Name: SomayehNasiri BahmanabadImmatriculateNo. 10016637 Main Factors in Biofilm Formation and colonization by Legionellapneumophila AbstractLegionella pneumophila (L.pneumophila) is a pathogenic gram-negative bacterium that can befound in both natural and human-made aquatic environments. It is responsiblefor legionellosis illness that can severely affect the lungs of humans.Although L.
pneumophila is generally hosted by protozoa, this pathogenis able to survive and replicate itself in natural environments as a freeorganism by forming biofilms. Now, some observations are reported that thepresence of this pathogen in biofilms may also lead to the legionellosisdisease. Hence, if biofilm formation isprevented, the spread of legionellosisillness will be avoided. However, there is very few information about severalfactors, such as chemical and biophysical conditions playing an important rolein the formation of biofilm by L. pneumophila. This review intends todeal with molecular basis of biofilm, the role of different bacterialcommunities, including protozoan and non-protozoan bacteria in biofilm, as wellas the endogenous factors responsible for the regulation of L.
pneumophila biofilmformation.1-IntroductionLegionella pneumophila (L.pneumophila) is a serious type of aquatic aerobic pathogen, found in bothhuman-made and aqueous environments. It causes legionellosis illness. This is asevere lung disease ( Yu et al., 2002), mostly infects people by inhalation of aerosol particles produced inwater structures, air conditioning, activated sludge systems, and so forth (Percival & Williams,2014).
In vitro, L. pneumophila produces one-species biofilms,which contains an extracellular matrix (Hindre et al., 2008). But, in theaqueous environment, L.
pneumophila can be found in different bacterialcommunities, including protozoa and other bacterial species. These multispeciesbacteria influence the colonization with L. pneumophila (Murga et al.
,2001). Among different species of bacteria, protozoa are believed to be themost dominant in which L. pneumophila can easily reside and replicateitself (Rowbotham et al., 1981). Moreover, L.
pneumophila is able toco-evolve with different types of protozoa and therefore develops mechanismsfor residing in a wide variety of host cells (Yan et al., 2004). Recent studieshave reported that the growth of L. pneumophila in biofilms can promotethe risk of legionellosis outbreaks (Hindre et al., 2008). A thoroughunderstanding of the living conditions of L. pneumophila in biofilmhabitat can lead to development of effective strategies for the control andprevention of the replication of L.
pneumophila in biofilms (Declerck et al., 2007). This review is aimed toprovide an overview of main factors, such as protozoan-host, biophysical andphysiochemical parameters, regulation of L. pneumophila endogenousfactors, and finally role of non-protozoan bacteria in the production andcolonization of biofilms by L. pneumophila. 2-Biofilm: A SafeHabitat for L. pneumophilaBiofilm is a community of attached microorganisms, sticking on bioticor abiotic surfaces in which trophic reactions occur. Biofilms have a dynamiccharacteristic.
It means that they can change in time and space in order toprovide a better survival and growth condition for the community ofmicroorganisms (Kolter et al., 2000). In order to understand how L. pneumophilacan survive and replicate within biofilms, we need to analyze the mainchemical and biophysical parameters, molecular basis, and also the role ofprotozoan and non-protozoan bacteria in the formation of L. pneumophila biofilmformation. Thus, this section is divided into four sections as follows:i) Protozoan-host biofilm formation by L.
pneumophilaii) Biophysicaland physiochemical parameters in biofilmformation and colonization by L. pneumophilaiii) Regulationof L. pneumophila endogenous factors influencing biofilm formationiv) TheRole of non-protozoa microbial species in L. pneumophila biofilm formation i) Formation of Protozoan- hostbiofilm by L. pneumophila Protozoaare important habitat that allow the pathogen to replicate itself intracellular(Rowbotham, 1981). Protozoa provide living conditions for the survival andreplication of Legionella species and therefore they are essential inthe survival of Legionella forms (Barker et al., 1993). There areseveral protozoa, such as amoeba species in which L.
pneumophila can behosted. Thus, protozoa are the most possible habitat for the growth of L.pneumophila (Valster et al., 2010). In other words, there is a correlationbetween the amount of L. pneumophila and biomass of protozoa (Liu etal., 2012). L.
pneumophila can also potentially grow off the deadprotozoa, so that may also encourage the multiplication of L. pneumophilaindirectly (Temmerman et al., 2006). Moreover, biofilms that are capable offloating, contain L.
pneumophila hosted by protozoa, suggesting that L.pneumophila can adhere to protozoa in floating biofilm in the absence ofavailable abiotic surfaces (Hsu et al., 2011). Protozoa play an important rolenot only as a medium for the multiplication of L. pneumophila, but also protects L. pneumophila from stresses,such as lack of nutrient, pH change, as well as biocides that are used todisinfect water system (Donlan et al., 2005). Figure 1 shows an infected protozoanspecies (amoeba) with L.
pneumophila. Figure1: an infected amoebae with L. pneumophila, expressing green fluorescentprotein (valster, 2010) ii) Biophysical and Physiochemical Parameters in L. pneumophila BiofilmFormation and colonizationFor the production of biofilm formation by L. pneumophila andalso its colonization, physiochemical parameters should be analyzed. Figure 2shows the replication of L.
pneumophila (indicated by Lpn and shown inorange) within protozoa (Wright, 1989). Figure 2: The replication ofL. pneumophila within environmental protozoa (Wright et al.,1989) Cations, knownas influential factors for the process of bacterial adhesion to the different surfacescan also contribute to biofouling (Geesy et al., 2000). Similarly, calcium andmagnesium facilitated the adhesion of L. pneumophila to artificialsurfaces. Also, when zinc and manganese are available, L.
pneumophila caneasily adhere to the surface. Moreover, zinc also promotes the ability of L.pneumophila to bind to cells of the surface (Yaradou et al., 2007). In addition of the availability of cations,carbon is the main source of energy for the formation of biofilm by L.pneumophila because it is a source of nutrients for the replication of the bacteria. However,it is seen that organic carbon can only influence the production of biofilm at20 ° C. In other words, this result is aproof of the influence of carbon in the production of biofilm at specifictemperatures (Pang et al.
, 2006).Another factor that influence the multiplication of L.pneumophila in the biofilm colonization is the state of flow.
For example,a steady state flow in human-made water systems can decrease the amount of L.pneumophila by preventing the adherence of bacteria to the surfaces, whilemotionless water provides a suitable condition for the growth of L.pneumophila (Makin et al., 2010).
Surprisingly, in turbulent flows, L.pneumophila is able to survive without decreasing in numbers. The reasonfor that is bacteria are able to reside themselves into the sediment in orderto survive from dynamic conditions and therefore they are less affected by theturbulence (Stout et al., 1985). Molecular factors also help L.
pneumophila to form biofilms. Collagen-like protein (Lcl), known as anadhesin is a cell-component that facilitate adhesion to the surface. Hence, itplays a major role in the formation of L.pneumophila biofilm (Vandersmissenet al., 2010).Also, Type IV pili (T4P), known as surface-exposed fibers contribute to thecolonization of L. pneumophila in the biofilm (Duncan et al.
, 2011).iii) Endogenous Factors Influencing Biofilm Community of L.pneumophila Biofilm formation is anenvironmental response for the survival L.
pneumophila. The production ofbiofilm is also affected by an important environmental prompt, iron. It plays amajor role in the growth of many organisms and can also influence L.
pneumophila replication (Cianciotto et al., 2007). For example, lactoferrin, an iron chelator, can kill L. pneumophila. Thus, it can beseen the importance of iron in L.
pneumophila viability (Orsi, 2004).Although iron is vital for the formation of biofilm, higher concentration ofiron can prevent biofilm formation.Quorum sensing, as a vitalprocess during biofilm production, is the response of bacteria to cell density. ?-hydroxy ketones(AHKs), are quorum sensing molecules that exist in L. pneumophila. Thesemolecules are more likely in charge of a wide variety of traits that may impactthe production of L. pneumophilabiofilm (Kessler, et al., 2013).
Temperature is also anotherimportant factor for biofilm colonization (Martinelli et al., 2000). Moreover,temperature affects the chracteristics of biofilms. In lab-scale experiments, attemperature between 37-42° C, biofilms mostly contain filamentous bacteriawhile rod shaped bacterial cells are the main community of biofilms at lower temperature (25 °C). In anexperiment, it was shown that the production of biofilms at higher temperature (37° C ) are much stronger thanat lower temperature (25 ° C) (Mampel et al., 2006). Interestingly, the biofilmsformed at temperature 25° C have adhesive characteristics (Piao et al., 2006).
These findings show that filamentation community of L. pneumophila are more likely influenced by temperature (Konishi et al.,2006). Figure 3: Confocal laser scanningmicrographs of GFP; expressing L. pneumophila Philadelphia-1 biofilmsformed under static conditions in BYE medium at 25°C and 37°C (Piao et al.
, 2006) iv)The Role of Non-Protozoa Microbial Species in L. pneumophila BiofilmFormationIn addition to protozoa, biofilms contain differenttypes of bacteria. These bacteria are more likely promoters to increase thelifespan of L. pneumophila (Messsi, et al.
, 2011).In an experiment, loss ofprotozoa due to the temperature rise did not change the replication of L.pneumophila. Thus, it suggests that L. pneumophila can survive andcontinue the process of multiplication without the presence of protozoa (Temmerman et al., 2006).P.
aeruginosa is a bacterial species, which caninfluence the colonization ability of L. pneumophila in biofilms.Although the replication and growth of L. pneumophila in naturalenvironmental biofilms is also attributed to P. aeruginosa, the presenceof other bacterial species together with P.
aeruginosa promote theincreasing growth of L. pneumophilain a multispecies medium(Swart et al., 2012). 3-ConclusionsL. pneumophila is apathogenic bacterium, which can be found in both human-made and naturalenvironments (Yu et al., 2002). Here, we studied the different factors andparameters that promote the production of biofilm and colonization by L. pneumophila.
, such as protozoan-host,physiochemical parameters, including cations, state of flow, molecular basis, temperature, as well as the role ofnon-protozoan bacterial community. The physiochemical parameters, known asimportant factors in biofilm formation attract a lot of attention. Recent research has gained insightinto these factors, which can control the replication of L.
pneumophilaand more likely to be useful for the prevention of legionellosis disease. Researchersare also more interested in studying the role of other bacterial species in theproduction of L. pneumophila biofilm (Temmerman et al., 2006). Another question that still has not been answeredis that how intercellular lifestyle of L. pneumophila helps to biofilmresistance to disinfection methods (Alleron et al.
, 2008). Finally, thisresearch gives precious information about the ecology of this pathogen in biofilmsthat can be used for future research in order to find preventive and protectivemethods to fight against L. pneumophila infections (Hindre et al., 2008).