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Main Factors in Biofilm Formation and colonization by Legionella



Legionella pneumophila (L.
pneumophila) is a pathogenic gram-negative bacterium that can be
found in both natural and human-made aquatic environments. It is responsible
for legionellosis illness that can severely affect the lungs of humans.
Although L. pneumophila is generally hosted by protozoa, this pathogen
is able to survive and replicate itself in natural environments as a free
organism by forming biofilms. Now, some observations are reported that the
presence of this pathogen in biofilms may also lead to the legionellosis
disease. Hence, if  biofilm formation is
prevented,  the spread of legionellosis
illness will be avoided. However, there is very few information about several
factors, such as chemical and biophysical conditions playing an important role
in the formation of biofilm by L. pneumophila. This review intends to
deal with molecular basis of biofilm, the role of different bacterial
communities, including protozoan and non-protozoan bacteria in biofilm, as well
as the endogenous factors responsible for the regulation of L. pneumophila biofilm


Legionella pneumophila (L.
pneumophila) is a serious type of aquatic aerobic pathogen, found in both
human-made and aqueous environments. It causes legionellosis illness. This is a
severe lung disease ( Yu et al., 2002), mostly infects people  by inhalation of aerosol particles produced in
water structures, air conditioning, 
activated sludge systems, and so forth (Percival & Williams,

In vitro, L. pneumophila produces one-species biofilms,
which contains an extracellular matrix (Hindre et al., 2008). But, in the
aqueous environment, L. pneumophila can be found in different bacterial
communities, including protozoa and other bacterial species. These multispecies
bacteria influence the colonization with L. pneumophila (Murga et al.,
2001). Among different species of bacteria, protozoa are believed to be the
most dominant in which L. pneumophila can easily reside and replicate
itself (Rowbotham et al., 1981). Moreover, L. pneumophila is able to
co-evolve with different types of protozoa and therefore develops mechanisms
for residing in a wide variety of host cells (Yan et al., 2004). Recent studies
have reported that the growth of L. pneumophila in biofilms can promote
the risk of legionellosis outbreaks (Hindre et al., 2008). A thorough
understanding of the living conditions of L. pneumophila in biofilm
habitat can lead to development of effective strategies for the control and
prevention of the replication of  L.
pneumophila in biofilms (Declerck et al., 2007). This review is aimed to
provide an overview of main factors, such as protozoan-host, biophysical and
physiochemical parameters, regulation of L. pneumophila endogenous
factors, and finally role of non-protozoan bacteria in the production and
colonization of biofilms by L. pneumophila.

2-Biofilm: A Safe
Habitat for L. pneumophila

Biofilm is a community of attached microorganisms, sticking on biotic
or abiotic surfaces in which trophic reactions occur. Biofilms have a dynamic
characteristic. It means that they can change in time and space in order to
provide a better survival and growth condition for the community of
microorganisms (Kolter et al., 2000). In order to understand how L. pneumophila
can survive and replicate within biofilms, we need to analyze the main
chemical and biophysical parameters, molecular basis, and also the role of
protozoan and non-protozoan bacteria in  the formation of L. pneumophila biofilm
formation. Thus, this section is divided into four sections as follows:

host biofilm formation by L. pneumophila

and  physiochemical parameters in biofilm
formation and colonization by L. pneumophila

of L. pneumophila endogenous factors influencing biofilm formation

Role of non-protozoa microbial species in L. pneumophila biofilm formation


Formation of  Protozoan- host
biofilm by L. pneumophila


are important habitat that allow the pathogen to replicate itself intracellular
(Rowbotham, 1981). Protozoa provide living conditions for the survival and
replication of Legionella species and therefore they are essential in
the survival of Legionella forms (Barker et al., 1993). There are
several protozoa, such as amoeba species in which L. pneumophila can be
hosted. Thus, protozoa are the most possible habitat for the growth of L.
pneumophila (Valster et al., 2010). In other words, there is a correlation
between the amount of L. pneumophila and biomass of protozoa (Liu et
al., 2012). L. pneumophila can also potentially grow off the dead
protozoa, so that may also encourage the multiplication of L. pneumophila
indirectly (Temmerman et al., 2006). Moreover, biofilms that are capable of
floating, contain L. pneumophila hosted by protozoa, suggesting that L.
pneumophila can adhere to protozoa in floating biofilm in the absence of
available abiotic surfaces (Hsu et al., 2011). Protozoa play an important role
not 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 to
disinfect water system (Donlan et al., 2005). Figure 1 shows an infected protozoan
species (amoeba) with L. pneumophila.



1: an infected amoebae with L. pneumophila, expressing green fluorescent
protein (valster, 2010)




Biophysical and Physiochemical Parameters in L. pneumophila Biofilm
Formation and colonization

For the production of biofilm formation by L. pneumophila and
also its colonization, physiochemical parameters should be analyzed. Figure 2
shows the replication of L. pneumophila (indicated by Lpn and shown in
orange) within protozoa (Wright, 1989).



Figure 2:  The replication of
L. pneumophila within environmental protozoa (Wright et al.,


Cations, known
as influential factors for the process of bacterial adhesion to the different surfaces
can also contribute to biofouling (Geesy et al., 2000). Similarly, calcium and
magnesium facilitated the adhesion of L. pneumophila to artificial
surfaces. Also, when zinc and manganese are available, L. pneumophila can
easily 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 at
20 ° C.  In other words, this result is a
proof of the influence of carbon in the production of biofilm at specific
temperatures (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, while
motionless 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 reason
for that is bacteria are able to reside themselves into the sediment in order
to survive from dynamic conditions and therefore they are less affected by the
turbulence (Stout et al., 1985).

Molecular factors also help L.
pneumophila to form biofilms. Collagen-like protein (Lcl), known as an
adhesin is a cell-component that facilitate adhesion to the surface. Hence, it
plays a major  role in the formation of L.
pneumophila biofilm (Vandersmissen
et al., 2010).
Also, Type IV pili (T4P), known as surface-exposed fibers contribute to the
colonization of L. pneumophila in the biofilm (Duncan et al., 2011).

iii)    Endogenous Factors Influencing Biofilm Community of L.


Biofilm formation is an
environmental response for the survival L. pneumophila. The production of
biofilm is also affected by an important environmental prompt, iron. It plays a
major 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 be
seen the importance of iron in L. pneumophila viability (Orsi, 2004).
Although iron is vital for the formation of biofilm, higher concentration of
iron can prevent biofilm formation.

Quorum sensing, as a vital
process during biofilm production, is the response  of bacteria to cell density. ?-hydroxy ketones
(AHKs), are quorum sensing molecules that exist in L. pneumophila. These
molecules are more likely in charge of a wide variety of traits that may impact
the production of  L. pneumophila
biofilm (Kessler, et al., 2013).

Temperature is also another
important factor for biofilm colonization (Martinelli et al., 2000). Moreover,
temperature affects the chracteristics of  biofilms. In lab-scale experiments, at
temperature between 37-42° C, biofilms mostly contain filamentous bacteria
while rod shaped bacterial cells are the main community of  biofilms at lower temperature (25 °C). In an
experiment, it was shown that the production of biofilms at  higher temperature (37° C ) are much stronger than
at lower temperature (25 ° C)  (Mampel  et al., 2006). Interestingly, the biofilms
formed 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.,


Figure 3: Confocal laser scanning
micrographs of GFP; expressing L. pneumophila Philadelphia-1 biofilms
formed under static conditions in BYE medium at 25°C and 37°C (Piao et al., 2006)


The Role of Non-Protozoa Microbial Species in L. pneumophila Biofilm

In addition to protozoa, biofilms contain different
types of bacteria. These bacteria are more likely promoters to increase the
lifespan of L. pneumophila (Messsi, et al., 2011).

In an experiment, loss of
protozoa due to the temperature rise did not change the replication of L.
pneumophila. Thus, it suggests that L. pneumophila can survive and
continue the process of multiplication without the presence of protozoa (Temmerman et al., 2006).

P. aeruginosa is a bacterial species, which can
influence the colonization ability of L. pneumophila in biofilms.
Although the replication and growth of L. pneumophila in natural
environmental biofilms is also attributed to P. aeruginosa, the presence
of other bacterial species together with P. aeruginosa promote the
increasing growth of  L. pneumophila
in a multispecies medium(Swart et al., 2012).



L. pneumophila is a
pathogenic bacterium, which can be found in both human-made and natural
environments (Yu et al., 2002). Here, we studied the different factors and
parameters 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 of
non-protozoan bacterial community. The physiochemical parameters, known as
important factors in biofilm formation attract a lot of  attention. Recent research has gained insight
into these factors, which can control the replication of L. pneumophila
and more likely to be useful for the prevention of legionellosis disease. Researchers
are also more interested in studying the role of other bacterial species in the
production of L. pneumophila biofilm (Temmerman et al., 2006). Another question that still has not been answered
is that how intercellular lifestyle of L. pneumophila helps to biofilm
resistance to disinfection methods (Alleron et al., 2008). Finally, this
research gives precious information about the ecology of this pathogen in biofilms
that can be used for future research in order to find preventive and protective
methods to fight against L. pneumophila infections (Hindre et al., 2008).














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