**1. Introduction**

*L. pneumophila*, the causative agent of Legionellosis, as being pathogenic to humans was following an outbreak of pneumonia at a convention of the American Legion in Philadelphia, USA in July 1976 [1]. This pathogen causes a severe form of pneumonia termed Legionnaires' disease (LD), and less frequently, Pontiac fever, a self-limited flu-like illness. Approximately 90% of LD cases are caused by *L. pneumophila*. Transmission of *L. pneumophila* occur primarily through the spread of contaminated aerosols present in cooling towers, condensers, faucets, showers, and hot tubs [2, 3]. Although stringent water quality examinations, the

formation of contaminated aerosols remains to be a major problem associated with disease spread [4].

Multiple mechanisms of persistence are harbored by *L. pneumophila* in various environmental conditions and in humans. Following invasion of amoeba or human macrophages, *L. pneumophila* form the *Legionella*-containing vacuole (LCV), which acquires vesicles from early and late endosomes, mitochondria and the endoplasmic reticulum (ER), thus escaping the microbicidal endocytic pathway. Hijacking the endocytic pathway by LCV is fundamental in initiating and maintaining a niche that secure *L. pneumophila* replication [5, 6]. Importantly, a battery of effector proteins produced by the Dot/Icm type IV secretion system of *L. pneumophila.* The Dot/Icm secreted effectors are required for successful intracellular replication of *L. pneumophila* [7–13]. Like other intracellular bacteria, *L. pneumophila* switch between a transmissive (virulent) and replicative (non-virulent) biphasic cycles. This switch is essential to ensure bacterial replication in nutrient starved or rich environments and transmit between different niches [14]. Nutrient rich environment is conducive of the replicative phase, where *L. pneumophila* express few virulence factors. While nutrient deprived environment is promotive of the transmissive phase, especially when the phagosome is unable to support the replication phase of *L. pneumophila*. Hallmark features of the transmissive phase include, increased motility, expression of plethora of virulence factors, resistance to stressors and egress from the infected host [14]. In the environment, *L. pneumophila* survive as free living (planktonic) or form bacterial biofilms with other organisms that adhere to surfaces [15–20]. Moreover, *L. pneumophila* is able to differentiate into inert, cyst-like but extremely infectious mature intracellular form (MIF) [21, 22]. Resilience of *L. pneumophila* extracellularly and under harsh environmental settings is attributed to its ability to exist in viable non-culturable (VBNC) state [23, 24]. Harboring a VBNC mode hinders the detection of many *Legionella* species. In nature, colonization and persistence is promoted via biofilm formation [25], and survival within freshwater amoeba and *C. elegans* [5, 26].

Herein, we review factors that mediate biofilm persistence, strategies utilized by the bacteria to become a member of the biofilm consortium and modes of eradicating *L. pneumophila* biofilm.

### **1.1 Constituents of** *L. pneumophila* **biofilm**

*L. pneumophila* is found as sessile cells associated with biofilms in freshwater environments, [19, 27, 28]. Biofilms mediate bacterial attachments to surfaces and to other pre-attached bacterial communities. Attachment is attained via forming an extracellular matrix (ECM) that is composed mainly of water, proteins, exopolysaccharides, lipids, DNA and RNA, and inorganic compounds [29–32]. Three developmental phases are required for biofilm formation. (I) initial attachments to a surface, (II) maturation and extracellular matrix formation, and (III) detachments and dispersion of the bacteria. Biofilms eventually develop into three-dimensional structures containing water channels, which allow bacteria to obtain nutrients, oxygen and get rid of waste products. The behavior of *L. pneumophila* has mainly been studied in the context of mono- or mixed species biofilms, due to the complexity of biofilm formed in natural environment [17–19, 33, 34]. Interestingly, *L. pneumophila* exhibit minor representation among other species in freshwater and environmental biofilms, [27, 28], and the existence of *L. pneumophila* may be influenced by other microorganisms in complex biofilms [35]. Some bacterial species positively or negatively affect the persistence of *L. pneumophila* biofilm [19]. Intriguingly, *Klebsiella pneumoniae (K. pneumoniae)*, *Flavobacterium* sp., *Empedobacter breve, Pseudomonas putida*

**263**

other bacteria.

*Biofilm, a Cozy Structure for* Legionella pneumophila *Growth and Persistence...*

can directly affect growth of *L. pneumophila* within biofilms.

*L. pneumophila* form biofilm when grown statically at 37°C for 7 days.

survival and association in the biofilm community [19, 42].

**1.2 Formation of biofilms as a survival niche in oligotrophic environment**

and *Pseudomonas fluorescens* positively associated with the long-term persistence of *L. pneumophila* in biofilms [18, 19, 36]. Other species within biofilms seem to be the provider of capsular polysaccharides, extracellular matrix that support the adherence [37–39], or the contributor of growth factors that stimulate growth of *L. pneumophila* [19]. *Pseudomonas aeruginosa* (*P. aeruginosa*), *Aeromonas hydrophila*, *Burkholderia cepacia*, *Acidovorax* sp., and *Sphingomonas* sp. [40] are among species that antagonize the persistence of *L. pneumophila* within the biofilm [19]. Inhibition of *L. pneumophila* biofilm by *P. aeruginosa* could be a consequence of the effect of homoserine lactone quorums sensing (QS) molecule [41], or production of bacteriocin [40]. Interestingly, *L. pneumophila* can coexist in biofilm formed by *P. aeruginosa* and *K. pneumoniae* indicating that the inhibition of *L. pneumophila* biofilm formation by *P. aeruginosa* can be alleviated by the permissive *K. pneumoniae* [19]. The authors suggest that the growth provided by *K. pneumoniae* to promote survival of *L. pneumophila* can at the same time lessen the inhibitory effect by *P. aeruginosa* [19]. Therefore, the identity, number and nature of interactions between bacterial species (commensalism or interference)

Biofilm formation of *L. pneumophila* in the laboratory is achieved by growing the bacteria under stringent conditions in nutrient-rich Buffered Yeast Extract medium (BYE) [18, 34]. Different temperatures correlated with different amount, degree of attachment and rate of biofilm formation. Mushroom like structure containing water channels is the hallmark features of biofilms formed at 25°C. In contrast, at 37°C *L. pneumophila* biofilm is thicker and deficient of water channels observed at 25°C. However, filamentous appearance with mat-like structure has been observed with *L. pneumophila* grown at 42°C. Studies in our laboratory showed that in contrast to the *dotA* mutant that lacks the type IV secretion, WT

Our knowledge is lacking regarding the factors encoded by *L. pneumophila* that promote the attachment and persistence within multispecies biofilms created by

Biofilm is extremely nutritious environment that harbors a mixture of living, dead organisms as well as protozoa and bacteria. To be a productive member of the microbial consortium, *L. pneumophila* has to compete with other bacteria for nutrients in a multispecies biofilm. Therefore, it is essential for *L. pneumophila* to strive in an environment adjacent to bacterial neighbors that best sustains their growth and survival [42]. Given the fastidious and auxotrophic nature of *L. pneumophila*, supplementation of the laboratory media with amino acids and iron is essential for growth [43, 44]. However, the ability of *L. pneumophila* to survive in oligotrophic environments is puzzling and suggests that the bacteria can live on a diet provided by other members in the biofilm community. To overcome the starvation mode in oligotrophic environment, *L. pneumophila* incorporate in two- and multispecies biofilms. Instead of attaching as a primary colonizer, *L. pneumophila* use a strategic mode where they dock to a pre-established biofilm, thus mediating bacterial

Obtaining the required carbon, nitrogen, and amino acids for replication of *L. pneumophila* seems to be primarily reliant on necrotrophic feeding on the products of dead bacteria and tissues within the biofilm [35, 36]. Moreover, heterotrophic bacteria support growth of *L. pneumophila* on media that does not usually support growth because it is deficient in L-cysteine and ferric pyrophosphate [45]. Consistent with this, *L. pneumophila* showed satellite colonies around some aquatic

*DOI: http://dx.doi.org/10.5772/intechopen.89156*

*Biofilm, a Cozy Structure for* Legionella pneumophila *Growth and Persistence... DOI: http://dx.doi.org/10.5772/intechopen.89156*

and *Pseudomonas fluorescens* positively associated with the long-term persistence of *L. pneumophila* in biofilms [18, 19, 36]. Other species within biofilms seem to be the provider of capsular polysaccharides, extracellular matrix that support the adherence [37–39], or the contributor of growth factors that stimulate growth of *L. pneumophila* [19]. *Pseudomonas aeruginosa* (*P. aeruginosa*), *Aeromonas hydrophila*, *Burkholderia cepacia*, *Acidovorax* sp., and *Sphingomonas* sp. [40] are among species that antagonize the persistence of *L. pneumophila* within the biofilm [19]. Inhibition of *L. pneumophila* biofilm by *P. aeruginosa* could be a consequence of the effect of homoserine lactone quorums sensing (QS) molecule [41], or production of bacteriocin [40]. Interestingly, *L. pneumophila* can coexist in biofilm formed by *P. aeruginosa* and *K. pneumoniae* indicating that the inhibition of *L. pneumophila* biofilm formation by *P. aeruginosa* can be alleviated by the permissive *K. pneumoniae* [19]. The authors suggest that the growth provided by *K. pneumoniae* to promote survival of *L. pneumophila* can at the same time lessen the inhibitory effect by *P. aeruginosa* [19]. Therefore, the identity, number and nature of interactions between bacterial species (commensalism or interference) can directly affect growth of *L. pneumophila* within biofilms.

Biofilm formation of *L. pneumophila* in the laboratory is achieved by growing the bacteria under stringent conditions in nutrient-rich Buffered Yeast Extract medium (BYE) [18, 34]. Different temperatures correlated with different amount, degree of attachment and rate of biofilm formation. Mushroom like structure containing water channels is the hallmark features of biofilms formed at 25°C. In contrast, at 37°C *L. pneumophila* biofilm is thicker and deficient of water channels observed at 25°C. However, filamentous appearance with mat-like structure has been observed with *L. pneumophila* grown at 42°C. Studies in our laboratory showed that in contrast to the *dotA* mutant that lacks the type IV secretion, WT *L. pneumophila* form biofilm when grown statically at 37°C for 7 days.

Our knowledge is lacking regarding the factors encoded by *L. pneumophila* that promote the attachment and persistence within multispecies biofilms created by other bacteria.

### **1.2 Formation of biofilms as a survival niche in oligotrophic environment**

Biofilm is extremely nutritious environment that harbors a mixture of living, dead organisms as well as protozoa and bacteria. To be a productive member of the microbial consortium, *L. pneumophila* has to compete with other bacteria for nutrients in a multispecies biofilm. Therefore, it is essential for *L. pneumophila* to strive in an environment adjacent to bacterial neighbors that best sustains their growth and survival [42]. Given the fastidious and auxotrophic nature of *L. pneumophila*, supplementation of the laboratory media with amino acids and iron is essential for growth [43, 44]. However, the ability of *L. pneumophila* to survive in oligotrophic environments is puzzling and suggests that the bacteria can live on a diet provided by other members in the biofilm community. To overcome the starvation mode in oligotrophic environment, *L. pneumophila* incorporate in two- and multispecies biofilms. Instead of attaching as a primary colonizer, *L. pneumophila* use a strategic mode where they dock to a pre-established biofilm, thus mediating bacterial survival and association in the biofilm community [19, 42].

Obtaining the required carbon, nitrogen, and amino acids for replication of *L. pneumophila* seems to be primarily reliant on necrotrophic feeding on the products of dead bacteria and tissues within the biofilm [35, 36]. Moreover, heterotrophic bacteria support growth of *L. pneumophila* on media that does not usually support growth because it is deficient in L-cysteine and ferric pyrophosphate [45]. Consistent with this, *L. pneumophila* showed satellite colonies around some aquatic

*Bacterial Biofilms*

with disease spread [4].

and *C. elegans* [5, 26].

ing *L. pneumophila* biofilm.

**1.1 Constituents of** *L. pneumophila* **biofilm**

formation of contaminated aerosols remains to be a major problem associated

Multiple mechanisms of persistence are harbored by *L. pneumophila* in various environmental conditions and in humans. Following invasion of amoeba or human macrophages, *L. pneumophila* form the *Legionella*-containing vacuole (LCV), which acquires vesicles from early and late endosomes, mitochondria and the endoplasmic reticulum (ER), thus escaping the microbicidal endocytic pathway. Hijacking the endocytic pathway by LCV is fundamental in initiating and maintaining a niche that secure *L. pneumophila* replication [5, 6]. Importantly, a battery of effector proteins produced by the Dot/Icm type IV secretion system of *L. pneumophila.* The Dot/Icm secreted effectors are required for successful intracellular replication of *L. pneumophila* [7–13]. Like other intracellular bacteria, *L. pneumophila* switch between a transmissive (virulent) and replicative (non-virulent) biphasic cycles. This switch is essential to ensure bacterial replication in nutrient starved or rich environments and transmit between different niches [14]. Nutrient rich environment is conducive of the replicative phase, where *L. pneumophila* express few virulence factors. While nutrient deprived environment is promotive of the transmissive phase, especially when the phagosome is unable to support the replication phase of *L. pneumophila*. Hallmark features of the transmissive phase include, increased motility, expression of plethora of virulence factors, resistance to stressors and egress from the infected host [14]. In the environment, *L. pneumophila* survive as free living (planktonic) or form bacterial biofilms with other organisms that adhere to surfaces [15–20]. Moreover, *L. pneumophila* is able to differentiate into inert, cyst-like but extremely infectious mature intracellular form (MIF) [21, 22]. Resilience of *L. pneumophila* extracellularly and under harsh environmental settings is attributed to its ability to exist in viable non-culturable (VBNC) state [23, 24]. Harboring a VBNC mode hinders the detection of many *Legionella* species. In nature, colonization and persistence is promoted via biofilm formation [25], and survival within freshwater amoeba

Herein, we review factors that mediate biofilm persistence, strategies utilized by the bacteria to become a member of the biofilm consortium and modes of eradicat-

*L. pneumophila* is found as sessile cells associated with biofilms in freshwater environments, [19, 27, 28]. Biofilms mediate bacterial attachments to surfaces and to other pre-attached bacterial communities. Attachment is attained via forming an extracellular matrix (ECM) that is composed mainly of water, proteins, exopolysaccharides, lipids, DNA and RNA, and inorganic compounds [29–32]. Three developmental phases are required for biofilm formation. (I) initial attachments to a surface, (II) maturation and extracellular matrix formation, and (III) detachments and dispersion of the bacteria. Biofilms eventually develop into three-dimensional structures containing water channels, which allow bacteria to obtain nutrients, oxygen and get rid of waste products. The behavior of *L. pneumophila* has mainly been studied in the context of mono- or mixed species biofilms, due to the complexity of biofilm formed in natural environment [17–19, 33, 34]. Interestingly, *L. pneumophila* exhibit minor representation among other species in freshwater and environmental biofilms, [27, 28], and the existence of *L. pneumophila* may be influenced by other microorganisms in complex biofilms [35]. Some bacterial species positively or negatively affect the persistence of *L. pneumophila* biofilm [19]. Intriguingly, *Klebsiella pneumoniae (K. pneumoniae)*, *Flavobacterium* sp., *Empedobacter breve, Pseudomonas putida*

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bacteria including *Flavobacterium breve*, *Pseudomonas spp*., *Alcaligenes spp*., and *Acinetobacter spp*. Further, *L. pneumophila* are able to obtain nutrients directly from algae and to grow on the extracellular products produced by cyanobacteria under laboratory conditions [46]. Further, several algae such as *Scenedesmus* spp., *Chlorella* spp., and *Gloeocystis* spp., supported the growth of *L. pneumophila* in basal salt media [28].

The second mechanism by which *L. pneumophila* obtain nutrient in biofilms is through amoeba. Amoeba serve as a secure niche that provides the environmental host for survival and replication of *Legionella* species in the environment [47, 48], and protect the bacteria from antibacterial agents [49]. Importantly, pathogenesis of *L. pneumophila* is correlated with persistence and adaptation of *L. pneumophila* in various amoebal hosts, and the nature of protozoal species can directly affect biofilm colonization with *L. pneumophila* [50, 51]. Indeed, *L. pneumophila* can parasitize more than 20 species of amoebae, three species of ciliated protozoa and one species of slime mold [52, 53]. Further, multiplication within amoeba mediated increase production of polysaccharides by *L. pneumophila,* thus enhancing its capacity to establish biofilm [54]. Further, debris from dead amoeba has been shown to support *L. pneumophila* growth [55], and the biomass of protozoa is directly correlated with outbreaks of *L. pneumophila*. Moreover, absence of amoeba did not result to an increase in the number of biofilm-associated *L. pneumophila*. Instead, *L. pneumophila* can enter the VBNC state to mediate their survival [28]. It has been suggested that metazoan such as the *C. elegans* could provide a natural host for *L. pneumophila* [56, 57]. Moreover, *L. pneumophila* survive within biofilm containing protozoan and *C. elegans* [58]. Therefore, harnessing nutrient from mixed species biofilms as well as survival in the amoeba and *C. elegans* enhances the persistence of *L. pneumophila*. Therefore, diversity of biofilm-associated organisms would provide a various means of nutrient acquisition in oligotrophic environment for such a fastidious organism.
