrabet et Al. 2012 ) . For illustration, Salmonella spp. interacts with protozoons such as ameba and the ciliophoran Tetrahymena pyriformis ( Tezcan-Merdol et al. 2004, Brandl et Al. 2005 ) . Miltner and Bermudez ( 2000 ) have suggested the possible function of Acanthamoeba castellanii as an environmental host for the infective Mycobacterium avium. Furthermore, protozoon cells that were harboring Legionella pneumophila were identified as the cause of a fatal eruption of Legionnaires ‘ disease eruption during a convention in Philadelphia in 1976.

While Acanthamoeba spp. is known to harbor a figure of bacterial pathogens, L.A monocytogenes has been late demonstrated to be phagocytosed and quickly degraded by the host ameba within merely 2 H of consumption ( Akya et al. 2010 ) . In position of this consequence, the inquiry arises whether Protozoa could move as a possible reservoir for L. monocytogenes.

Interactions between Bacteria and Protozoa

Protozoa are unicellular eucaryotic micro-organisms that are ubiquitously present in diverse home grounds. They feed heterotrophically and are by and large recognised as the major consumers of bacteriums in the environment. Protozoan cells can be present either singly or as settlements of cells ( eg. Volvox spp. ) , may swim freely ( Paramecium spp. ) , or are parasitic for other animate beings ( eg. Trypanosoma spp. ) .

Briefly, there are three chief groups of bacterivorous Protozoa: ameba, ciliophorans and mastigophorans. Amoebae provender on algae, bacteriums, works cells, and smaller protozoons. Amoebae move by organizing pseudopods ( impermanent foot-like constructions ) with diverse morphologies. Ciliates can be found about everyplace there is H2O, such as lakes, rivers, oceans and besides dirt. They are characterized by big Numberss of hair-like cell organs ( called cilia ) that are involved in motion of the cells, chemotaxis, every bit good as predation of bacteriums ( Fenchel 1987 ) . Flagellates have whip-like extremities ( called scourge ) for the chief intents of motive power every bit good as to direct nutrient atoms or cells into its mouth-like gap.

The major cause of bacterial mortality in the environment is suggested to be chiefly as a consequence of eating of bacteriums by protozoons ( Pernthaler 2005 ) . A bulk of Protozoas feed by phagocytosis, a procedure by which they engulf bacteriums and digest them within a nutrient vacuole. Briefly, one time the bacterial quarry is captured, it is packaged into a nutrient vacuole. Once inside the protozoon nutrient vacuole, the procedure of digestion commences ( Fenchel 1987 ) . This is carried out through the release of host peptidases and lysozymes into the nutrient vacuole in order to interrupt down the bacterium within the nutrient vacuole. This will provide the Protozoa with energy and foods for its growing. The acidic environment within the nutrient vacuoles assists the Protozoa in disenabling the bacterial quarry for digestion. The merchandises of the digestion procedure are so released into the cytol. However, old surveies have clearly demonstrated that non all bacteriums are digested as nutrient beginning. Some types of bacteriums survive within the Protozoa in order to prevail and use those protozoal cells as a host. The major results in a bacteria-protozoa interaction ( Mistake: Reference beginning non found ) include:

Upon phagocytosis, the bacterium multiplies to high Numberss within the vacuoles, ensuing in monolithic expansion of these vacuoles that will finally do lysis of the host, let go ofing free bacteriums into the extracellular environment, e.g. Legionella pneumophila ( Rowbotham 1983 ) .

The same procedure as ( 1 ) , except that following lysis of host, free bacterium is released alongside integral vacuoles incorporating infective bacteriums, e.g. L. pneumophila ( Rowbotham 1983 ) .

Ingested bacteriums multiply within the host but non able to do lysis of the host, e.g. Coxiella burnetti ( La Scola and Raoult 2001 ) .

Ingested bacteriums survive within encysted Protozoa, e.g. L. pneumophila ( Rowbotham 1983 ) .

A figure of of import surveies have antecedently shown that infective bacteriums that are able to last within protozoons can be protected from external emphasiss such as chemical germicides 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 figure of intracellular bacterial pathogens to defy killing by its host protozoon cells may hold resulted in their development as pathogens of the mammalian land. Indeed it is possible that protozoon cells are the nexus between bacteriums that inhabits the environment and the bacteriums that cause diseases in mammals such as worlds.

Protozoa as Model Organisms for Study of Pathogenesis

Protozoan cells have been antecedently utilized as theoretical account beings for surveies in assorted Fieldss such as development and ecology ( Friman et al. 2008 ) , population and community biological science ( Holyoak and Lawler 2005 ) , the function of cell organs ( Smith et al. 2007 ) every bit good as toxicity surveies ( Stefanidou et al. 2008 ) . The usage of Protozoa in the survey of host-pathogen interactions has its advantages and has progressively become more common in recent old ages, most significantly in infective diseases surveies. Infection surveies by and large utilised mammalian species such as mice and sometimes even worlds as the host systems. By utilizing the mammals as the host system, the analysis is non merely expensive, drawn-out and capable to extended ethical reappraisal, it is besides technically ambitious and complex. In contrast, similar surveies in protozoons are more convenient, quicker and besides cost-efficient ( Montagnes et al. 2012 ) .

As theoretical account systems, protozoon cells can besides assist in understanding better the mechanisms of infective diseases within mammalian cells ( Montagnes et al. 2012 ) . Intracellular bacterial pathogens have been antecedently shown to get away the phagolysosomes of Protozoa and mammalian phagocytic cells by using similar mechanisms. Hence, Protozoa are utile theoretical accounts for analyzing the pathogenesis of timeserving, human pathogens. In footings of development, one-celled beings such as protozoons are older than multi-celled beings such as mammals. Hence the possibility that several mammalian infective bacteriums would hold evolved from intracellular pathogens within protozoon cells, can non be ruled out ( Montagnes et al. 2012 ) . For illustration, the interactions of L. pneumophila-mammalian cells and L. pneumophila-protozoa sharing a figure of phenotypical and molecular similarities between them show this fact ( Barker and Brown 1994, Fields 1996 ) .

It is now clear that protozoan-bacterial pathogen interactions play an of import function in transmittal of human disease. This was particularly apparent when Protozoa harboring L.A pneumophila were identified as the cause of a Legionnaires ‘ disease eruption during an American Legion convention in Philadelphia in 1976. A sum of 34 human deaths out of 221 instances were reported during that eruption. Therefore, it is inevitable that surveies on bacteria-protozoa interactions can supply important stairss into possible bar of infective diseases.

The amoeba Dictyostelium discoideum is an illustration of a utile theoretical account in the survey of human pathogens, including L. pneumophila ( Solomon et al. 2000 ) , Neisseria meningitidis ( Colucci et al. 2008 ) every bit good as Salmonella enterica serovar Typhimurium ( Annesley and Fisher 2009 ) . Other protozoon theoretical accounts normally studied include the ciliary Tetrahymena spp. ( Friman et al. 2008 ) , the marine mastigophoran Oxyrrhis spp. ( Montagnes et al. 2011 ) , and besides the choanoflagellate Monosiga spp. ( Behringer et al. 2009 ) . There are a figure of other advantages with utilizing Protozoa as a theoretical account system, including the easiness with the protozoons can turn in big sums every bit good as the simple storage and care techniques. In general, protozoa civilizations can be maintained on simple, cheap media, such as bacterial suspensions. Protozoa civilizations may be easy isolated from a assortment of natural and unreal environments. Protozoan cells can besides be stored as stock civilizations over a long period of clip, for illustration by suspending concentrated suspensions of protozoon cells in DMSO with storage at -20°C. These stock civilizations can be revived with merely a small attempt.

Interactions between Pathogenic Bacteria and Protozoa

Intracellular bacterial pathogens of worlds that parasitize protozoons exist within a privileged environment, protected from external emphasiss. Therefore bacteria-protozoa interactions are likely to hold of import ecological every bit good as public wellness effects.

The association of pathogens and Protozoa may hold contributed to the endurance and continuity of bacterial pathogens in assorted natural and unreal environments. Encapsulation of bacterial pathogens within protozoon cells and provides a protective consequence against environmental emphasis, such as predation, famishment, germicides and high temperatures. A figure of pathogens can last for drawn-out periods of clip within cysts of protozoon cells, and can non be detected by methodological analysiss based in civilization and can non be killed by the normal anti-bacterial methods and other inauspicious environmental conditions ( Greub and Raoult 2004 ) . A figure of surveies have shown that following internalisation within protozoons, the pathogens have increased virulency and demonstrate addition pathogenicity following infection of mammalian cells ( Rasmussen et al. 2005, Steinberg and Levin 2007, Adiba et Al. 2010 ) . The definite grounds for this happening remain to be explained. The function of protozoons as possible reservoirs of infective bacterium has been widely studied, yet small is understood about the true nature of the interaction at cellular degree ( Barker and Brown 1994 ) . Besides moving as a possible reservoir for the care of infective bacteriums in the environment, these eucaryotic beings are besides vectors for the transmittal of homo and carnal disease. How this occurs and the function of that procedure in development of these pathogens is non to the full known. Following uptake by Protozoa, several well-known intracellular bacterial pathogens of worlds have been shown to hedge digestion by the host and multiply within vacuoles ( Rowbotham 1983, Barker and Brown, 1994, Tezcan-Merdol et Al. 2004 ) . Care of virulency cistrons in environmental pathogens has been progressively understood to be a consequence of interactions between infective bacteriums and soil-borne micro-organisms, such as protozoons ( 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 broad scope of environments, including processed nutrients ( Vazquez-Boland et al. 2001 ) . Thus ingestion of L. monocytogenes-contaminated nutrient consequences in human listeria meningitis, a disease associated with a high mortality rate, peculiarly in immune-compromised persons ( Schlech 2000 ) . For this ground, nutrient merchandises contaminated by L.A monocytogenes are a major concern for the nutrient industry and public wellness governments likewise. However, while it is accepted that L. monocytogenes is omnipresent, small is known of the ecology of this being in natural environments. In peculiar, the function of protozoons as a natural reservoir for L. monocytogenes is non good studied, even though this bacteria has evolved an array of virulency factors that are critical for consumption and constitution of an intracellular life style within mammalian cells ( Cossart et al. 2003 ) .

Protozoans are known to feed on bacteriums through consumption by phagocytosis ( Barker and Brown 1994 ) . This interaction yields different results depending on the type of bacteriums involved. For illustration, Rowbotham ‘s ( 1980 ) landmark survey demonstrated that Acanthamoeba polyphaga could harbor retroflexing Legionella pneumophila. Since that survey, a figure of published studies have examined the relationship between L.A pneumophila and a broad figure of nonparasitic protozoons. The virulency of L.A pneumophila was enhanced by intracellular growing in A. castellanii ( Cirillo et al. 1999 ) . Furthermore, resuscitation of feasible but non-culturable L. pneumophila cells was triggered following consumption by A.A castellanii ( Steinert et al. 1997 ) . The ability of L. pneumophila to multiply intracellularly within mammalian cells has besides been well-documented ( Horwitz and Silverstein 1980 ) .

In a separate survey, co-culture of Helicobacter pylori with A. castellanii non merely resulted in a 100 fold addition of bacterial counts, integral and metabolically active H. pylori were besides found located within amebic vacuoles ( Winiecka-Krusnell et al. 2002 ) . Brandl et Al. ( 2005 ) later showed endurance of Green Fluorescent Protein ( GFP ) labelled S. enterica serovar Thompson cells located in vesicle-like constructions secreted by both starved and fed Tetrahymena cells. Similarly Mycobacterium avium was shown to be able to last within the outer walls of Acanthamoeba polyphaga cysts ( Steinert et al. 1998 ) . Interestingly, S.A enterica and E. coli O157: H7 cells encapsulated within expelled protozoon cysts are able to multiply and go out from these constructions ( Gourabathini et al. 2008 ) . These observations suggested that the vesicle-like constructions may supply a protected environmental reservoir for infective bacteriums.

Zhou et Al. ( 2007 ) showed that L. monocytogenes had no marauding consequence on A.A castellanii, and the presence of ameba can really heighten the growing of the bacterium. However, a survey by Akya et Al. ( 2009 ) provided informations that showed co-cultures of L.A monocytogenes with Acanthamoeba spp. resulted in active phagocytosis and violent death of the bacteriums by these amebas within 5 H of eating.

In both natural and unreal home grounds such as rivers, drainage systems and H2O distribution systems, bacteriums are typically present in planktonic and biofilm communities ( Barker and Brown 1994 ) . Biofilms represent a manner of growing for bacteriums including mammalian bacterial pathogens. This environment comprises extracellular merchandises, and inorganic and organic dust that promote biofilm bacteriums endurance during exposure to stressors, such as UV exposure ( Espeland and Wetzel 2001 ) , desiccation and salt ( LeMagrex-Debar et al. 2000 ) , antibiotics ( Mah and O’Toole 2001 ) every bit good as graze by Protozoa ( Snelling et al. 2006 ) . Natural bacterial biofilms are typically colonized by ameba, mastigophorans and ciliophorans ( Weitere et al. 2003, Parry 2004 ) . However, it is non known whether different strains of bacteriums respond otherwise at the physiological degree to graze by Protozoa. Huws et Al. ( 2005 ) showed that A. castellanii and the ciliophoran Colpoda maupasi, were able to crop on biofilm stuff. Croping Protozoa can hold a important impact on the unity and composing of biofilm communities. Grazing, for illustration, consequences in decrease of biofilm biomass and may do rapid alterations in morphological and systematic community composing in countries where planktonic bacteriums are present ( Hahn and Hofle 2001, Jurgens and Matz 2002 ) . In this context, Jackson and Jones ( 1991 ) showed that ameba were able to crop biofilm bacteriums to a point where the biofilm break was accompanied by sheding of the biofilm construction.

When Protozoas are non present, L. pneumophila has been shown to prevail and stay feasible for up to 15 vitamin D within unreal biofilms that were made utilizing filter-sterilized tap H2O or distilled H2O ( Lau and Ashbolt 2009 ) . However, other surveies have provided grounds that 30 % – 40 % of biofilm samples isolated from beginnings such as hospital H2O supplies, dental units and lights-outs showed presence of Acanthamoeba spp. ( Barbeau and Buhler 2001, Carlesso et Al. 2007 ) . Though biofilms have by and large been shown to supply protection against predation by protozoons, one survey has demonstrated that the consequence of protozoon graze on the biofilm community is a important factor that controls the biofilm composing in aquatic ecosystems ( Pedersen 1990 ) . Furthermore, biofilms may besides be able to curtail predation by protozoons as biofilm cells have been shown to release defensive factors that inhibit predation by either protozoon or bacterial rivals ( Matz and Kjelleberg 2005 ) .

A figure of surveies have demonstrated that chondriosomes are recruited to vacuoles incorporating intracellular pathogens ( Horwitz 1983, Sinai et Al. 1997, Chong et Al. 2009 ) . Recruitment of chondriosome to macrophage phagosomes ensuing in an upregulation of mitochondrial reactive O species ( mROS ) for bacterial violent death by the mitochondrial tract was shown in a old survey ( West et al. 2011 ) . Factors that might advance survival include traits of the bacterium every bit good as of the host. Other factors may be serotype specific. It is known that different serotypes of L. monocytogenes exhibit different degrees of pathogenicity. Of the 13 serotypes of L. monocytogenes that can do disease, more than 90 % of human isolates belong to merely three serotypes: 1/2a, 1/2b, and 4b. Serotype 4b isolates are responsible for 33 % – 50 % of sporadic human instances worldwide ( Ward et al. 2004 ) . Sandgren et Al. ( 2005 ) has shown that following vaccination of mice theoretical accounts with six serotypes of Streptococcus pneumoniae known to do invasive disease in worlds, differing degrees of host immune response was observed, proposing that invasive disease caused by different serotypes may ensue in different grades of host response. Different serotypes of S.A pneumoniae even exhibit fluctuations in opposition to phagocytosis ( Guckian et Al. 1980 ) every bit good as vary in the activation of the alternate complement tract ( All right 1975 ) . The nature of the disease caused by S. Typhimurium is suggested to be mostly dependent on the specific serotype-host combination ( Watson et al. 1999 ) . Another survey has shown that even among strains of S. pneumoniae of the same multilocus sequence type ( ST ) and serotype, differences in the virulency of these strains in a mouse infection theoretical account were observed ; and these differences were attributed to the familial differences between the bacterial strains of the same serotype ( Silva et al. 2006 ) . Listeria monocytogenes is a facultative intracellular pathogen capable of endurance and proliferation within mammalian cells such as macrophages and epithelial cells ( Vazquez-Boland et al. 2001 ) . As a food-borne pathogen, L. monocytogenes is the causative agent of human listeria meningitis that occurs following ingestion of contaminated nutrients such as dairy and processed nutrient. Upon come ining a mammalian host cell via a inactive or host-induced phagocytosis, L. monocytogenes is enclosed within phagosomes. At this phase, the host acidifies the phagosome to extinguish incursive bacteriums. 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 flight into the nutrient-rich cytosol of the host cell. Upon come ining the host cytosol, L. monocytogenes Begins to split and show ActA ( Tilney and Portnoy 1989 ) , which in bend leads to enlisting of host cell proteins that are indispensable in the formation of actin dress suits and actin-based motility ( Vazquez-Boland et al. 2001 ) . Actin-based motility within the cytol consequences in migration of bacterial cells to adjacent clean host cells via listeriopods ( double- membraned phagosome ) . An infective rhythm is completed upon flight of L. monocytogenes from the listeriopods within the new host cell ( Vazquez-Boland et al. 2001 ) .

Mammalian hosts employ a broad array of anti-microbial mechanisms to kill intracellular pathogens. Examples of these mechanisms include phagosome-lysosome merger and vacuole acidification ( Flannagan et al. 2009 ) , peptidases ( Pham 2006 ) and production of azotic 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 get away host defense mechanism mechanisms in order for intracellular endurance within host mammalian cells. For illustration, Legionella pneumophila is known to last and retroflex within professional scavenger cells by airting the ripening of phagosomes to make an intracellular environment that is contributing for intracellular bacterial reproduction ( Hart et al. 1987 ) , whereas several species of mycobacteria are capable of forestalling phagosome-lysosome merger within macrophages ( Bruggemann et al. 2006 ) . Furthermore there is grounds that L. monocytogenes inhibits host-induced programmed cell death in macrophages ( Barsig and Kaufmann 1997 ) , induces a hold in host phagosome ripening ( Henry et al. 2006 ) , every bit good as suppressing phagosome-lysosome merger within macrophages ( Bouvier et al. 1994 ) . These studies indicate that L. monocytogenes engages several protective mechanisms to avoid, or hold, killing by host cells.

Phagosome-lysosome Fusion mediates killing of L. monocytogenes

The ability of L. monocytogenes to last within intracellular compartments within mammalian cells has been clearly demonstrated ( Dramsi et al. 1993, Wood et Al. 1993, Francis and Thomas 1996 ) . However, this survey has demonstrated that Colpoda RR expeditiously inactivates L. monocytogenes within hours of feeding on this bacterial pathogen. Indeed, Colpoda RR are able to feed on planktonic DRDC8 cells every bit good as graze expeditiously on biofilms of DRDC8 ( Chapter 2 ) . Furthermore, the fact that DRDC8 cells are unable to occupy ColpodaA RR cells when phagocytosis is inhibited by the K+ outflow inhibitor tetraethylammonium chloride ( see Chapter 2 ) indicated that internalisation of these bacteriums within vacuoles was mediated by Colpoda RR. This decision contrasts with the well established InlA and InlB mediated invasion of mammalian cells by DRDC8.

Phagosome-lysosome merger and vacuole acidification are of import in debasement 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 ripening procedure. During ripening, acidification of the vacuoles occurs due to presence of V-ATPases, following phagosome-lysosome merger ( Huynh and Grinstein 2007 ) . Acidification of vacuoles consequences in the lytic activity of a assortment of degradative enzymes such as peptidases, every bit good as advancing the coevals of H peroxide ( Huynh and Grinstein 2007 ) . In this survey, acridine orange every bit good as the fluid stage investigation Lysosensora„? Blue DND-167 were used to qualify the DRDC8-containing vacuoles within Colpoda RR. Acridine orange[ 1 ]has been shown to concentrate within lysosomes, going protonated and sequestered, doing them to look as bright orange granules in fluorescence microscopy, whereas Lysosensora„? Blue DND-167 is a non-fluorescent investigation compound that freely diffuses through the membrane and becomes extremely fluorescent at an acidic pH ( ca. & lt ; 5.0 ) . Consequently this investigation is utile for placing trafficking of acidic vacuoles. In the context of this survey, this investigation was used to demo that DRDC8-containing vacuoles within Colpoda RR fused with little acidic cysts ( presumptively lysosomes ) , an result mirrored in Colpoda RR stained with acridine orange. As expected, the content of the larger cysts became acidic shortly after phagosome-lysosome merger. These consequences support the thought that for Colpoda at least, lysosome mediated acidification of vacuoles incorporating bacterium is a likely precursor to killing of bacteriums and digestion. In this regard, the procedure mimics well-described cellular procedures used by mammalian phagocytic cells. The fact that killing of intra-ciliate DRDC8 cells, was impaired by inhibitors that target phagosome-lysosome merger ( NH4Cl ) and vacuole acidification ( bafilomycin A1 and monensin ) , lends strong support for the function of these cellular procedures in Colpoda.

Ammonium chloride is a weak base that interferes with the ripening of the phagosome and as a effect leads to suppression of phagosome-lysosome merger ( Hart and Young 1991 ) . However there are cases of contradictory results reported that concern endurance of bacteriums within NH4Cl-treated host cells. For illustration, Harley and Drasar ( 1989 ) reported that L.A pneumophila within NH4Cl-treated HeLa cells were able to last and retroflex within intracellular compartments, but Byrd and Horwitz ( 1991 ) subsequently showed that L.A pneumophila survived without reproduction within treated human monocytes. Nevertheless, in the current survey, this weak base clearly blocked ripening of the phagolysosome to the point where DRDC8 was able to last within Colpoda vacuoles, although no grounds for growing and cell division was detected.

The engagement of acidification of the phagosome following merger with lysosomes, in killing of intra-ciliate DRDC8 was confirmed by usage 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 ensuing in the addition of the vacuolar pH ( Nakazato and Hatano 1991 ) . The consequence of these inhibitors on endurance of DRDC8 within Colpoda cells is straight comparable with surveies that report impaired killing of bacteriums within macrophage cell lines. For illustration, killing of intra-amoebic L. monocytogenes by A. polyphaga was impaired following interventions with both bafilomycin A1 and monensin ( Akya et al. 2009 ) . Furthermore, bafilomycin A1 intervention of murine RAW 264.7 macrophages inhibited killing and debasement of phagocytosed E. coli ( Frankenberg et al. 2008 ) . Similarly, suppression of acidification of Staphylococcus aureus-containing vacuoles besides resulted in impaired bacterial violent death by alveolar macrophages ( Bidani et al. 2000 ) . Di et Al. ( 2006 ) has demonstrated the usage of alveolar macrophages that carried cystic fibrosis transmembrane conductance regulator chloride channel mutants to straight implicate the function of vacuole acidification in the violent death of bacteriums.

Precisely why DRDC8 is able to last within inhibitor-treated Colpoda RR, but is unable to retroflex, is ill-defined. Several studies have suggested that endurance without reproduction of M.A avium or L. pneumophila within activated macrophages is due to replication limitation mediated by enhanced phagosome-lysosome merger ( Schaible et Al. 1998, Santic et Al. 2005 ) . However this is an improbable account for Colpoda cells treated with inhibitors designed to forestall merger of lysosomes with the phagosome. An alternate, and more likely ground to explicate this observation is that suppression by NH4Cl, bafilomycin and monensin was sufficient to let endurance, but insufficiently complete to let growing of subsister L.A monocytogenes cells within the phagosome. Other integral antibacterial mechanisms may besides hold been sufficient to quash growing without impacting endurance. Furthermore, as Listeriolysin O look is reduced at temperatures used for co-culture ( Datta and Kothary 1993 ) , L.A monocytogenes would non be able to intercede devastation of the ciliophoran vacuole membrane to get away and retroflex within the cytosol of Colpoda RR.

Proteases assist killing of L. monocytogenes

In add-on to the function of lysosome merger with phagocytic vacuoles and vacuolar acidification, a assortment of endopeptidases ( cysteine and aspartate peptidases ) , exopeptidases ( cysteine and serine peptidases ) and hydrolases located within phagosomes are besides reported to degrade bacterial constituents within the phagosomes ( Pillay et al. 2002 ) . Substrates for the exopeptidases are generated by the C1 household of cysteine peptidases of endopeptidases ( Pillay et al. 2002 ) .

Since the experimental informations presented confirmed that damage of peptidases of Colpoda RR by peptidase inhibitors allowed the endurance of DRDC8 within the ciliophorans, this result indicates that endo- and exo-peptidase of Colpoda RR are likely to be a factor involved in the violent death and debasement of internalized DRDC8. Since the serine and cysteine peptidase inhibitors in the cocktail used in this survey would hold probably impaired the endopeptidases, the activity of exopeptidases would hold besides been limited as the endopeptidase activity is required to bring forth substrates for the exopeptidases. Although the consequence of peptidase inhibitors on endurance of DRDC8 within Colpoda cells is fresh for work with bacteriophagic protozoons, old surveies have highlighted the antibacterial function of serine peptidases within mammalian cells. For illustration, Rosenberger et Al. ( 2004 ) showed that peptidase ordinance of macrophage cathelicidin-related antimicrobic peptide ( CRAMP ) look and activity impaired the reproduction of intracellular S.A Typhimurium. Similarly, Standish and Weiser ( 2009 ) demonstrated that suppression of neutrophil granule peptidases by serine peptidase inhibitors resulted in endurance of Streptococcus pneumoniae within neutrophils. Whether serine peptidases ( or other peptidases ) in peculiar are involved in restricting the bacteriocidal response to L. monocytogenes within Colpoda cells is unknown. This inquiry could easy be resolved by intervention of Colpoda cells used in co-cultures with specific peptidase inhibitors.

L. monocytogenes is insensitive to Nitric Oxide

Nitric oxide is a toxic extremist synthesised from L-arginine by inducible azotic oxide synthase ( iNOS ) . It is a cardinal constituent of the mammalian innate unsusceptibility and an of import bactericidal agent ( Nathan and Shiloh 2000 ) cardinal to killing ingested pathogens by activated macrophages ( Nathan 1997 ) . Intracellular pathogens such as Mycobacterium TB and Leishmania major are killed by a azotic oxide-dependent mechanism ( Vouldoukis et al. 1995, Bekker et al. 2001 ) . Many other published surveies implicate azotic oxide produced by mammalian host cells in the violent death of internalized bacteriums. One of these surveies has for illustration, showed that L-NMMA suppression of azotic oxide synthesis within activated macrophages correlatives with loss of control of intracellular reproduction of Rhodococcus equi ( Darrah et al. 2000 ) . This type of survey implicates azotic oxide as a go-between of the violent death of intracellular R. equi by activated macrophages. Furthermore, an increased S.A Typhimurium load was detected within reticuloendothelial variety meats of iNOS mutant mice ( Mastroeni et al. 2000 ) . Increased degrees of azotic oxide production were detected within peritoneal macrophages in response to interferon gamma in a separate survey ( Vazquez-Torres et al. 2000 ) . In that survey, the elevated degrees of NO appeared to increase antibacterial activity against S.A Typhimurium.

A recent study by von Bargen et Al. ( 2011 ) indicated that following infection with R. equi, macrophages produced ca. 10 AµM NO at 24 H station infection. Summersgill et Al. ( 1992 ) showed L.A pneumophila were killed by azotic oxide released by murine macrophages, and the degree of nitrite measured at 24 H station infection was ca. 58.6 AµM. However, the informations presented in this present survey mirrors the result of a separate survey by Beckerman et Al. ( 1993 ) that showed violent death of L. monocytogenes occurred even after intervention of activated macrophages with L-NMMA. The result of this present survey could be an indicant that though Colpoda RR does bring forth low degrees of NO, the ciliophoran does non use it as a mechanism of defense mechanism against phagocytosed pathogens.

Superoxides

NADPH oxidase within hosts is activated upon phagocytosis, ensuing in the coevals of intracellular superoxide ( Takeya and Sumimoto 2003 ) . The superoxide plays an of import function in the oxygen-dependent antibacterial mechanisms of phagocytic cells ( Fang 2004 ) . Phagocytic cells are known to utilize a combination of oxidative mechanisms, including superoxide in add-on to non-oxidative mechanisms such as pH, to support against the broad scope of phagocytosed infective bacteriums ( Roos and Winterbourn 2002 ) . It has been antecedently established that the NADPH oxidase is of import for extinguishing L.A monocytogenes within host cells ( Shiloh et al. 1999 ) . The results of the present survey that showed less superoxide production by Colpoda RR following feeding with DRDC8 in comparing to command co-cultures with E. coli DH5I± and heat-killed DRDC8 controls mirrored the result of a separate survey that demonstrated macrophages produced less superoxide following Burkholderia cenocepacia infection as compared to heat-inactivated B.A cenocepacia and E. coli controls ( Keith et al. 2009 ) . Interestingly, a survey provided grounds of Listeriolysin O suppressing host NADPH oxidase by forestalling its localisation to phagosomes therefore leting the bacteria to get away the phagosome while avoiding the microbicidal respiratory explosion ( Lam et al. 2011 ) . Inhibitor mediated consumption of L. monocytogenes by Colpoda

Phagocytosis is the primary mechanism used by ciliophorans for the consumption of bacteriums. An introversion pinches off to organize a big endocytic cyst ( phagosome ) incorporating the ingested stuff ( Alberts et al. 1994 ) . This procedure is called endocytosis. The phagosome travels within the cell and finally fuses with a lysosome. The ingested substance is so digested ( Alberts et al. 1994 ) . A significantly reduced consumption of bacteriums by all inhibitor-treated ciliophorans ( except L-NMMA ) compared to untreated ciliophorans seemed to bespeak that phagocytosis of DRDC8 by treated Colpoda RR was affected by the repressive effects of the chemicals. This phenomenon has been a characteristic of a figure of old surveies. One of the surveies showed that besides suppressing vacuole acidification, bafilomycin A1 besides conferred repressive effects on the consumption of E. coli by alveolar macrophages ( Bidani and Heming 1995 ) . Treatment of A. polyphaga with monensin besides resulted in a decreased consumption of L.A monocytogenes by the ameba compared to untreated control ameba cells ( Akya et al. 2009 ) . One ground for this consequence may be related to reduced Ca2+ degrees within Colpoda cells. Calcium ( Ca2+ ) is regarded as a demand for phagocytosis ( Peck and Duborgel 1985 ) and vacuole acidification ( Downey et al. 1999 ) , although a few other surveies have reported at odds results refering the importance of Ca2+ for phagosome-lysosome merger ( Jaconi et al. 1990, Zimmerli et Al. 1996, Dewitt and Hallett 2002 ) . Importantly, additions in vacuole pH within macrophages, ensuing from intervention by either NH4Cl or bafilomycin A1 consequence in several fold lessenings in Ca2+ concentrations within the macrophage cells ( Christensen et al. 2002 ) . Therefore, it seems likely that decreased degrees of endogenous Ca2+ brought about by NH4Cl, bafilomycin and monensin intervention, are responsible for decreased consumption of L. monocytogenes by treated Colpoda cells. The AS buffer used to suspend Colpoda cells has sufficient concentrations of Ca2+ to advance active phagocytosis and phagosome-lysosome merger 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 ephemeral and dependant on continued presence of those inhibitors. However, it is non known why intervention of the ciliophorans with peptidase inhibitor cocktail besides resulted in a decreased bacteriums uptake, although it is interesting to theorize that this consequence may be caused by the consequence of peptidases on signalling Cascadess necessary for phagocytosis.

Decisions

When phagocytic cells “ provender ” on bacterial cells, the host phagosome undergoes a ripening procedure in which actin undergoes depolymerisation ( Bengtsson et al. 1993 ) , enabling merger of the phagosomes with lysosomes ( Flannagan et al. 2009 ) . The phagosome-lysosome construction formed is an acidic, hydrolytic compartment in which the bacterial cell may be killed and digested. Defensins, cathelicidins, muramidases, and a assortment of peptidases are recruited by the phagosome to kill the pathogen ( Flannagan et al. 2009 ) . It was interesting to observe that suppression of a individual mechanism within Colpoda RR resulted endurance of DRDC8 within the ciliophoran. This observation indicated that killing of L.A monocytogenes by Colpoda RR can non be attributed to any one individual mechanism. Phagocytosis and violent death of bacteriums by mammalian cells is a tract that consists of a cascade of defined procedures. Survival of bacteriums within mammalian cells following suppression of either one of these procedures has been good established for mammalian cells. Intervention with the consumption of bacteriums such as Pseudomonas aeruginosa and Yersinia enterocolitica by macrophages and neutrophils by direct damage of the phagocytic machinery of the host has been antecedently demonstrated ( Garrity-Ryan et Al. 2000, Grosdent et Al. 2002 ) . Other types of bacteriums such as Mycobacterium TB and S. Typhimurium have schemes to either antagonize the acid accretion within phagolysosomes, or have acquired cistrons that encode proteins that help the bacteriums to defy 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 antimicrobic peptides such as peptidases that are recruited by the phagosome ( Schmidtchen et al. 2002 ) , whereas Staphylococcus aureus are able to defy the effects of phagosome-recruited antimicrobic peptides ( Peschel et al. 2001 ) . Hence it is non unusual to observe the intra-ciliate endurance of DRDC8 when one of the ciliophoran defense mechanism mechanisms was inhibited in this present survey. Furthermore, it besides shows that the Colpoda RR defense mechanism mechanism machinery is similar to that seen in mammalian cells.

As a pathogen of both worlds and animate beings, Listeria monocytogenes is one of the most deadly and omnipresent pathogens as this facultative intracellular bacteria is good adapted to last in the environment. As the aetiologic agent for listeria meningitis, L. monocytogenes causes infection when contaminated nutrient is ingested ( Cossart and Bierne 2001 ) . By comparing with the big figure of published studies refering L.A monocytogenes, there have been few surveies dedicated to the ecology of L. monocytogenes in natural environments. In peculiar, the function of protozoons as a possible reservoir for L.A monocytogenes has non been good studied, even though this bacteria has evolved an array of virulency factors that are of import for invasion of cells and constitution of an intracellular life style within mammalian cells ( Cossart et al. 2003 ) . L. monocytogenes has shown to hold parasitizing effects on mammalian cells ( e.g. HeLa and Caco-2 cell lines ) and is infective in mice ( Francis and Thomas 1996 ) . By contrast, antecedently demonstrated results of co-cultures of L.A monocytogenes with Acanthamoeba spp. have shown otherwise. Akya et Al. ( 2010 ) showed that ameba are able to actively phagocytose and kill bacteriums located in phagolysosomal compartments within 2 Hs post consumption. Interestingly, several groups have reported secernment of infective quarries bacteriums from protozoon cells within membrane edge fecal pellets. For illustration, Rowbotham ( 1980 and 1986 ) described release of vesicle-like constructions ( fecal pellets ) that contained an infective dosage of L. pneumophila. Following predation and phagocytosis by ameba, the bacteriums were released within pellets. These pellet associated bacterial cells were in add-on to those released from lysed ameba cells. Encapsulation of feasible L. pneumophila cells within fecal pellets has besides been confirmed for other protozoon hosts, such as Tetrahymena spp. ( McNealy 2001 ) every bit good as A. polyphaga and A. castellanii ( Berk et al. 1998 ) . A separate survey went farther to demo that L. pneumophila located within fecal pellets of A. castellanii were able to last to up to 6 months within medium that had a hapless alimentary handiness ( Bouyer et al. 2007 ) . Furthermore, co-cultures of T. pyriformis with strains of Escherichia coli resulted in secernment of fecal pellet-like constructions that contained feasible, culturable bacteriums ( Schlimme et al. 1997 ) . Brandl et Al. ( 2005 ) besides showed that up to 50 feasible S. enterica cells are present per fecal pellet secreted by Tetrahymena sp. However, Gourabathini et Al. ( 2008 ) demonstrated that while Tetrahymena sp. released fecal pellets incorporating E. coli O157: H7 and S. enterica following graze on these bacteriums, this procedure did non happen when the ciliophorans grazed on L. monocytogenes.

Encapsulation of bacterial cells within membrane bound pellet-like constructions presents these cells with a important advantage in footings of endurance in the presence of antibacterial agents. Berk et Al. ( 1998 ) showed that fecal pellet encapsulated L.A pneumophila cells retained viability following exposure to biocides ( e.g. free Cl, and which others ) every bit good as rhythms of freeze-thaw intervention. Similarly, S. enterica cells contained within Tetrahymena sp. fecal pellets exhibit higher opposition to low concentrations of Cl in comparing to unattached bacteriums ( Brandl et al. 2005 ) . However, to day of the month there is no univocal grounds to back up the contention that L. monocytogenes is either secreted within fecal pellet constructions by predatory protozoons, or that fecal pellet-associated cells are more immune to bactericidal agents than single cells.

Rowbotham ( 1980 ) was the first to supply grounds of ameba as a vector of Legionella spp. More late, Berk et Al. ( 1998 ) demonstrated Acanthamoeba spp. expelled fecal pellets that contained bacterial cells into the extracellular environment. Since so, merely a few published surveies have demonstrated fecal pellet production by other protozoons following co-culture with different species of bacterial pathogens.

The genus Colpoda belongs to the category Colpodea, and is normally 20 – 50 I?m in length with a reniform form. Colpoda ciliates feed on bacteriums as the major nutrient beginning. Like L.A monocytogenes, Colpoda can be found in a assortment of environments, particularly in dirt and flora. For this ground, Colpoda may be a good theoretical account being to analyse interactions between L. monocytogenes and protozoons. In this survey, we have demonstrated that Colpoda ciliates secrete fecal pellets incorporating unrecorded L.A monocytogenes DRDC8 cells following co-culture. This result is alone, as old surveies have shown the inability of other protozoa species to release fecal pellets incorporating bacteriums following co-cultures with L.A monocytogenes. The amoeba A.A palestinensis and the ciliophoran T. pyriformis did non bring forth any fecal pellets following feeding with L. monocytogenes ( Brandl et al. 2005, Gourabathini et Al. 2008 ) . The ciliophoran Glaucoma sp. produced merely a few fecal pellets following co-culture with L. monocytogenes ( Gourabathini et al. 2008 ) . Brandl et Al. ( 2005 ) showed that Tetrahymena sp. strain MB125 expelled merely few fecal pellets following co-culture with L.A monocytogenes, and these fecal pellets contained merely few cells of L. monocytogenes. It is interesting that although the present survey provided clear grounds to demo Colpoda RR and MLS-5 secreted fecal pellets incorporating bacteriums following co-cultures with L.A monocytogenes and S. Typhimurium, Gourabathini et Al. 2008 reported that Colpoda steinii did non release any fecal pellets incorporating bacteriums following co-culture with

these bacteria-containing fecal pellets should still be considered as a possible clinically of import vector of disease transmittal.

It has been good documented that host protozoons may heighten endurance of bacterial pathogens within protozoons by protecting bacteriums from exposure to germicides and sanitizers. Pathogens such as S. Typhimurium and Campylobacter jejuni have demonstrated opposition to free Cl following consumption by T. pyriformis ( King et al. 1988 ) . C. jejuni nowadays within T.A pyriformis and A. castellanii besides exhibit increased opposition to Virudine, a germicide normally used in the domestic fowl industry ( Snelling et al. 2005 ) . Furthermore, Adekambi et Al. ( 2006 ) reported that every bit many as 26 species of water-associated mycobacteriums cells that were encapsulated within cysts of A.A polyphaga were able to last up to 24 H exposure to free Cl. Bacteria encapsulated within fecal pellets secreted by protozoons have besides been demonstrated to be immune to biocidal agents. Brandl et Al. ( 2005 ) for illustration, reported that S. Thompson cells located within fecal pellets of Tetrahymena sp. were protected during intervention with Ca hypochlorite ( Ca ( ClO ) 2 ) concentrations of 0.13 and 0.42 % . Similarly, Berk et Al. ( 1998 ) showed that L.A pneumophila cells within fecal pellets of Acanthamoeba spp. are immune to external emphasiss such as chilling tower biocides, freeze-thawing and sonication procedures. Consequently it is non surprising that DRDC8 cells encapsulated within fecal pellets of Colpoda RR and MLS-5 are immune to concentrations of gentamycin ( 0 – 100 Aµg mL-1 ) and NaOCl ( 0 – 10 % ) that are usually deadly to rinse cell suspensions of this pathogen. The membranes that make up the fecal pellets may either act as a barrier to forestall the pervasion of the biocides into the pellets, and/or organic stuffs present within the pellets may demobilize or forestall the biocide molecules damaging the bacterial cells. The singular opposition of these pellet-located DRDC8 cells to these otherwise biocidal concentrations of germicides indicates that the fecal pellets are likely to be an first-class protective shield for the bacteriums in the presence of germicides and cleaning agents used in nutrient processing environments.

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