ArticleLiterature Review

Biofilms: The environmental playground of Legionella pneumophila

Authors:
To read the full-text of this research, you can request a copy directly from the author.

Abstract

Legionella pneumophila, the aetiological agent of 90% of legionellosis cases, is a common inhabitant of natural and anthropogenic freshwater environments, where it resides in biofilms. Biofilms are defined as complex, natural assemblages of microorganisms that involve a multitude of trophic interactions. A thorough knowledge and understanding of Legionella ecology in relation to biofilm communities is of primary importance in the search for innovative and effective control strategies to prevent the occurrence of disease cases. This review provides a critical update on the state-of-the-art progress in understanding the mechanisms and factors affecting the biofilm life cycle of L. pneumophila. Particular emphasis is given to discussing the different strategies this human pathogen uses to grow and retain itself in biofilm communities. Biofilms develop not only at solid-water interfaces (substrate-associated biofilms), but also at the water-air interface (floating biofilms). Disturbance of the water surface can lead to liberation of aerosols derived from the floating biofilm into the atmosphere that allow transmission of biofilm-associated pathogens over considerable distances. Recent data concerning the occurrence and replication of L. pneumophila in floating biofilms are also elaborated and discussed.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the author.

... 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. ...
... 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]. ...
... 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]. ...
... Unsurprisingly, most of these field-based studies are grounded in Legionella screenings in the water, while exploring the potential of new inputs from molecular tools [29]. However, they often ignore the role of ecological niches, such as protozoa and biofilms, as critical spots for Legionella settlement, adaptation, and infectivity [30,31]. Biofilm sampling is actually within the scope of routine Legionella environmental surveillance by many reference documents [23][24][25], yet biofilm sampling and analysis lack standard sampling and analytical practices [32,33] that can give consistent and representative outputs. ...
... The first aspect is linked to the ecology of the bacteria. Legionella is a bacteria that, despite its fastidious nutritional requirements, survives and adapts to different conditions [22,31]. The parasitic lifestyle with protozoa [100] and the synergies established in biofilms [103,104], as well as their ability to enter the viable-but-non-culturable cell (VBNC) state [105], seem to be key to Legionella's successful persistence under harsh external stresses. ...
... As suggested by hypothesis (a), L. pneumophila can survive as a free-living organism, yet its ability to grow to significant concentrations without a host seems to be very limited [25]. Growing L. pneumophila in a laboratory is a difficult task, involving an unusual set of nutrient requirements [31,107] that are not commonly found in fresh water [22]. This seems to contradict the wide spread of the bacteria and their ability to proliferate in such oligotrophic (nutrient-scarce) environments. ...
Article
Full-text available
Legionella is responsible for the life-threatening pneumonia commonly known as Legionnaires’ disease or legionellosis. Legionellosis is known to be preventable if proper measures are put into practice. Despite the efforts to improve preventive approaches, Legionella control remains one of the most challenging issues in the water treatment industry. Legionellosis incidence is on the rise and is expected to keep increasing as global challenges become a reality. This puts great emphasis on prevention, which must be grounded in strengthened Legionella management practices. Herein, an overview of field-based studies (the system as a test rig) is provided to unravel the common roots of research and the main contributions to Legionella’s understanding. The perpetuation of a water-focused monitoring approach and the importance of protozoa and biofilms will then be discussed as bottom-line questions for reliable Legionella real-field surveillance. Finally, an integrated monitoring model is proposed to study and control Legionella in water systems by combining discrete and continuous information about water and biofilm. Although the successful implementation of such a model requires a broader discussion across the scientific community and practitioners, this might be a starting point to build more consistent Legionella management strategies that can effectively mitigate legionellosis risks by reinforcing a pro-active Legionella prevention philosophy.
... pneumophila in the built environment. Biofilms are defined as microbial aggregates adhered to a biotic or abiotic surface and enclosed in a self-generated extracellular matrix (89). Biofilms are noted for being highly resistant to chemical treatment, antibiotics, and other biocides (90). ...
... Pseudomonas aeruginosa, Legionella spp. do not appear to form monospecies biofilms in the natural environment (89). Rather, it is likely that planktonic L. pneumophila colonize pre-existing biofilms formed by other aquatic bacteria. ...
... Legionella pneumophila is a gram-negative bacterium commonly found in aquatic environments, where it replicates within protozoan hosts and persists within biofilms (89). When inhaled, contaminated water droplets transmit L. pneumophila to the human lung, where this opportunistic pathogen can infect alveolar macrophages. ...
Thesis
Legionella pneumophila is a waterborne pathogen and the causative agent of the severe pneumonia Legionnaires’ Disease. This bacterium is an intracellular parasite of both human lung macrophages, as well as amoeba and ciliated protists, its natural host. Within the environment, L. pneumophila exists as either an intracellular parasite of protozoa, within biofilms, or as free-living, planktonic cells. Unfortunately, remediation of contaminated water systems has proven an ongoing challenge, mostly due to the ability of L. pneumophila to differentiate into an environmentally resistant, highly infectious but spore-like cell type called a Mature Infectious Form (MIF). The genetic pathways and regulatory mechanisms that promote MIF cell development remain largely unknown. A few proteins have been identified as abundant in MIF cells, however, including Lpg0279. In this dissertation, I explore the impact of Lpg0279 on L. pneumophila cellular differentiation and its interaction with a two-component system encoded by lpg0278-lpg0277. I show that lpg0279 is co-transcribed with lpg0278 and lpg0277, expression is controlled by the stationary phase sigma factor RpoS, and that transcription is enhanced in nutrient-limited conditions. The two-component system encoded by lpg0278-lpg0277 modulates the turnover of the second messenger molecule cyclic-di-GMP. This ubiquitous molecule directs lifestyle changes in multiple bacterial species. The L. pneumophila genome codes for multiple proteins that make and break cyclic-di-GMP, but few studies have been conducted examining the impact of this second messenger molecule on the L. pneumophila life cycle. Lpg0277 is a bifunctional enzyme and the response regulator of the two-component system. Using isogenic mutants and a series of phenotypic assays, I show that the diguanylate cyclase activity of Lpg0277 (and therefore an increase in c-di-GMP) promotes transition of L. pneumophila to the stationary phase of growth, the generation of pyomelanin pigment and poly-3-hydroxybutyrate granules, and contributes to long-term survival in low-nutrient conditions. In addition, I provide evidence to support a model in which Lpg0279 initially acts as a negative regulator of this enzymatic activity. This research provides important insight into how c-di-GMP contributes both to L. pneumophila differentiation, and to persistence of this bacterium in low-nutrient environments. This information can inform future surveillance methods, detection of MIF cells, and remediation efforts to control spread of this pathogen in the built environment.
... La matrice d'EPS n'est pas une structure précisément définie et sa composition dépend des espèces microbiennes se trouvant à l'intérieur du biofilm (Wingender et al. , 1999). En plus de polysaccharides, cette matrice est composée de protéines, d'acides nucléiques, de lipides et d'autres biopolymères telles que des substances humiques qui participent au maintien de la structure et de la morphologie du biofilm (Flemming et Wingender, 2001;2010;Wingender et al. , 1999). Enfin, les EPS représentent une source de nutriment pour les microorganismes et par conséquent, jouent un rôle essentiel en écologie microbienne (Flemming et Wingender, 2010). ...
... Dans les environnements d'eau douce, L. pneumophila peut être retrouvée sous forme planctonique ou au sein de biofilms (Declerck, 2010;Declerck et al. , 2009). La présence de Legionella dans les biofilms environnementaux peut également être affectée par d'autres microorganismes Taylor et al. , 2009). ...
... They are part of the natural flora in many 13 freshwater environments (i.e. rivers, streams) where they occur in relatively low numbers 14 (Declerck, 2010;Declerck et al., 2009). L. pneumophila is recognized as responsible for 15 ...
Thesis
Les circuits et réseaux d’eau subissent épisodiquement des problèmes de contamination par des microorganismes tels que Legionella pneumophila, conduisant à la dégradation de la qualité microbiologique de l’eau circulante. De nouveaux moyens de traitements doivent être mis en place de façon notamment à limiter l’usage de biocides chimiques. Ce travail de thèse s’inscrit dans cette problématique et a pour objectif d’identifier de nouveaux composés actifs contre L. pneumophila en tenant compte de son comportement et de ses interactions au sein de son microenvironnement. De manière à obtenir des composés produits par des souches bactériennes appartenant à la même niche écologique que L. pneumophila, une campagne de prélèvement d’eau a été réalisée. Un total de 273 isolats environnementaux ont été isolés et testés contre L. pneumophila. Les résultats obtenus indiquent que 178 de ces isolats (65%) présentent une activité anti-Legionella. Quatre souches ont ensuite été sélectionnées (Aeromonas bestiarum SW257, Rahnella aquatilis SW265, Flavobacterium spp. PW52 et Pseudomonas spp PW329) et les composés actifs produits ont été caractérisés. A. bestiarum SW257 produit un peptide anti-Legionella. Flavobacterium sp. PW52 produit un mélange de composés anti-Legionella ayant des propriétés tensioactives, les flavolipides. Pseudomonas sp. PW329 produit des composés organiques volatiles, identifiés par SPME-GC-MS, actifs contre L. pneumophila. Enfin, R. aquatilis SW265 produit un sidérophore à activité anti-Legionella.
... Since Legionellales are common in the environment, an increased local abundance may thus quickly lead to fatal local disease outbreaks (Morton et al., 1986;van Loenhout et al., 2012). Under natural conditions, Legionellales bacteria rely on a eukaryotic host to proliferate (Fields et al., 2002;Declerck et al., 2009;Declerck, 2010). Accordingly, fluctuations in the abundance of Legionellales bacteria are strongly linked to fluctuations of the abundance of their hosts in the environment (Scheikl et al., 2014;Amaro et al., 2015). ...
... To our knowledge, it is still questionable whether Legionellales replicate under natural conditions without a eukaryotic host (Fields et al., 2002). It was shown that a lack of amoebae prevents growth of the substrate-attached Legionella pneumophila (Declerck et al., 2009;Declerck, 2010). Legionella can be grown axenically in the lab, but for this, exact conditions have to be met. ...
Article
Full-text available
Legionellales-infected water is a frequent cause of local outbreaks of Legionnaires’ disease and Pontiac fever. Decontaminations are difficult because Legionellales reproduce in eukaryotic microorganisms (protists). Most often, Legionellales have been isolated from amoebae; however, the culture-based sampling methods are taxonomically biased. Sequencing studies show that amoebae in the cercozoan class Thecofilosea are dominant in soils and wastewater treatment plants, prompting us to screen their capability to serve as potential hosts of endosymbiotic bacteria. Environmental isolates of Thecofilosea contained a surprising richness of endosymbiotic Legionellales, including Legionella . Considering the widespread dispersal of Legionellales in apparently unrelated amoeboid protist taxa, it appears that the morphotype and not the evolutionary origin of amoebae determines their suitability as hosts for Legionellales. We further provide a protocol for gnotobiotic cultivation of Legionellales and their respective hosts, facilitating future genomic and transcriptomic research of host–symbiont relationships.
... These bacteria are members of the complex microbial communities found in drinking water systems, and interactions with other microorganisms can sometimes promote or inhibit their growth (Figure 1). For example, Legionella proliferate within protozoan hosts (e.g., Acanthamoeba spp.), exploiting their intracellular environment to replicate and gain protection against external stressors (Declerck, 2010, Taylor et al. 2009). Also, several laboratory-scale studies have shown bacterial isolates that directly benefit or inhibit Legionella growth on pure culture agar plates ). ...
... When evaluating feasibility of B. bacteriovorus as a predatory biocontrol agent against L. pneumophila in engineered aquatic ecosystems, multiple aspects have to be taken into consideration. First is the efficacy in reducing multispecies biofilms, as this is a primary niche for Legionella (Declerck 2010). In general, B. bacteriovorus has shown promising efficacy to prevent and reduce biofilms colonized by a wide range of Gram-negative pathogens in a laboratory setting (Dashiff et al. 2011, Kadouri and O'Toole 2005, Sun et al. 2017). ...
Article
Full-text available
Opportunistic pathogens belonging to the genus Legionella are among the most reported waterborne-associated pathogens in industrialized countries. Legionella colonize a variety of engineered aquatic ecosystems and persist in biofilms where they interact with a multitude of other resident microorganisms. In this review, we assess how some of these interactions could be used to develop a biological-driven “probiotic” control approach against Legionella. We focus on: (1) mechanisms limiting the ability of Legionella to establish and replicate within some of their natural protozoan hosts; (2) exploitative and interference competitive interactions between Legionella and other microorganisms; and (3) the potential of predatory bacteria and phages against Legionella. This field is still emergent, and we therefore specifically highlight research for future investigations, and propose perspectives on the feasibility and public acceptance of a potential probiotic approach.
... Different aspects of the interactions of Lp with biofilms or floating biofilms have been previously reviewed (e.g. Lau & Ashbolt, 2009;Declerck, 2010;Abdel-Nour et al., 2013), but here I will present aspects relevant to the discussion about the extracellular growth of Lp in freshwater environments. The general consensus seems to be that planktonic Lp colonizes pre-existing biofilms and survives within their complex microbial community, albeit Lp replication is dependent on the presence of amoeba. ...
... Another aspect of Lp and biofilms, is growth in floating biofilms (reviewed by Declerck, 2010). As explained above, contrary to the most common form of biofilms (which grow on a solid-water interface), floating biofilms are formed at the water-air interface. ...
... Legionella are known to survive at temperatures between 5 and 63 C and have a pH range between 5.0 and 9.2. 19 Should the bacteria be internalized within a protozoan host, when that host enters into a cyst stage, Legionella too will be protected from unfavourable conditions such as extreme heat, biocides, osmotic stress etc. 19,20 This survival mechanism most likely has a major impact on Legionella's ability to persist in the environment, when considered along with its ability to form biofilms. ...
... Legionella are known to survive at temperatures between 5 and 63 C and have a pH range between 5.0 and 9.2. 19 Should the bacteria be internalized within a protozoan host, when that host enters into a cyst stage, Legionella too will be protected from unfavourable conditions such as extreme heat, biocides, osmotic stress etc. 19,20 This survival mechanism most likely has a major impact on Legionella's ability to persist in the environment, when considered along with its ability to form biofilms. ...
Article
The risk of Legionella transmission from rain water harvesting (RWH)-fed water toilet flushing was investigated. Experiments monitored RWH tanks to determine Legionella spp. presence which was successfully isolated from three of four of RWH tanks (site 1: 3600 CFU/dm³ in February and 3600 CFU/dm³ in May; site 2: not detected; site 3: <940 GU/dm³; site 4: 44,000 GU/dm³), and to determine whether toilets could generate aerosol droplets capable of harbouring bacteria. The concentrations of particles measuring 10 µm or less in diameter (PM10, capable of reaching the alveolar region of the lungs and causing disease) were monitored following flushes. Aerosols were detectable in an enclosed toilet cubicle (PM10 concentration increased in one experiment from 0.038 mg/m³ when t = 1–600 s to 0.057 mg/m³ when t = 600–800 s). Recovery of Lactobacillus plantarum (surrogate for Legionella) from a seeded toilet cistern (108 CFU ml/dm³) indicated that bacteria were expelled, as demonstrated by recovery on MRS plates placed around the toilet unit. Legionella could be dispersed via aerosols from a toilet flushed with water from an RWH system and the effect would be more pronounced in smaller, enclosed areas, but this is unlikely to pose a risk to human health.
... Long stagnation in premise plumbing led to the absence of disinfectant and promotes the growth of biofilm on the pipe internal surface (Lautenschlager et al., 2010;Ling et al., 2018;Rhoads et al., 2016). The presence of biofilms in the premise plumbing has been found to facilitate the survival of L. pneumophila (Buse et al., 2012;Cooper and Hanlon, 2010;Declerck, 2010;Declerck et al., 2009;Wang et al., 2017). However, the complex role of stagnation, disinfectant residual, biofilms, and opportunistic pathogens released from biofilms on public health has not been well characterized. ...
... Specifically, we used experimental measurements of the chlorine decay by biofilms grown in simulated premise plumbing (Xu et al., 2018) and the kinetics of biofilm-derived L. pneumophila release and inactivation (Shen et al., 2017). We chose to focus on the role of stagnation on residual chlorine decay by biofilms because stagnation was a key factor in promoting the growth of microbes in distribution systems (Prest et al., 2016) and biofilm-derived L. pneumophila are known to be more resistant to disinfectants than planktonic cells (Cooper and Hanlon, 2010;Declerck, 2010;Shen et al., 2017). The findings on infection risk under different stagnation durations and chlorine residual conditions may facilitate the decision on how to reduce premise plumbing infection risk from L. pneumophila. ...
Article
Full-text available
The ubiquitous presence of biofilms in premise plumbing and stagnation, which commonly occurs in premise plumbing, can exacerbate the decay of chlorine residual in drinking water. Using biofilms grown in a simulated premise plumbing setup fed directly with freshly treated water at two full-scale water treatment plants, we previously determined the mass transfer coefficients for chlorine decay in premise plumbing. These coefficients coupled with inactivation kinetics of L. pneumophila released from biofilms reported previously were integrated into a Monte Carlo framework to estimate the infection risk of biofilm-derived L. pneumophila from 1 to 48 h of stagnation. The annual infection risk was significantly higher when water stayed stagnant for up to 48 h in pipes covered internally with biofilms, compared to clean pipes without biofilms. The decay of residual chlorine due to biofilms during 48-hour stagnation led to up to 6 times increase in the annual infection risk compared to the case where biofilms was absent. Global sensitivity analysis revealed that the rate of L. pneumophila detachment from biofilms and the decay of chlorine residual during stagnation are the two most important factors influencing the infection risks. Stagnation caused by water use patterns and water-saving devices in the premise plumbing can lead to increased infection risk by biofilm-derived L. pneumophila. Overall, this study's findings suggested that biofilms could induce chlorine decay and consequently increase L. pneumophila infection risk. Thus, reducing stagnation, maintaining residual chlorine, and suppressing biofilm growth could contribute to better management of L. pneumophila infection risk.
... These conditions have been found to be conducive to the formation of biofilms, which often leads to the establishment and maintenance of chronic water system colonisation by Legionella. 14,15,16 Furthermore, the capacity of these bacteria to live in free-living protozoa such as amoebae, their natural host, can protect them from adverse conditions or biocide treatments. [17][18][19][20][21][22] Several decontamination and/or disinfection techniques are available to control the risk of Legionella infections. ...
... Using the haploid social soil amoeba Dictyostelium discoideum, genetic and pharmacological disruption of class I PI3Ks indicated that these kinases are largely dispensable for uptake of wild-type L. pneumophila, but required for uptake of an icm/dot mutant strain (51,64). Moreover, using D. discoideum producing a fluorescent probe for PtdIns (3,4,5)P 3 , live-cell microscopy revealed that this PI lipid accumulated at bacterial entry sites and was cleared within approximately 40 s after uptake, regardless of whether the amoebae were infected with wild-type or icm/dot mutant L. pneumophila. In parallel, plasma membrane PtdIns(4,5)P 2 disappeared from the uptake sites (65). ...
Article
Full-text available
Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. The environmental bacterium replicates in free-living amoebae as well as in lung macrophages in a distinct compartment, the Legionella-containing vacuole (LCV). The LCV communicates with a number of cellular vesicle trafficking pathways and is formed by a plethora of secreted bacterial effector proteins, which target host cell proteins and lipids. Phosphoinositide (PI) lipids are pivotal determinants of organelle identity, membrane dynamics and vesicle trafficking. Accordingly, eukaryotic cells tightly regulate the production, turnover, interconversion, and localization of PI lipids. L. pneumophila modulates the PI pattern in infected cells for its own benefit by (i) recruiting PI-decorated vesicles, (ii) producing effectors acting as PI interactors, phosphatases, kinases or phospholipases, and (iii) subverting host PI metabolizing enzymes. The PI conversion from PtdIns(3)P to PtdIns(4)P represents a decisive step during LCV maturation. In this review, we summarize recent progress on elucidating the strategies, by which L. pneumophila subverts host PI lipids to promote LCV formation and intracellular replication.
... The presence of some microbial species in such a multispecies community positively promotes and improves inter and intra-species communications as well as the overall integrity of the formed biofilm. A typical example of synergistic interaction in the aquatic environment is the occurrence of Legionella pneumophila (which is often a minority member) in multispecies biofilm in freshwater and other natural environments [35]. Some bacterial species constituting the multispecies biofilm readily promotes L. pneumophila survival and persistence in such community [36]. ...
Article
Full-text available
In the aquatic environment, microorganisms are predominantly organized as biofilms. Biofilms are formed by the aggregation of microbial cells and are surrounded by a matrix of extracellular polymeric substances (EPS) secreted by the microbial cells. Biofilms are attached to various surfaces, such as the living tissues, indwelling medical devices, and piping of the industrial potable water system. Biofilms formed from a single species has been extensively studied. However, there is an increased research focus on multispecies biofilms in recent years. It is important to assess the microbial mechanisms underlying the regulation of multispecies biofilm formation to determine the drinking water microbial composition. These mechanisms contribute to the predominance of the best-adapted species in an aquatic environment. This review focuses on the interactions in the multispecies biofilms, such as coaggregation, co-metabolism, cross-species protection, jamming of quorum sensing, lateral gene transfer, synergism, and antagonism. Further, this review explores the dynamics and the factors favoring biofilm formation and pathogen transmission within the drinking water distribution systems. The understanding of the physiology and biodiversity of microbial species in the biofilm may aid in the development of novel biofilm control and drinking water disinfection processes.
... It is tempting to conclude that most of Legionella was in the water phase in the system. The presence of Legionella in biofilms within water distribution systems has been reported in several studies (Abdel-Nour et al., 2013;Abu Kweek and Amer, 2018;Armon et al., 1997;Buse et al., 2014;Buse et al., 2012;Declerck, 2010;Lau and Ashbolt, 2009;Moritz et al., 2010;van der Kooij et al., 2005). Several factors influence the formation of biofilm by Legionella and its ability to integrate biofilms (Buse et al., 2017;Piao et al., 2006;Rhoads et al., 2017;Rogers et al., 1994a). ...
Article
Legionella pneumophila is a waterborne bacterium known for causing Legionnaires' Disease, a severe pneumonia. Cooling towers are a major source of outbreaks, since they provide ideal conditions for L. pneumophila growth and produce aerosols. In such systems, L. pneumophila typically grow inside protozoan hosts. Several abiotic factors such as water temperature, pipe material and disinfection regime affect the colonization of cooling towers by L. pneumophila. The local physical and biological factors promoting the growth of L. pneumophila in water systems and its spatial distribution are not well understood. Therefore, we built a lab-scale cooling tower to study the dynamics of L. pneumophila colonization in relationship to the resident microbiota and spatial distribution. The pilot was filled with water from an operating cooling tower harboring low levels of L. pneumophila. It was seeded with Vermamoeba vermiformis, a natural host of L. pneumophila, and then inoculated with L. pneumophila. After 92 days of operation, the pilot was disassembled, the water was collected, and biofilm was extracted from the pipes. The microbiome was studied using 16S rRNA and 18S rRNA genes amplicon sequencing. The communities of the water and of the biofilm were highly dissimilar. The relative abundance of Legionella in water samples reached up to 11% whereas abundance in the biofilm was extremely low (≤0.5%). In contrast, the host cells were mainly present in the biofilm. This suggests that L. pneumophila grows in host cells associated with biofilm and is then released back into the water following host cell lysis. In addition, water temperature shaped the bacterial and eukaryotic community of the biofilm, indicating that different parts of the systems may have different effects on Legionella growth.
... Environmental conditions affect the proliferation of Legionella bacteria in lakes, streams, and estuaries [11,12], and the contamination events that may lead to disease transmission [13,14]. The bacteria are abundant in aqueous environments [15] and survive by parasitizing amoebae, including many that persist in environmental biofilms [16,17]. The bacteria optimally grow in wet, warm conditions (between 25 and 42 °C) and flourish in sessile biofilm communities [18,19] in the natural and built environment [20,21]. ...
Article
Full-text available
Background An increasing severity of extreme storms and more intense seasonal flooding are projected consequences of climate change in the United States. In addition to the immediate destruction caused by storm surges and catastrophic flooding, these events may also increase the risk of infectious disease transmission. We aimed to determine the association between extreme and seasonal floods and hospitalizations for Legionnaires’ disease in 25 US states during 2000–2011. Methods We used a nonparametric bootstrap approach to examine the association between Legionnaires’ disease hospitalizations and extreme floods, defined by multiple hydrometeorological variables. We also assessed the effect of extreme flooding associated with named cyclonic storms on hospitalizations in a generalized linear mixed model (GLMM) framework. To quantify the effect of seasonal floods, we used multi-model inference to identify the most highly weighted flood-indicator variables and evaluated their effects on hospitalizations in a GLMM. Results We found a 32% increase in monthly hospitalizations at sites that experienced cyclonic storms, compared to sites in months without storms. Hospitalizations in months with extreme precipitation were in the 89th percentile of the bootstrapped distribution of monthly hospitalizations. Soil moisture and precipitation were the most highly weighted variables identified by multi-model inference and were included in the final model. A 1-standard deviation (SD) increase in average monthly soil moisture was associated with a 49% increase in hospitalizations; in the same model, a 1-SD increase in precipitation was associated with a 26% increase in hospitalizations. Conclusions This analysis is the first to examine the effects of flooding on hospitalizations for Legionnaires’ disease in the United States using a range of flood-indicator variables and flood definitions. We found evidence that extreme and seasonal flooding is associated with increased hospitalizations; further research is required to mechanistically establish whether floodwaters contaminated with Legionella bacteria drive transmission.
... Legionella spp. can exist within biofilms or amoebal hosts in freshwater systems, transitioning between a replicative and a transmissive/virulent phase life cycle [17,18]. Nutrient deprivation within a biofilm or host triggers the upregulation of genes encoding virulence traits such as motility, osmotic stress resistance, pigmentation production, and multiple virulence factors [17]. ...
Chapter
Full-text available
Manipulation of host phosphoinositide lipids has emerged as a key survival strategy utilized by pathogenic bacteria to establish and maintain a replication-permissive compartment within eukaryotic host cells. The human pathogen, Legionella pneumophila, infects and proliferates within the lung’s innate immune cells causing severe pneumonia termed Legionnaires’ disease. This pathogen has evolved strategies to manipulate specific host components to construct its intracellular niche termed the Legionella-containing vacuole (LCV). Paramount to LCV biogenesis and maintenance is the spatiotemporal regulation of phosphoinositides, important eukaryotic lipids involved in cell signaling and membrane trafficking. Through a specialized secretion system, L. pneumophila translocates multiple proteins that target phosphoinositides in order to escape endolysosomal degradation. By specifically binding phosphoinositides, these proteins can anchor to the cytosolic surface of the LCV or onto specific host membrane compartments, to ultimately stimulate or inhibit encounters with host organelles. Here, we describe the bacterial proteins involved in binding and/or altering host phosphoinositide dynamics to support intracellular survival of L. pneumophila.
... Both full-scale reactors and commercial point-of-use UV sterilizers are designed to provide doses that achieve 3-5-log inactivation of planktonic bacteria, protozoa, and viruses; unfortunately, these same doses are likely inadequate to treat many OBPs to the same extent under realistic conditions. This is due to their intercalation into complex biological matrices capable of shielding from UVC photons, including biofilm exopolymer particles (Priscilla, 2010), protozoan hosts (Brieland et al., 1997;Cirillo et al. 1997Cirillo et al. , 1999Steinert et al., 1998;Thomas et al., 2004), and expelled vacuoles and vesicles from the latter (Berk et al., 1998;Chen et al., 2004). Currently, there is a need for more detailed study of bacterial UV dose responses in these matrices, particularly for Legionella and Mycobacteria. ...
Article
Respiratory infections from opportunistic bacterial pathogens (OBPs) have heightened research interests in drinking water distribution systems, premise plumbing, and point-of-use technologies. In particular, biofilm growth in showerheads increases OBP content, and inhalation of shower aerosols is a major exposure route for Legionellae and Mycobacteria infections. Incorporation of UVC LEDs into showerheads has thus been proposed as a point-of-use option for healthcare facilities. Herein we have examined incongruities between the nature of OBP contamination in shower water and the hypothetical application of conventional UV disinfection engineering concepts. Effective UV dosing within showerheads must overcome significant shielding effects imparted by the biological matrices in which common OBPs reside, including biofilm particles and protozoan hosts. Furthermore, prevention of biofilm growth in showerhead interiors requires a different UV irradiation approach and is lacking in established design parameters. Development of showerhead devices is also likely to face a trade-off between bathing functionality and simpler form factors that are more conducive to internal UV irradiation.
... Речь идет о дезинфекции больших объемов воды, в которых необходимо добиться положительного эффекта не только в отношении планктонных форм легионелл, но и природных биопленок, содержащих легионеллы. Известно, что природные биопленки являются основными «накопителями» легионелл в водных системах и в их составе отличаются значительно более высоким уровнем устойчивости к дезинфектантам [13,16]. Данные, полученные в отношении легионелл в лабораторных модельных системах с использованием моновидовых биопленок, недостаточно информативны для экстраполяции их на реальные потенциально опасные водные объекты [3,10]. ...
Article
Full-text available
Aim. Comparative study of the different methodical approaches directed on the elimination of plankton forms and Legionella biofilms from potentially dangerous water systems. Materials and methods. Evaluation of short-term heatingof water to 70OC implemented in water supply module by volume 290 L with regulated temperature profile. Evaluation of catalytic cleansing of water implemented in the pool for water birds by volume 10 000 L. The initial, intermediate and final level of water objects colonization by L. pneumophila determined accordance MUK 4.2.2217-07. Presence of biofilms associated with L .pneumophila on the surface of the equipment, water surface and ball valves of water supply system determined visual with subsequent bacteriological confirmation by L. pneumophila isolation from biofilms. Results. The high level of bactericidal activity against L. pneumophila plankton forms and biofilms was shown by both methods. Short-term heating to 70O during 24 hours prevented Legionella growth for 2 months of management. The catalytic cleansing of water against high initial level of contamination of water object by L. pneumophila manifested not earlier 3 weeks of maintenance and continued during 2 months. Conclusion. The presented methodical approaches can be used for development of efficient strategy of prevention of legionellosis during management of the different potentially dangerous water systems in public buildings.
... Legionella, the causative agent of Legionnaires' disease, is a facultative intracellular Gram-negative bacteria that is ubiquitous in natural and man-made water systems (Rowbotham, 1980). These bacteria live parasitically in protozoa and can be found within biofilms (Rowbotham, 1980;Fields et al., 2002;Declerck, 2010;Stewart et al., 2012). Legionella is transmitted through inhaled aerosols and subsequently, enters and grows within human monocytes and alveolar macrophages, which can lead to a fatal form of pneumonia known as Legionnaires' disease (Isberg et al., 2009). ...
Article
Full-text available
Approximately 85% of cases of Legionnaires' disease are caused by Legionella pneumophila serogroup 1. In this study, we analyzed the distribution of lag-1 alleles, ORF 7 and ORF 8 genes of lipopolysaccharide (LPS) and sequence-based types of 616 L. pneumophila serogroup 1 strains isolated in Japan (206 clinical, 225 environmental) and China (13 clinical and 172 environmental). The lag-1 gene was harbored by significantly more of the clinical isolates compared with the environmental isolates (90.3 vs. 19.1% and 61.6 vs. 3.0%, respectively; both P < 0.001). ORF 7 genes were detected in 51.0% of Japanese clinical and 36.0% of Japanese environmental (P = 0.001) isolates, as well as 15.3% of Chinese clinical and 9.9% of Chinese environmental isolates (P = 0.544). ORF 8 genes were detected in 12.1% of Japanese clinical and 5.8% of Japanese environmental (P = 0.017) isolates, as well as 7.7% of Chinese clinical and 3.4% of Chinese environmental isolates (P = 0.388). The Japanese and Chinese isolates were assigned to 203 and 36 different sequence-types (ST), respectively. ST1 was predominant. Most isolates with the same ST also had the same lag-1, ORF 7, and ORF 8 gene subgroups. In conclusion, the lag-1 was present in most of the clinical isolates, but was absent from most of the environmental isolates from both China and Japan, regardless of the water source and SBT type. PCR-based serotyping and subgrouping methods can be used to define a hierarchy of virulence genotypes that require stringent surveillance to prevent human disease.
... The bacterium Legionella pneumophila (Lp) naturally inhabits nutrient-poor aquatic environments where it undergoes a bi-phasic lifestyle, alternating between replication in host eukaryotes and residence in multi-species biofilms (Lau and Ashbolt, 2009;Declerck et al., 2007;Declerck, 2010;Taylor et al., 2013). If Lp undergoes this lifecycle within man-made structures such as cooling towers, the bacteria can become aerosolized and cause outbreaks of a severe, pneumonia-like disease in humans, called Legionnaires' disease McDade et al., 1977;Fields et al., 2002). ...
Article
Full-text available
To persist in microbial communities, the bacterial pathogen Legionella pneumophila must withstand competition from neighboring bacteria. Here, we find that L. pneumophila can antagonize the growth of other Legionella species using a secreted inhibitor: HGA (homogentisic acid). Unexpectedly, L. pneumophila can itself be inhibited by HGA secreted from neighboring, isogenic strains. Our genetic approaches further identify lpg1681 as a gene that modulates L. pneumophila susceptibility to HGA. We find that L. pneumophila sensitivity to HGA is density-dependent and cell intrinsic. This resistance is not mediated by the stringent response nor the previously described Legionella quorum-sensing pathway. Instead, L. pneumophila cells secrete HGA only when they are conditionally HGA-resistant, which allows these bacteria to produce a potentially self-toxic molecule while restricting the opportunity for self-harm. We propose that established Legionella communities may deploy molecules such as HGA as an unusual public good that can protect against invasion by low-density competitors.
... In the environment, L. pneumophila is thought to most efficiently replicate within free-living amoebae, leading to the release of highly virulent bacteria primed for the infection of humans (21,24). Consistent with this, L. pneumophila was found to cooccur with free-living amoebae in various aquatic environments (5,10,25,26). ...
Article
Full-text available
Bacterial pathogens are generally investigated in the context of disease. To prevent outbreaks, it is essential to understand their lifestyle and interactions with other microbes in their natural environment. Legionella pneumophila is an important human respiratory pathogen that survives and multiplies in biofilms or intracellularly within protists, such as amoebae. Importantly, transmission to humans occurs from these environmental sources. Legionella infection generally leads to rapid host cell lysis. It was therefore surprising to observe that amoebae, including fresh environmental isolates, were well protected during Legionella infection when the bacterial symbiont Protochlamydia amoebophila was also present. Legionella was not prevented from invading amoebae but was impeded in its ability to develop fully virulent progeny and were ultimately cleared in the presence of the symbiont. This study highlights how ecology and virulence of an important human pathogen is affected by a defensive amoeba symbiont, with possibly major consequences for public health.
... Unlike Pseudomonas, L. pneumophila is a facultative intracellular bacterium and is also able to grow in biofilms, although it will most likely colonize existing biofilms rather than producing its own (Declerck, 2010). Therefore, a mechanism for cleaving surface adhesin RtxA in L. pneumophila can probably regulate both infection potential and biofilm formation due to implication of RtxA in host attachment as discussed earlier. ...
Thesis
Legionella pneumophila is the causative agent of a form of pneumonia called legionellosis or Legionnaires’ disease. Between 2012 and 2015, the reported European cases of legionellosis increased from 5,848 to 7,069 cases per year where France, Germany, Italy and Spain accounted for 69% of the reported cases. Worryingly, the case fatality of incidents was 8.2% making this disease a considerable health concern. One virulence factor produced by this bacterium is a large protein (~700 kDa) belonging to the RTX (Repeats in ToXin) family called RtxA secreted by the type 1 secretion system. The hereby work reveals that, in vitro, LapG periplasmic protease cleaves RtxA N-terminus in the middle of a di-alanine motif (a.a. 108-109). We also show using lapG and lapD mutant strains, that RtxA release is controlled by these two proteins similar to Pseudomonas fluorescenes LapA. We observed that a strain lacking LapG protease maintains RtxA on the cell surface, while a strain lacking LapD does not exhibit cell surface RtxA. Interestingly, we identified the presence of homologous potential T1SS/LapDG systems in many Legionella species and other Gammaproteobacteria. Regarding L. pneumophila virulence, our work showed that mutants for L. pneumophila T1SS (lssBD/tolC) were more disruptive to its virulence than lapG/lapD mutants. We also hypothesize, by challenging infection, that L. pneumophila might be actively targeting its host via RtxA. Additionally, by observing rtxA mutants as well as detecting RtxA on host surface briefly after inoculation and attenuating virulence by using anti RtxA antibodies, we assume an important but not limiting role for this protein in the infection process
... It is tempting to conclude that most of Legionella was in the water phase in the system. The presence of Legionella in biofilms within water distribution systems has been reported in several studies (Abdel-Nour et al., 2013;Abu Kweek and Amer, 2018;Armon et al., 1997;Buse et al., 2014;Buse et al., 2012;Declerck, 2010;Lau and Ashbolt, 2009;Moritz et al., 2010;van der Kooij et al., 2005). Several factors influence the formation of biofilm by Legionella and its ability to integrate biofilms (Buse et al., 2017;Piao et al., 2006;Rhoads et al., 2017;Rogers et al., 1994a). ...
Preprint
Full-text available
Legionella pneumophila (Lp) is a waterborne bacterium known for causing Legionnaires’ Disease, a severe pneumonia. Cooling towers are a major source of outbreaks, since they provide ideal conditions for Lp growth and produce aerosols. In such systems, Lp typically grow inside protozoan hosts. Several abiotic factors such as water temperature, pipe material and disinfection regime affect the colonization of cooling towers by Lp. The local physical and biological factors promoting the growth of Lp in water systems and its spatial distribution are not well understood. Therefore, we built a lab-scale cooling tower to study the dynamics of Lp colonization in relationship to the resident microbiota and spatial distribution. The pilot was filled with water from an operating cooling tower harboring low levels of Lp . It was seeded with Vermamoeba vermiformis , a natural host of Lp , and then inoculated with Lp. After 92 days of operation, the pilot was disassembled, the water was collected, and biofilm was extracted from the pipes. The microbiome was studied using 16S rRNA and 18S rRNA genes amplicon sequencing. The communities of the water and of the biofilm were highly dissimilar. The relative abundance of Legionella in water samples reached up to 11% whereas abundance in the biofilm was extremely low (≤0.5 %). In contrast, the host cells were mainly present in the biofilm. This suggest that Lp grows in host cells associated with biofilm and is then released back into the water following host cell lysis. In addition, water temperature shaped the bacterial and eukaryotic community of the biofilm, indicating that different parts of the systems may have different effects on Legionella growth.
... Another factor that affects the survival of L. pneumophila is its ability to grow and proliferate intracellularly inside biofilms of other organisms such as fresh water protozoa which is formed at the solid-liquid interfaces or at the liquid-air interface [13]. Also factors such as, the presence of sediment materials, stagnation, roughness of surfaces, flow rate, materials from which surface is made and disinfectants' concentration affect the accumulation of L. pneumophila in the pipelines, and sometimes support its colonization of the water systems in a way that makes it hardly eradicated [14]. ...
Article
Full-text available
Aims: To determine the prevalence of Legionella spp. in domestic hot water systems and evaluate the molecular diversity among these Legionella spp. Isolates. Place and Duration of Study: Sample collection area was the city of Aqaba, Jordan, between May and December 2012. Sample analysis was done in Ben-Hayyan international laboratories, Aqaba city, and the molecular microbiology laboratories, Taibah University, Saudi Arabia. Methodology: Two hundred (200) water samples were collected randomly from hot water tanks of private apartments, and were tested for the occurrence of Legionella spp. using direct membrane filtration method followed by species identification using Gram stain, the API 20NE biochemical system and the Legionella species latex agglutination test. Genotype characterizations of the Legionella isolates was carried out using DNA extraction followed by RAPD-PCR amplification with OP-A3 primer and analysis of the resulting patterns. Results: Of the 200 samples, 17 (8.5%) were positive for the presence of Legionella spp. A 307 total of 15 (88.2%) out the 17 positive samples were confirmed as Legionella pneumophila, 10 of them were of serogroup 1 and 5 isolates were of serogroup 2-14, the remaining two isolate were Legionella species other than L. pneumophila. RAPD-PCR analysis classified all 17 Legionella isolates into three groups. Serogroup 1 isolates were classified into group A, serogroup 2-14 isolates in group B and Legionella spp. isolates in group C. Group A was further sub-clustered into two subgroups, genotype A 1 containing isolates collected from hot water tanks of a temperature set at 25-30°C and A 2 containing isolates collected from hot water tanks of a temperature set at 55-80°C. Conclusion: This study showed the colonization of the plumbing systems of private houses by Legionella spp. and demonstrated that the temperature of the water tanks maybe one of the most important factors that affect the genotypic behavior of Legionella pneumophila.
... Legionella cell length is associated with ppGpp signaling. A biofilm at 37 • C is much more solid than at 25 • C, and interestingly, at 25 • C, it is more prone to better adhesion potential [29,82]. Specific genes may also regulate biofilm formation, such as the putative twin-arginine translocation pathway involving the tatB and tatC genes [83]. ...
Article
Full-text available
Legionella pneumophila is defined as a bacterium that can cause severe pneumonia. It is found in the natural environment and in water, and is often found in water tanks. It can be an integral part of biofilms in nature, and the protozoa in which it can live provide it with food and protect it from harmful influences; therefore, it has the ability to move into a sustainable but uncultured state (VBNC). L. pneumophila has been shown to cause infections in dental practices. The most common transmission route is aerosol generated in dental office water systems, which can negatively affect patients and healthcare professionals. The most common way of becoming infected with L. pneumophila in a dental office is through water from dental instruments, and the dental unit. In addition to these bacteria, patients and the dental team may be exposed to other harmful bacteria and viruses. Therefore, it is vital that the dental team regularly maintains and decontaminates the dental unit, and sterilizes all accessories that come with it. In addition, regular water control in dental offices is necessary.
... [1][2][3][4][5] They also play an important role in opportunistic pathogen survival and propagation in both large-scale DWDS and small-scale premise plumbing systems. 6 About 60% of infections by Legionella, an opportunistic pathogen commonly found in premise plumbing, was attributed to the lack of disinfectants, which can be depleted during contact with the biofilm matrix. 7 The presence of biofilms in DWDS can also cause color and taste problems in the drinking water. 2 Multiple factors in the distribution systems contribute to biofilm growth in drinking water. ...
Article
Understanding the effects of biofilm structural and mechanical properties, which can influence biofilm cohesiveness and detachment under physical stress, is critical for biofilm and biofilm-associated pathogen control. In this study, we used optical coherence tomography (OCT) and nanoindentation to determine the role of silicate and tin (two experimental nonphosphate corrosion inhibitors) on the porous structure and stiffness of three types of multispecies biofilms. These biofilms were grown from groundwater (a drinking water source), and this groundwater was amended with either tin or silicate corrosion inhibitor (0.5 mg/L as Sn and 20 mg/L as SiO2). Based on the elastic moduli of these biofilms, tin biofilms and groundwater biofilms were the stiffest, followed by silicate biofilms. The thickness normalized by the growth time for silicate biofilms was highest at 38 ± 7.1 μm/month, compared to 21 ± 3.2 and 11 ± 2.4 μm/month for tin biofilms and groundwater biofilms, respectively. The silicate biofilms had the greatest overall porosities and were thickest among the three biofilms. Based on the pore network modeling (PNM) of OCT images, larger pores and connections were found in the silicate biofilms compared to those in tin and groundwater biofilms. Our analysis showed that the thicker and more porous biofilms (silicate biofilms) were potentially less resistant to deformation than the thinner and denser biofilms (tin and groundwater biofilms).
... This is in part due to the fact source tracking can be difficult for legionellosis (Addiss et al., 1989). Biofilm development in cooling towers can also make control and detection of L. pneumophila in circulating water difficult (Declerck, 2010). The effectiveness of regulatory, monitoring, and health issues related to Legionella risk has been reviewed by Whiley et al. (2014). ...
Article
Full-text available
At the U.S. Department of Energy’s Savannah River Site (SRS) in Aiken, SC, cooling tower water is routinely monitored for Legionella pneumophila concentrations using a direct fluorescent antibody (DFA) technique. Historically, 25–30 operating SRS cooling towers have varying concentrations of Legionella in all seasons of the year, with patterns that are unpredictable. Legionellosis, or Legionnaires’ disease (LD), is a pneumonia caused by Legionella bacteria that thrive both in man-made water distribution systems and natural surface waters including lakes, streams, and wet soil. Legionnaires’ disease is typically contracted by inhaling L. pneumophila , most often in aerosolized mists that contain the bacteria. At the SRS, L. pneumophila is typically found in cooling towers ranging from non-detectable up to 10 ⁸ cells/L in cooling tower water systems. Extreme weather conditions contributed to elevations in L. pneumophila to 10 ⁷ –10 ⁸ cells/L in SRS cooling tower water systems in July–August 2017. L. pneumophila concentrations in Cooling Tower 785-A/2A located in SRS A-Area, stayed in the 10 ⁸ cells/L range despite biocide addition. During this time, other SRS cooling towers did not demonstrate this L. pneumophila increase. No significant difference was observed in the mean L. pneumophila mean concentrations for the towers ( p < 0.05). There was a significant variance observed in the 285-2A/A Tower L. pneumophila results ( p < 0.05). Looking to see if we could find “effects” led to model development by analyzing 13 months of water chemistry and microbial data for the main factors influencing the L. pneumophila concentrations in five cooling towers for this year. It indicated chlorine and dissolved oxygen had a significant impact ( p < 0.0002) on cooling tower 785A/2A. Thus, while the variation in the log count data for the A-area tower is statistically greater than that of the other four towers, the average of the log count data for the A-Area tower was in line with that of the other towers. It was also observed that the location of 785A/2A and basin resulted in more debris entering the system during storm events. Our results suggest that future analyses should evaluate the impact of environmental conditions and cooling tower design on L. pneumophila water concentrations and human health.
... Streptococcus iniae may also associate with the existing aquatic biofilms, which are generally complex communities of bacteria and other microorganisms (Flemming et al., 2016;King et al., 2004;Neagu et al., 2017). This incorporation of fastidious pathogens has been observed in other species, allowing enhanced survival and longer persistence of infectious organisms in a system (Declerck, 2010;Donlan, 2002;Jacques et al., 2010). The documented natural and experimental co-infections of S. iniae with other bacteria, viruses, fungi, and parasites (Abdel-Latif et al., 2020;Soto et al., 2017) make this possibility of intraspecies communities intriguing. ...
Article
Full-text available
The globally distributed bacterium Streptococcus iniae is responsible for outbreaks of disease resulting in high mortality in a wide range of economically important freshwater and marine fish species. Despite the significance of S. iniae, our understanding of its transmission and infection dynamics remains incomplete. Biofilms are important for the survival and pathogenesis of many bacteria, but there is a paucity of information on their role in the ex-host persistence of S. iniae. This study aimed to compare biofilm formation by isolates representing different S. iniae genotypes and to investigate the effect of biofilm formation on environmental persistence and resistance to common disinfectants and antimicrobials. Eleven clinical isolates of S. iniae representing 4 distinct genetic groups and diverse host types were assessed for their ability to form biofilms. Planktonic bacteria or mature biofilms were exposed to in vitro aquatic microcosms of different temperatures to quantify the number of culturable bacteria in each system over time. The minimum biofilm eradication concentration (MBEC) assay® system was used to determine biofilm resistance to 18 antimicrobials and 4 disinfectants commonly used in food producing animals and aquaculture, respectively. All isolates formed biofilms within 72 h. Bacteria remained culturable notably longer in the biofilm form compared to the planktonic, with a significant impact from temperature and salinity (p < 0.05). The MBEC was higher than the planktonic minimal inhibitory concentration (MIC) for at least one isolate in 15 out of the 18 antimicrobials tested. The MBEC was also higher than the minimum biocidal concentration (MBC) for 11 out of 18 tested, including oxytetracycline and florfenicol, the two most common antimicrobials used against S. iniae infections in fish. While both forms were susceptible to disinfection by bleach, hydrogen peroxide and Virkon® Aquatic, treatment with povidone‑iodine did not eliminate biofilms. The ability of S. iniae to form resilient biofilms provides an effective mechanism for their persistence in the environment, which must be considered and further researched to control this widespread pathogen.
... The abundance of Mycobacterium and Legionella was significantly higher on microplastics than in incubation water (p < 0.01). Mycobacterium and Legionella are common inhabitants of freshwater environments and biofilms are their prevailing microbial lifestyle (Declerck, 2010;Esteban & García-Coca, 2018;Taylor et al., 2009). The gene copies of Legionella on microplastics were significantly influenced by microplastic size (p < 0.05). ...
Article
Full-text available
Due to inefficient removal of microplastics by drinking water treatment process, widely distributed microplastics in drinking water sources may pose risks to drinking water safety. To explore the factors influencing the colonization of bacteria on microplastics and biological risks posed by microplastics in drinking water sources, we exposed microplastics with different sizes and polymer types to water from Yangtze River and Jialing River for 21 days under controlled conditions. High-throughput sequencing and qPCR were conducted to qualitatively and quantitatively analyze the bacterial community on microplastics. The results showed that bacteria tended to adhere to the surfaces of microplastics, resulting in higher community richness and diversity and different community structure on microplastics compared with those in incubation water. The number of bacteria on microplastics increased with decreasing particle size. It was the nature of incubation water rather than microplastics determined the bacterial community structure on microplastics. Some potential pathogens were discovered on microplastics, especially Mycobacterium which accounted for a high proportion. Overall, this study provided an insight into microplastic biofilm and the challenges brought by them in drinking water sources.
... Legionella pneumophila is a facultative intracellular bacterium, which also colonizes and persists in complex biofilms composed of various microorganisms, including bacteria and protozoa (Lau and Ashbolt, 2009;Declerck, 2010;Abdel-Nour et al., 2013;Hoffmann et al., 2014;Swart et al., 2018). Predatory protozoa, such as the amoeba Acanthamoeba castellanii, migrate and 'graze' on the bacterial biofilm communities (Huws et al., 2005). ...
Article
Full-text available
The environmental bacterium Legionella pneumophila causes the pneumonia Legionnaires’ disease. L. pneumophila forms biofilms and employs the Icm/Dot type IV secretion system (T4SS) to replicate in amoebae and macrophages. A regulatory network comprising the Legionella quorum sensing (Lqs) system and the transcription factor LvbR controls bacterial motility, virulence, and biofilm architecture. Here we show by comparative proteomics that in biofilms formed by the L. pneumophila ΔlqsR or ΔlvbR regulatory mutants the abundance of proteins encoded by a genomic “fitness island”, metabolic enzymes, effector proteins and flagellar components (e.g., FlaA) varies. ∆lqsR or ∆flaA mutants form “patchy” biofilms like the parental strain JR32, while ∆lvbR forms a “mat‐like” biofilm. Acanthamoeba castellanii amoebae migrated more slowly through biofilms of L. pneumophila lacking lqsR, lvbR, flaA, a functional Icm/Dot T4SS (∆icmT), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ∆lvbR or ∆icmT biofilms, but not in ∆lqsR or ∆flaA biofilms. The amoeba‐adherent bacteria induced promoters implicated in motility (PflaA) or virulence (PsidC, PralF). Taken together, the Lqs‐LvbR network (quorum sensing), FlaA (motility) and the Icm/Dot T4SS (virulence) regulate migration of A. castellanii through L. pneumophila biofilms, and – apart from the T4SS – govern bacterial cluster formation on the amoebae. This article is protected by copyright. All rights reserved.
... At this time, however, the study is limited by the fact that many data related to the operation of facilities, such as car washes, high-pressure water cleaners, and wastewater treatment systems (Legionella flow, dispersion), are not sufficiently accurate. We also did not take into consideration the possibility that Legionella may have been present in biofilms, a parameter that may be affected according to the type of facility and that could play a role in Legionella survival and transmission [31,49,50]. Nevertheless, this type of analysis can help us to learn more about these phenomena, and can be used to quantitatively assess risk for a given facility so that appropriate control methods can be implemented [47,51]. ...
Article
Full-text available
Based on the data from a French outbreak of legionellosis, a probabilistic approach was developed to analyze and assess the potential role of several suspected sources of contamination. Potential dates of exposure of all cases were determined using back-calculation, using two probability distribution functions to model incubation period. A probabilistic analysis and risk assessment were then used to determine the most probable sources of contamination for each wave of the outbreak. The risk assessment was based on parameters representing emission and dispersion of Legionella: level and duration of emission; aerosol dispersion capacity; and probability of potential exposure for each patient. Four types of facilities containing the Legionella epidemic strain were analyzed: cooling towers, aerated wastewater basins, high pressure water cleaners, and car wash stations. The results highlighted the potential role of an aerated wastewater basin in the outbreak in addition to cooling towers. The role of high-pressure water cleaners and car wash stations appeared to be non-significant. This study also reveals the lack of knowledge on facility parameters that can be useful for microbial risk assessments. This type of probabilistic analysis can be used to quantitatively assess the risk for various facilities in order to manage a legionellosis outbreak.
... These biofilmassociated populations are protected from disinfectants, which enables their long-term residence, and the detachment of L. pneumophila from biofilms degrades water quality, which poses health risks to the public. 32 3.4. P. f luorescens Biofilm Formation in the Presence of the Surrogate and L. pneumophila. ...
... Since their discovery as the causative agent of Legionnaire's disease in 1976 [1], bacteria of the Legionella genus have become a major source of drinking water-related disease outbreaks [2]. Naturally occurring in water [3] and soil [4], Legionella possess traits that enable their survival in a wide variety of environmental conditions [5], including protozoan host parasitization [6]. Though numerous transmission sources have been implicated in the spread of legionellosis, transmission is thought to occur primarily through the inhalation of cells, commonly via aerosolized water from engineered systems [7]. ...
Article
Full-text available
Legionella is an environmental pathogen that is responsible for respiratory disease and is a common causative agent of water-related outbreaks. Due to their ability to survive in a broad range of environments, transmission of legionellosis is possible from a variety of sources. Unfortunately, a disproportionate amount of research that is devoted to studying the occurrence of Legionella in environmental reservoirs is aimed toward cooling towers and premise plumbing. As confirmed transmission of Legionella has been linked to many other sources, an over-emphasis on the most common sources may be detrimental to increasing understanding of the spread of legionellosis. This review aims to address this issue by cataloguing studies which have examined the occurrence of Legionella in less commonly investigated environments. By summarizing and discussing reports of Legionella in fresh water, ground water, saltwater, and distribution system drinking water, future environmental and public health researchers will have a resource to aid in investigating these pathogens in relevant sources.
Article
Full-text available
Legionella pneumophila (Lp), responsible for a severe pneumonia called Legionnaires’ disease, represents an important health burden in Europe. Prevention and control of Lp contamination in warm water systems is still a great challenge often due to the failure in disinfection procedures. The aim of this study was to evaluate the in vitro activity of Terpinen-4-ol (T-4-ol) as potential agent for Lp control, in comparison with the essential oil of Melaleuca alternifolia (tea tree) (TTO. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of T-4-ol were determined by broth micro-dilution and a micro-atmosphere diffusion method to investigate the anti-Lp effects of T-4-ol and TTO vapors. Scanning Electron Microscopy (SEM) was adopted to highlight the morphological changes and Lp damage following T-4-ol and TTO treatments. The greatest antimicrobial activity against Lp was shown by T-4-ol with a MIC range of 0.06–0.125% v/v and MBC range of 0.25–0.5% v/v. The TTO and T-4-ol MIC and MBC decreased with increasing temperature (36 °C to 45 ± 1 °C), and temperature also significantly influenced the efficacy of TTO and T-4-ol vapors. The time-killing assay showed an exponential trend of T-4-ol bactericidal activity at 0.5% v/v against Lp. SEM observations revealed a concentration- and temperature- dependent effect of T-4-ol and TTO on cell surface morphology with alterations. These findings suggest that T-4-ol is active against Lp and further studies may address the potential effectiveness of T-4-ol for control of water systems.
Article
Background: Legionella is a fastidious Gram-negative bacterium that is responsible for Legionnaires’ disease. Legionella is a ubiquitous aquatic bacterium, especially in cooling systems. Several studies have investigated Legionella contamination in natural and man-made water resources. Legionella is resistant to chlorine and other disinfectants; thus, it is important to consider it in places where people with immunodeficiency are kept. Objectives: The aim of this study was to detect the Legionella pneumophila mip gene in clinical samples, kidney transplants, and dialysis wards by the polymerase chain reaction (PCR) method. Methods: In this study, 156 samples were taken from the kidney transplant and dialysis wards. DNA extraction was done. After confirmation of primers, PCR was performed to amplify 16srRNA and mip genes. The PCR product was electrophoresed on agarose gel 1%. Results: Among the samples, 23 samples were infected with Legionella (14.7%), of which 7 samples were identified for the mip gene (4.5%) and 16 samples for 16srRNA (10.2%). The confirmation of the presence of these genes was done by sequencing. In serum, tissue, urine, hot water, and cold water samples were positive for the 16srRNA gene (7.5%, 26.66%, 7.14%, 20%, and 6.66%, respectively). Among these samples, 50% of tissue samples, 25% of urine, and 33.33% of hot water were positive for the mip gene. Conclusions: The presence of L. pneumophila in aqueous samples of transplant and dialysis wards is important. Therefore, rapid detection of this bacterium or the mip gene by a molecular method can play an important role in reducing infection and transplant rejection.
Article
Although an intracellular pathogen, L. pneumophila has developed mechanisms to ensure long-term survival in low-nutrient aqueous conditions. Eradication of L. pneumophila from contaminated water supplies has proven challenging, as outbreaks have been traced to previously remediated systems. Understanding the genetic determinants that support L. pneumophila persistence in low-nutrient environments can inform design and assessment of remediation strategies. Here we characterize a genetic locus that encodes a two-component signaling system ( lpg0278-lpg0277 ) and a putative regulator protein ( lpg0279 ) that modulates the production of the messenger molecule cyclic di-GMP. We show that this locus promotes both L. pneumophila cell differentiation and survival in nutrient-limiting conditions, thus advancing the understanding of the mechanisms that contribute to L. pneumophila environmental resilience.
Article
Purpose of Review Legionella bacteria cause Legionnaires’ disease (LD), a severe and potentially fatal pneumonia. Legionellosis has been increasing in the USA and in Europe. We reviewed the literature to describe host factors predisposing to LD, environmental factors facilitating Legionella proliferation, and transmission modes leading to legionellosis cases and outbreaks. Findings Men, smokers, and persons 50 years and older, or with chronic pulmonary or immunocompromising conditions, are at risk for LD. Stagnated water and warm water temperature promote Legionella growth. Most legionellosis cases result from exposure to aerosolized contaminated water from man-made water systems, including hot tubs and whirlpool spas; hot water systems in large buildings, hotels, and hospitals; and cooling towers. Summary Water management plans for hot water systems at hospitals, long-term care facilities, and large buildings are important to help prevent LD, and careful epidemiologic investigations remain essential in controlling and preventing legionellosis outbreaks.
Article
Purpose To perform epidemiological surveillance of Legionella pneumophila in recreational swimming pools in the city of Valladolid (Spain), an area with a continental climate and low incidence of legionella-associated infections. Additionally, wild-type minimum inhibitory concentration (MIC) distributions for eight antibiotics commonly used for the treatment of legionellosis were calculated from the isolates obtained. Methods Twelve recreational pools were enrolled between June 2003 and December 2016 and 7221 water samples were taken from three different points of the water network (tank, tap and shower). Legionella culture was performed according to ISO 11731 and 11731-2 standards. MICs of antibiotics were obtained by a gradient test. Results 1.44% of the water samples were positive for L. pneumophila. 60 strains (57.69%) were isolated from showers, 26 (25.00%) from tanks and 18 (17.31%) from taps. L. pneumophila counts were <100 CFU/L in 75 samples (72.12%), 100–1000 CFU/L in 17 (16.35%) and >1000 CFU/L in 12 (11.54%). The MIC90 values obtained were for Rifampicin 0.125 mg/L; Trimethoprim-Sulfamethoxazole 0.25 mg/L; Azithromycin and Levofloxacin 0.5 mg/L; Clarithromycin and Ciprofloxacin 1.0 mg/L; Doxycycline and Tigecycline 4.0 mg/L. Conclusions The use of showers in recreational pools can become a potential pathway for exposure to L. pneumophila, even in cold climates. The wild-type MIC distributions presented in this article may be useful for a better detection of antibiotic resistance and can contribute to improvements in the choice of the antibiotic treatment of legionellosis.
Book
Full-text available
Avant-propos Les microorganismes sont une composante essentielle de tout écosystème. Ils occupent des micro-habitats spécifiques convenant à leurs besoins. Dans ces micro-habitats, les microorganismes ont établi différents types de relations entre eux et avec d'autres organismes : neutres, symbiotiques, pathogènes et antagonistes. De par ces relations, ils affectent ou non, positivement ou négativement, le développement des autres membres de la communauté biotique. Ils sont donc de véritables ouvriers nécessaires aux fonctionnements des écosystèmes telluriques et aquatiques, qui peuvent accomplir des tâches aussi importantes que méconnues indispensables au bon fonctionnement des cycles biogéochimiques de la matière, comme la transformation des déchets, l'oxydation, la réduction, la précipitation et la solubilisation des ions minéraux, la production des composés organiques, la fixation de l'azote moléculaire, l'altération de la roche mère, …etc. Dans l'environnement, les microorganismes jouent le rôle de producteurs ou de décomposeurs. Les microorganismes producteurs sont photolithotrophes ou chimiolithotrophes. Ils tirent leur énergie de la lumière ou des composés inorganiques et synthétisent des matières organiques, en raison de ces propriétés, ils constituent le point de départ de nombreuses chaînes alimentaires. Les microorganismes décomposeurs sont chimiolithotrophes. Par leurs activités métaboliques, ces derniers dégradent les matières organiques en matières minérales. Ce recyclage permanent entretient la vie en rendant les éléments nutritifs constamment disponibles bien qu'ils soient en quantités limitées dans l'environnement. A partir de la microbiologie environnementale, les chercheurs ont développé des applications tendant à utiliser des microorganismes dans les différents domaines d'intérêt de l'homme : industrie agro-alimentaire, pharmacologie, agriculture, cosmétologie, bioremédiation, épuration des eaux usées…etc. C'est ce que nous allons développer dans ce document. Dans ce manuscrit, nous allons d'abord comprendre les méthodes d'étude des microorganismes en passant par l'écologie des virus, puis comprendre l'état actuel des connaissances sur le cycle des matériaux dans l'environnement, les bioréacteurs et les biofilms afin de montrer des exemples d'application des microorganismes environnementaux. Par conséquent, le but de ce travail est d'examiner le rôle des microorganismes dans le cycle biochimique, et de nommer et classer les microorganismes d'intérêt. Cet ouvrage a été rédigé de telle manière concordante aux programmes de Master en microbiologie appliquée selon le canevas du ministère de l'enseignement supérieure algérien. -Ce document ne dispense pas les étudiants de la responsabilité d'assister aux cours-Auteure P
Article
Full-text available
Legionella pneumophila can cause a potentially fatal form of humane pneumonia (Legionnaires’ disease), which is most problematic in immunocompromised and in elderly people. Legionella species is present at low concentrations in soil, natural and artificial aquatic systems and is therefore constantly entering man-made water systems. The environment temperature for it’s ideal growth range is between 32 and 42°C, thus hot water pipes represent ideal environment for spread of Legionella. The bacteria are dormant below 20°C and do not survive above 60°C. The primary method used to control the risk from Legionella is therefore water temperature control. There are several other effective treatments to prevent growth of Legionella in water systems, however current disinfection methods can be applied only intermittently thus allowing Legionella to grow in between treatments. Here we present an alternative disinfection method based on antibacterial coatings with Cu-TiO2 nanotubes deposited on preformed surfaces. In the experiment the microbiocidal efficiency of submicron coatings on polystyrene to the bacterium of the genus Legionella pneumophila with a potential use in a water supply system was tested. The treatment thus constantly prevents growth of Legionella pneumophila in presence of water at room temperature. Here we show that 24-hour illumination with low power UVA light source (15 W/m² UVA illumination) of copper doped TiO2 nanotube coated surfaces is effective in preventing growth of Legionella pneumophila. Microbiocidal effects of Cu-TiO2 nanotube coatings were dependent on the flow of the medium and the intensity of UV-A light. It was determined that tested submicron coatings have microbiocidal effects specially in a non-flow or low-flow conditions, as in higher flow rates, probably to a greater possibility of Legionella pneumophila sedimentation on the coated polystyrene surfaces, meanwhile no significant differences among bacteria reduction was noted regarding to non or low flow of medium.
Article
Legionella species are facultative intracellular pathogens, which cause a life-threatening pneumonia termed Legionnaires’ disease. Legionella pneumophila employs the Legionella quorum sensing (Lqs)-LvbR network to regulate virulence and motility, but its role for growth in media is ill-defined. Here we report that compared to the parental L. pneumophila strain JR32, a Δ lqsR mutant showed a reduced lag phase at 30°C and reached a higher cell density at 45°C, while the Δ lqsA , Δ lqsS and Δ lqsT mutants showed a longer lag phase and reached only a lower cell density. A Δ lvbR mutant resumed growth like the parental strain at 30°C, but exhibited a substantially reduced cell density at 45°C. Thus, LvbR is an important cell density regulator at elevated temperatures. Environmental and clinical L. pneumophila strains grew in AYE medium after distinct lag phases with similar rates at 30°C, reached different cell densities at the optimal growth temperature of 40°C, and no longer grew at 50°C. Legionella longbeachae reached a rather low cell density at 40°C and did not grow at and beyond 45°C. Genes encoding components of the Lqs-LvbR network were present in the genomes of the environmental and clinical L. pneumophila isolates, and upon growth at 30°C or 45°C the P lqsR , P lqsA , P lqsS and P lvbR promoters from strain JR32 were expressed in these strains with distinct patterns. Taken together, our results indicate that the Lqs-LvbR network governs the temperature-dependent growth onset and cell density of the L. pneumophila reference strain JR32, and possibly also of environmental and clinical L. pneumophila isolates. Importance Environmental bacteria of the genus Legionella are the causative agents of the severe pneumonia Legionnaires’ disease, the incidence of which is worldwide on the rise. Legionella pneumophila and Legionella longbeachae are the clinically most relevant species. The opportunistic pathogens are inhaled through contaminated aerosols and replicate in human lung macrophages with a similar mechanism as in their natural hosts, free-living amoebae. Given their prevalence in natural and technical water systems, an efficient control of Legionella spp. by physical, chemical or biological means will reduce the incidence of Legionnaires’ disease. Here we show that the Legionella quorum sensing (Lqs) system and the pleiotropic transcription factor LvbR govern the temperature-dependent growth onset and cell density of bacterial cultures. Hence, the growth of L. pneumophila in water systems is not only determined by the temperature and nutrient availability, but also by quorum sensing, i.e., density- and signaling molecule-dependent gene regulation.
Article
Full-text available
The West Bank can be considered a high-risk area for Legionnaires’ disease (LD) due to its hot climate, intermittent water supply and roof storage of drinking water. Legionella, mostly L. pneumophila, are responsible for LD, a severe, community-acquired and nosocomial pneumonia. To date, no extensive assessment of Legionella spp and L. pneumophila using cultivation in combination with molecular approaches in the West Bank has been published. Two years of environmental surveillance of Legionella in water and biofilms in the drinking water distribution systems (DWDS) of eight hospitals was carried out; 180 L. pneumophila strains were isolated, mostly from biofilms in DWDS. Most of the isolates were identified as serogroup (Sg) 1 (60%) and 6 (30%), while a minor fraction comprised Sg 8 and 10. Multilocus Variable number of tandem repeats Analysis using 13 loci (MLVA-8(12)) was applied as a high-resolution genotyping method and compared to the standard Sequence Based Typing (SBT). The isolates were genotyped in 27 MLVA-8(12) genotypes (Gt), comprising four MLVA clonal complexes (VACC 1; 2; 5; 11). The major fraction of isolates constituted Sequence Type (ST)1 and ST461. Most of the MLVA-genotypes were highly diverse and often unique. The MLVA-genotype composition showed substantial regional variability. In general, the applied MLVA-method made it possible to reproducibly genotype the isolates, and was consistent with SBT but showed a higher resolution. The advantage of the higher resolution was most evident for the subdivision of the large strain sets of ST1 and ST461; these STs were shown to be highly pneumonia-relevant in a former study. This shows that the resolution by MLVA is advantageous for back-tracking risk sites and for the avoidance of outbreaks of L. pneumophila. Overall, our results provide important insights into the detailed population structure of L. pneumophila, allowing for better risk assessment for DWDS.
Article
Wastewater is treated by concerted actions of the microbial communities within bioreactors. Although protists (unicellular eukaryotes) are good bioindicators and important players influencing denitrification, nitrification, and flocculation, they are the least known organisms in WWTPs. The few recent environmental surveys of the protistan diversity in WWTPs show that the most abundant protistan sequences in WWTPs belong to Thecofilosea (Rhizaria). We re-investigated previously published environmental sequencing data and gathered strains from seven WWTPs to determine which species dominate WWTPs worldwide. We found that all highly abundant thecofilosean sequences represent a single species – Rhogostoma minus. Considering that Thecofilosea are frequent hosts for Legionellales, i.e. bacteria linked to waterborne diseases, we confirm that Rhogostoma minus functions as a host for Legionellales in WWTPs. Whether the highly abundant Rhogostoma minus also serves as a host for known human pathogenic Legionellales requires further attention.
Chapter
Full-text available
Introduction Legionella spp. are ubiquitous aquatic organisms found to be associated with community-acquired pneumoniae (CAP) as well as hospital-acquired pneumonia (HAP). Direct inhalation of aerosols from environmental colonisation is typically the source of infection. The aim of this study was to determine the level of colonisation in hospital water supply systems in order to assess the criticality of the water distribution network and strengthen preventive measures. Methods From 2009 to 2018, 769 water samples were collected and then analysed according to the standard methods indicated in ISO11731-2:2004 and ISO11731:2017 for Legionella detection. Results The samples were positive in 37.1% cases (n. 285) and negative in 62.9% cases (n. 484). The threshold of 10,000 CFU/L was exceeded in 15.1% cases and led to decolonisation as indicated by Italian and European ECDC guidelines. In the autumn-winter period SG1 showed a positivity of 41.2% (n. 40) with a decrease in the spring-summer period with 9.6% (n. 18) of positivity. In contrast, SG2-15 showed a positivity of 30.9% (n. 30) in autumn-winter, which tends to increase to 56.9% (n. 112) in spring-summer (p < 0.001). Conclusion Surprisingly, besides showing a seasonal trend already described previously in the literature, the positivity of our sample was not balanced even for serogroups in the two periods. This could be due to genetic differences and ecological niches to be further investigated that could also have links with the greater pathogenicity of SG1. Environmental microbiological surveillance and risk assessment should be performed more frequently and disinfection must be carried out, especially in health facilities where people are more susceptible to infections.
Article
Full-text available
The aim of this study was to ascertain whether the combined action of metal nanoparticles (silver, copper, zinc oxide, iron oxide) would ensure the appropriate biocidal properties oflow-density polyethylene (LDPE) against pathogenic microorganisms. According to the research hypothesis, appropriately selected concentrations of the applied metal nanoparticles allow for a high level of biocidal activity of polymeric materials against both model and pathogenic bacterial strains (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Legionella pneumophila, Salmonella enterica subsp. enterica) and fungi (Aspergillus brasiliensis, Saccharomyces cerevisiae, Candida albicans, Penicilium expansum), whilst ensuring the safety of use due to the lack of migration of particles to the surrounding environment. Studies have shown that adding 4% of a biocide containing Ag, Cu, ZnO, and Fe2O3 nanoparticles is the most optimal solution to reduce the number of S. aureus, S. enterica and P. aeruginosa by over 99%. The lowest effectiveness was observed against L. pneumophila bacteria. As for E. coli, a higher biocide content did not significantly increase the antibacterial activity. The results showed a high efficiency of the applied biocide at a concentration of 2% against fungal strains. The high efficiency of the obtained biocidal results was influenced by the uniform dispersion of nanoparticles in the material and their low degree of agglomeration. Furthermore, a slight migration of components to the environment is the basis for further research in the field of the application of the developed materials in industry.
Article
Background Recently, MALDI-TOF has emerged as a quick tool for bacterial typing. The aim was to evaluate if MALDI-TOF based typing of Legionella pneumophila can achieve the same discriminatory power as that of the Sequence Based Typing (SBT) method. Methods The Sequence Type (ST) was obtained from the 90 strains included in the training set and an in-house MALDI-TOF library based on the Main Spectra Profile (MSP) was generated for the identification of such ST. Then, our library was validated by three procedures: a) creating a dendrogram, b) searching for specific peaks present exclusively in each MSP entry, and c) analysing a validation set composed of 14 strains with known ST. Fully characterized L. pneumophila ATCC 33152, which belongs to ST 36, was used as a control strain. Results: In the training set, 17 strains belonged to ST 1, 1 to ST 20, 63 to ST 22, 1 to ST 146, 6 to ST 578, and 2 to ST 1086. Specific peaks present in each MSPs spectrum, which are considered type-specific biomarkers, ranged from 2 to 11; more concretely, MSP for ST 1 identification shows 2 specific peaks; MSP for ST 20 identification: 9 specific peaks; MSP for ST 22 and ST 36 identification: 11 specific peaks; MSP for ST 146 identification: 5 specific peaks; and MSP for ST 578 and ST 1086 identification: 3 specific peaks. Using the validation set (nine strains belonging to ST 22 and five to ST 1), MALDI-TOF assigned accurately the ST in 30 min per tested strain with a full match. Conclusions The ST of L. pneumophila can be identified and reported in few minutes directly from colonies grown on BCYE agar using MALDI-TOF.
Thesis
Les macrolides sont recommandés seuls ou en association dans le traitement des légionelloses. Cependant, malgré une antibiothérapie adaptée et l'absence de souches résistantes décrites, des échecs thérapeutiques sont régulièrement observés. L'isolement de souches de Legionella à partir de prélèvements respiratoires est indispensable à la réalisation d'antibiogrammes mais il n'est obtenu que dans 24% des cas en France. Au cours de ce travail, nous avons tout d'abord amélioré le rendement d'isolement des souches cliniques, notamment par des techniques de co-culture amibienne, avant de nous intéresser aux mécanismes pouvant expliquer les échecs thérapeutiques (antagonisme entre antibiotiques, sélection de résistance). Nous avons pu montrer, dans un modèle de croissance intracellulaire de Legionella pneumophila, une absence d'antagonisme entre macrolides et fluoroquinolones ou rifampicine invalidant la première hypothèse. Nous avons ensuite généré des lignées de mutants hautement résistants aux macrolides. Nous avons mis en évidence des mutations dans les gènes codant les protéines ribosomales L4 et L22 associées à un faible niveau de résistance. Des mutations dans les gènes codant l'ARN ribosomal 23S étaient associées à un niveau plus élevé de résistance, dépendant de la nature de la mutation et du nombre de copies du gène mutées. La facilité d'obtention de mutants résistants aux macrolides in vitro suggère une potentielle acquisition de résistance in vivo au décours de l'antibiothérapie et justifie une recherche systématique de cette résistance dans les prélèvements cliniques
Article
Intracellular bacterial pathogens harbor genes, the closest homologues of which are found in eukaryotes. Regulator of chormosome condensation 1 (RCC1) repeat proteins are phylogenetically widespread and implicated in protein‐protein interactions, such as the activation of the small GTPase Ran by its cognate guanine nucleotide exchange factor, RCC1. Legionella pneumophila and Coxiella burnetii , the causative agents of Legionnaires’ disease and Q fever, respectively, harbor RCC1 repeat coding genes. L. pneumophila secretes the RCC1 repeat “effector” proteins LegG1, PpgA and PieG into eukaryotic host cells, where they promote the activation of the pleiotropic small GTPase Ran, microtubule stabilization, pathogen vacuole motility and intracellular bacterial growth as well as host cell migration. The RCC1 repeat effectors localize to the pathogen vacuole or the host plasma membrane and target distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself. C. burnetii translocates the RCC1 repeat effector NopA into host cells, where the effector localizes to nucleoli. NopA binds to Ran GTPase and promotes the nuclear accumulation of Ran(GTP), thus pertubing the import of the transcription factor NF‐κB and innate immune signaling. Hence, divergent evolution of bacterial RCC1 repeat effectors defines the range of Ran GTPase cycle targets and likely allows fine‐tuning of Ran GTPase activation by the pathogens at different cellular sites This article is protected by copyright. All rights reserved.
Article
The water-borne bacterium Legionella pneumophila is the causative agent of Legionnaires' disease. In the environment, the opportunistic pathogen colonizes different niches, including free-living protozoa and biofilms. The physiological state(s) of sessile Legionella in biofilms and their functional consequences are not well understood. Using single-cell techniques and fluorescent growth rate probes as well as promoter reporters, we show here that sessile L. pneumophila exhibits phenotypic heterogeneity and adopts growing and nongrowing ("dormant") states in biofilms and microcolonies. Phenotypic heterogeneity is controlled by the Legionella quorum sensing (Lqs) system, the transcription factor LvbR, and the temperature. The Lqs system and LvbR determine the ratio between growing and nongrowing sessile subpopulations, as well as the frequency of growth resumption ("resuscitation") and microcolony formation of individual bacteria. Nongrowing L. pneumophila cells are metabolically active, express virulence genes and show tolerance toward antibiotics. Therefore, these sessile nongrowers are persisters. Taken together, the Lqs system, LvbR and the temperature control the phenotypic heterogeneity of sessile L. pneumophila, and these factors regulate the formation of a distinct subpopulation of nongrowing, antibiotic tolerant, virulent persisters. Hence, the biofilm niche of L. pneumophila has a profound impact on the ecology and virulence of this opportunistic pathogen.
Article
Aims: Legionella pneumophila (Lp), a human pathogen, has been detected in windscreen wiper fluid reservoirs (WWFRs) where commercial screenwashes (CSWs) are commonly added. Limited information is available on CSWs against planktonic Lp; however, responses of sessile Lp and planktonic Lp pre-acclimated in nutrient-limited water to CSWs remain unknown. This study thus investigates the antibacterial effects of CSWs on sessile and starved planktonic Lp, in comparison with unstarved Lp. Methods and results: Lp biofilms were produced on glass and WWFR materials of high-density polyethylene (HDPE) and polypropylene (PP). Planktonic Lp with and without acclimation in tap water were prepared. Log reductions in cell counts averaged 0.4-5.0 for ten brands of CSWs against sessile Lp and 1.0-3.9 and 0.9-4.9, respectively, against starved and unstarved planktonic Lp for five CSWs. Both biofilm formation and acclimation in tap water enhanced Lp resistance to CSWs. Significantly different log-reduction values among CSW brands were observed for sessile Lp on HDPE and planktonic Lp regardless of acclimation (p<0.05). Conclusions: Biofilm formation, starvation acclimation, and CSW brand are crucial factors influencing Lp response to CSWs. Significance and impact of study: This study advances the knowledge of Lp reaction in anthropogenic water systems with CSWs.
Article
Full-text available
Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD ) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored infectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.
Article
Full-text available
In the ecology of Legionella pneumophila a crucial role may be played by its relationship with the natural flora; thus we investigated the interactions between Legionella and other aquatic bacteria, particularly within biofilms. Among 80 aquatic bacteria screened for the production of bacteriocin-like substances (BLSs), 66.2% of them were active against L. pneumophila. The possible effect of some of these aquatic bacteria on the development and stability of L. pneumophila biofilms was studied. Pseudomonas fluorescens, the best BLS producer, showed the greatest negative effect on biofilm formation and strongly enhanced the detachment of Legionella. Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Aeromonas hydrophila, and Stenotrophomonas maltophilia, although producing BLSs at different levels, were less active in the biofilm experiments. Acinetobacter lwoffii did not produce any antagonistic compound and was the only one able to strongly enhance L. pneumophila biofilm. Our results highlight that BLS production may contribute to determining the fate of L. pneumophila within ecological niches. The interactions observed in this study are important features of L. pneumophila ecology, which knowledge may lead to more effective measures to control the persistence of the germ in the environment.
Article
Full-text available
An explosive, common-source outbreak of pneumonia caused by a previously unrecognized bacterium affected primarily persons attending an American Legion convention in Philadelphia in July, 1976. Twenty-nine of 182 cases were fatal. Spread of the bacterium appeared to be air borne. The source of the bacterium was not found, but epidemiologic analysis suggested that exposure may have occurred in the lobby of the headquarters hotel or in the area immediately surrounding the hotel. Person-to-person spread seemed not to have occurred. Many hotel employees appeared to be immune, suggesting that the agent may have been present in the vicinity, perhaps intermittently, for two or more years.
Article
Full-text available
Biofilms containing diverse microflora were developed in tap water on glass and polybutylene surfaces. Legionella pneumophila within the biofilms was labelled with monoclonal antibodies and visualized with immunogold or fluorescein isothiocyanate conjugates. Development of a differential interference contrast technique in an episcopic mode enabled simultaneous visualization of the total biofilm flora and gold-labelled legionellae. The legionellae occurred in microcolonies within the biofilm in the absence of amoebae, suggesting that the bacterial consortium was supplying sufficient nutrients to enable legionellae to grow extracellularly within the biofilm.
Article
Full-text available
The 16S ribosomal RNA sequences of Legionella pneumophila, L. erythra, L. hackeliae, L. spiritensis, L. longbeachae, L. bozemanii (Fluoribacter bozemanae) and L. micdadei (Tatlockia micdadei) were determined using reverse transcriptase. The sequences were compared with published sequences for Gram-negative bacteria and phylogenetic trees were constructed. The data confirm previous work which showed that the family Legionellaceae forms a monophyletic subgroup within the gamma subdivision of the Proteobacteria. The data show that all of the legionellae studied are highly related (greater than 95%) on the basis of 16S rRNA sequences and do not support the division of the family Legionellaceae into three genera.
Article
Full-text available
Legionella pneumophila serogroup 1 was observed to satellite around colonies of Flavobacterium breve on an L-cysteine-deficient medium which did not support growth of legionellae. Both isolates were recovered from the hot water tanks of hospitals. Ferric PPi stimulated satellite growth between 0.01 and 0.1%.
Article
Full-text available
Survival and growth of Legionella pneumophila in both biofilm and planktonic phases were determined with a two-stage model system. The model used filter-sterilized tap water as the sole source of nutrient to culture a naturally occurring mixed population of microorganisms including virulent L. pneumophila. At 20 degrees C, L. pneumophila accounted for a low proportion of biofilm flora on polybutylene and chlorinated polyvinyl chloride, but was absent from copper surfaces. The pathogen was most abundant on biofilms on plastics at 40 degrees C, where it accounted for up to 50% of the total biofilm flora. Copper surfaces were inhibitory to total biofouling and included only low numbers of L. pneumophila organisms. The pathogen was able to survive in biofilms on the surface of the plastic materials at 50 degrees C, but was absent from the copper surfaces at the same temperature. L. pneumophila could not be detected in the model system at 60 degrees C. In the presence of copper surfaces, biofilms forming on adjacent control glass surfaces were found to incorporate copper ions which subsequently inhibited colonization of their surfaces. This work suggests that the use of copper tubing in water systems may help to limit the colonization of water systems by L. pneumophila.
Article
Full-text available
Two Acanthamoeba species, fed at three temperatures, expelled vesicles containing living Legionella pneumophila cells. Vesicles ranged from 2.1 to 6.4 microns in diameter and theoretically could contain several hundred bacteria. Viable L. pneumophila cells were observed within vesicles which had been exposed to two cooling tower biocides for 24 h. Clusters of bacteria in vesicles were not dispersed by freeze-thawing and sonication. Such vesicles may be agents for the transmission of legionellosis associated with cooling towers, and the risk may be underestimated by plate count methods.
Article
Full-text available
Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation ofS. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants ofS. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.
Article
Full-text available
The formation of adherent multilayered biofilms embedded into a glycocalyx represents an essential factor in the pathogenesis of Staphylococcus epidermidis biomaterial-related infections. Using biofilm-producing S. epidermidis 1457 and transposon Tn917 carried on plasmid pTV1ts, we isolated nine isogenic biofilm-negative transposon mutants. Transduction by S. epidermidis phage 71 was used to prove the genetic linkage of transposon insertions and altered phenotypes. Mapping of the different transposon insertions by Southern hybridization and pulsed-field gel electrophoresis indicated that these were inserted in four unlinked genetic loci. According to their phenotypes, including quantitative differences in biofilm production in different growth media, in the amount of the polysaccharide intercellular adhesin (PIA) produced, in the hemagglutination titers, and in the altered colony morphology, the mutants could be separated into four phenotypic classes corresponding with the genetic classes. Synthesis of PIA was not detectable with class I and II mutants, whereas the amount of PIA produced reflected the residual degree of biofilm production of class III and IV mutants in different growth media. Chromosomal DNA flanking the transposon insertions of five class I mutants was cloned and sequenced, and the insertions were mapped to different locations of icaADBC, representing the synthetic genes for PIA. Expression of icaADBC from a xylose-dependent promoter in the different isogenic mutant classes reconstituted biofilm production in all mutants. In a Northern blot analysis no icaADBC-specific transcripts were observed in RNA isolated from mutants of classes II, III, and IV. Apparently, in addition to icaADBC, three other gene loci have a direct or indirect regulatory influence on expression of the synthetic genes for PIA on the level of transcription.
Article
Full-text available
Burkholderia cepacia and Pseudomonas aeruginosa often co-exist as mixed biofilms in the lungs of patients suffering from cystic fibrosis (CF). Here, the isolation of random mini-Tn5 insertion mutants of B. cepacia H111 defective in biofilm formation on an abiotic surface is reported. It is demonstrated that one of these mutants no longer produces N-acylhomoserine lactones (AHLs) due to an inactivation of the cepR gene. cepR and the cepI AHL synthase gene together constitute the cep quorum-sensing system of B. cepacia. By using a gene replacement method, two defined mutants, H111-I and H111-R, were constructed in which cepI and cepR, respectively, had been inactivated. These mutants were used to demonstrate that biofilm formation by B. cepacia H111 requires a functional cep quorum-sensing system. A detailed quantitative analysis of the biofilm structures formed by wild-type and mutant strains suggested that the quorum-sensing system is not involved in the regulation of initial cell attachment, but rather controls the maturation of the biofilm. Furthermore, it is shown that B. cepacia is capable of swarming motility, a form of surface translocation utilized by various bacteria to rapidly colonize appropriate substrata. Evidence is provided that swarming motility of B. cepacia is quorum-sensing-regulated, possibly through the control of biosurfactant production. Complementation of the cepR mutant H111-R with different biosurfactants restored swarming motility while biofilm formation was not significantly increased. This result suggests that swarming motility per se is not essential for biofilm formation on abiotic surfaces.
Article
Full-text available
Detachment from biofilms is an important consideration in the dissemination of infection and the contamination of industrial systems but is the least-studied biofilm process. By using digital time-lapse microscopy and biofilm flow cells, we visualized localized growth and detachment of discrete cell clusters in mature mixed-species biofilms growing under steady conditions in turbulent flow in situ. The detaching biomass ranged from single cells to an aggregate with a diameter of approximately 500 μm. Direct evidence of local cell cluster detachment from the biofilms was supported by microscopic examination of filtered effluent. Single cells and small clusters detached more frequently, but larger aggregates contained a disproportionately high fraction of total detached biomass. These results have significance in the establishment of an infectious dose and public health risk assessment.
Article
Full-text available
A central feature of all adaptive radiations is morphological divergence, but the phenotypic innovations that are responsible are rarely known. When selected in a spatially structured environment, populations of the bacterium Pseudomonas fluorescens rapidly diverge. Among the divergent morphs is a mutant type termed "wrinkly spreader" (WS) that colonizes a new niche through the formation of self-supporting biofilms. Loci contributing to the primary phenotypic innovation were sought by screening a WS transposon library for niche-defective (WS(-)) mutants. Detailed analysis of one group of mutants revealed an operon of 10 genes encoding enzymes necessary to produce a cellulose-like polymer (CLP). WS genotypes overproduce CLP and overproduction of the polymer is necessary for the distinctive morphology of WS colonies; it is also required for biofilm formation and to maximize fitness in spatially structured microcosms, but overproduction of CLP alone is not sufficient to cause WS. A working model predicts that modification of cell cycle control of CLP production is an important determinant of the phenotypic innovation. Analysis of >30 kb of DNA encoding traits required for expression of the WS phenotype, including a regulatory locus, has not revealed the mutational causes, indicating a complex genotype-phenotype map.
Article
Full-text available
This international collaborative survey identified culture-confirmed legionellosis in 508 patients with sporadic community-acquired legionellosis. Legionella pneumophila constituted 91.5% of the isolates. Serogroup 1 was the predominant serogroup (84.2%), and serogroups 2–13 (7.4%) accounted for the remaining serogroups. The Legionella species most commonly isolated were L. longbeachae (3.9%) and L. bozemanii (2.4%), followed by L. micdadei, L. dumoffii, L. feeleii, L. wadsworthii and L. anisa (2.2% combined). L. longbeachae constituted 30.4% of the community-acquired Legionella isolates in Australia and New Zealand
Article
A central feature of all adaptive radiations is morphological divergence, but the phenotypic innovations that are responsible are rarely known. When selected in a spatially structured environment, populations of the bacterium Pseudomonas fluorescens rapidly diverge. Among the divergent morphs is a mutant type termed “wrinkly spreader” (WS) that colonizes a new niche through the formation of self-supporting biofilms. Loci contributing to the primary phenotypic innovation were sought by screening a WS transposon library for niche-defective (WS-) mutants. Detailed analysis of one group of mutants revealed an operon of 10 genes encoding enzymes necessary to produce a cellulose-like polymer (CLP). WS genotypes overproduce CLP and overproduction of the polymer is necessary for the distinctive morphology of WS colonies; it is also required for biofilm formation and to maximize fitness in spatially structured microcosms, but overproduction of CLP alone is not sufficient to cause WS. A working model predicts that modification of cell cycle control of CLP production is an important determinant of the phenotypic innovation. Analysis of >30 kb of DNA encoding traits required for expression of the WS phenotype, including a regulatory locus, has not revealed the mutational causes, indicating a complex genotype-phenotype map.
Article
Material accumulates at the water-air interface of all natural water bodies to form a surface film. The interface is a dynamic environment, so surface films are altered by water movements, solar radiation, and biological processes. These films consist of a complex, of organic matter and microorganisms, some of which are harmful. Researchers have often overlooked surface films when studying water bodies, and their importance is only now being recognized.
Article
Biofilms can develop on almost any surface exposed to an aqueous environment. The biofilm systems that result can be used beneficially, as exemplified by fixed-film wastewater treatment processes (for example, trickling filters and rotating biological contactors). In addition, biofilms play a major positive role in stream purification processes. However, biofilms can be quite troublesome in certain engineering systems. In water distribution systems and heat transfer equipment, for example, biofilms can cause substantial energy losses resulting from increased fluid frictional resistance and increased heat transfer resistance. The significance of biofilm development on various processes is summarized. (JMT)
Article
Material accumulates at the water–air interface of all natural water bodies to form a surface film. The interface is a dynamic environment, so surface films are altered by water movements, solar radiation, and biological processes. These films consist of a complex of organic matter and microorganisms, some of which are harmful. Researchers have often overlooked surface films when studying water bodies, and their importance is only now being recognized.
Article
During the third Icebreaker ODEN expedition to the North Pole, Arctic Ocean 2001, the surface of open leads between the pack ice was investigated for physical parameters. The major purpose was to evaluate the occurrence of a surfactant microlayer in the leads. This was done by estimating sea-surface tension and collecting sea-surface microlayers in open leads (SMOL). Three collection methods used for SMOL were rotating drums covered with hydrophilic Teflon, thin hydrophobic Teflon sheets, and glass plates. Collections were successfully made at 14 stations between 21 July and 21 August 2001, at geo-positions between 86°29‘ N and the geographical North Pole. Surface tensions resembled surfactant-free seawater surfaces during the first 2 weeks of August but were depressed by 6−9 mN m-1 during the third week of August. Variations in SMOL physical properties were large between stations. Weather conditions and collection equipment functionality are discussed in relation to results.
Article
Bacterial biofilm is a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface, which constitutes a protected mode of growth that allows survival in hostile environment. The biofilm-forming microorganisms have been shown to elicit specific mechanisms for initial attachment to a surface, formation of microcolony leading to development of three-dimensional structure of mature biofilm. They differ from their free-living counterparts in their growth rate, composition and increased resistance to biocides, antibiotics and antibodies by virtue of up regulation and/or down regulation of approximately 40 per cent of their genes. This makes them highly difficult to eradicate with therapeutic doses of antimicrobial agents. A greater understanding of mechanism of their formation and survival under sessile environments may help in devising control strategies.
Article
Aims: Stagnation is widely believed to predispose water systems to colonization by Legionella. A model plumbing system was constructed to determine the effect of flow regimes on the presence of Legionella within microbial biofilms. Methods and results: The plumbing model contained three parallel pipes where turbulent, laminar and stagnant flow regimes were established. Four sets of experiments were carried out with Reynolds number from 10,000 to 40,000 and from 355 to 2,000 in turbulent and laminar pipes, respectively. Legionella counts recovered from biofilm and planktonic water samples of the three sampling pipes were compared with to determine the effect of flow regime on the presence of Legionella. Significantly higher colony counts of Legionella were recovered from the biofilm of the pipe with turbulent flow compared with the pipe with laminar flow. The lowest counts were in the pipe with stagnant flow. Conclusions: We were unable to demonstrate that stagnant conditions promoted Legionella colonization. Significance and impact of the study: Plumbing modifications to remove areas of stagnation including deadlegs are widely recommended, but these modifications are tedious and expensive to perform. Controlled studies in large buildings are needed to validate this unproved hypothesis.
Article
Studies on Legionella show a continuum from environment to human disease. Legionellosis is caused by Legionella species acquired from environmental sources, principally water sources such as cooling towers, where Legionella grows intracellularly in protozoa within biofilms. Aquatic biofilms, which are widespread not only in nature, but also in medical and dental devices, are ecological niches in which Legionella survives and proliferates and the ultimate sources to which outbreaks of legionellosis can be traced. Invasion and intracellular replication of L. pneumophila within protozoa in the environment play a major role in the transmission of Legionnaires' disease. Protozoa provide the habitats for the environmental survival and reproduction of Legionella species. L. pneumophila proliferates intracellularly in various species of protozoa within vacuoles studded with ribosomes, as it also does within macrophages. Growth within protozoa enhances the environmental survival capability and the pathogenicity (virulence) of Legionella. The growth requirements of Legionella, the ability of Legionella to enter a viable non-culturable state, the association of Legionella with protozoa and the occurrence of Legionella within biofilms complicates the detection of Legionella and epidemiological investigations of legionellosis. Polymerase chain reaction (PCR) methods have been developed for the molecular detection of Legionella and used in environmental and epidemiological studies. Various physical and chemical disinfection methods have been developed to eliminate Legionella from environmental sources, but gaining control of Legionella in environmental waters, where they are protected from disinfection by growing within protozoa and biofilms, remains a challenge, and one that must be overcome in order to eliminate sporadic outbreaks of legionellosis.
Article
An examination of cellular extracts ofLegionella pneumophila (Philadelphia 1 and Knoxville 1) was undertaken and key enzymes of the Embden-Meyerhof-Parnas (EMP) and pentose phosphate (PP) pathways, and the Krebs cycle were found. No enzymatic evidence of the ED pathway was obtained. In regard to carbon flow in the EMP pathway, the activities of fructose-1,6-biphosphatase (6–7.3 nmol/min/mg protein) and of phosphofructokinase (0.67–0.8 nmol/min/mg protein) suggested a gluconeogenic role. In further support of this direction, good activities were detected for phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase. While an energized membrane was required for glutamate uptake by whole cells, an energized mechanism for glucose uptake could not be demonstrated. The Krebs cycle was essentially complete and, despite high specific activities for isocitrate and malate dehydrogenases, whole cells failed to oxidize these substrates, suggesting a transport deficiency. The major carbon and energy sources serine and glutamate were catabolized vial-serine dehydratase and glutamate-aspartate transaminase, respectively. This study confirmed that amino acids are catabolized via the Krebs cycle and that sugars are synthesized by the gluconeogenic enzymes of the EMP pathway.
Article
The ability of heterotrophic plate count bacterial strains isolated from chlorinated drinking water on low-nutrient media to inhibit the growth ofLegionella species was examined. Between 16% and 32% of these strains were able to inhibit the growth ofLegionella species when tested on buffered charcoal yeast extract agar. The exact proportion of inhibiting strains varied with the individualLegionella species. Two strains that inhibited the growth of severalLegionella species could also stimulate the growth of the same species when both the test strain and theLegionella species were grown on buffered charcoal yeast extract agar that lacked the essential amino acidl-cysteine.
Article
Observations of surface microlayer material collected from freshwaters demonstrate its ability to assemble rapidly at the water-air interface following experimental disruption. This property is exploited to provide a means for studying and manipulating the in situ locomotory behaviour of some surface microlayer amoebae (Acanthamoeba, Naegleria, Vannella) under laboratory conditions. Amoeboid movement is substratum-dependent and so it is important to know the means by which these organisms attach to a water-air interface. Reflection interference microscopy (RIM) demonstrates that during locomotion Vannella adheres to a glass coverslip by means of an unstable platform of associated contact (parallel to, but ∼100-nm off it) within which stable, focal contacts form attaching to the substratum. These two key features of cell-substratum interactions occur also in Acanthamoeba and Naegleria. Direct examination of cell-substratum interactions during amoeboid movement at the water-air interface by RIM proves that the associated contact is also established here.
Article
The reservoir for hospital-acquired Legionnaires disease has been shown to be the potable water distribution system. We investigated the influence of the natural microbial population and sediment (scale and organic particulates) found in water systems as growth-promoting factors for Legionella pneumophila. Our in vitro experiments showed that: (i) water from hot-water storage tank readily supported the survival of L. pneumophila, (ii) the concentration of sediment was directly related to the survival of L. pneumophila, (iii) the presence of environmental bacteria improved the survival of L. pneumophila via nutritional symbiosis, (iv) the combination of sediment and environmental bacteria acted synergistically to improve the survival of L. pneumophila, and (v) the role of sediment in this synergistic effect was determined to be nutritional. Sediment was found to stimulate the growth of environmental microflora, which in turn stimulated the growth of L. pneumophila. These findings confirm the empiric observations of the predilection of L. pneumophila for growth in hot-water tanks and its localization to sediment. L. pneumophila occupies an ecological niche within the potable water system, with interrelationships between microflora, sediment, and temperature.
Article
Legionella pneumophila (Legionnaires disease bacterium) of serogroup 1 was isolated from an algal-bacterial mat community growing at 45 degrees C in a man-made thermal effluent. This isolate was grown in mineral salts medium at 45 degrees C in association with the blue-green alga (cyanobacterium) Fischerella sp. over a pH range of 6.9 to 7.6. L. pneumophila was apparently using algal extracellular products as its carbon and energy sources. These observations indicate that the temperature, pH, and nutritional requirements of L. pneumophila are not as stringent as those previously observed when cultured on complex media. This association between L. pneumophila and certain blue-green algae suggests an explanation for the apparent widespread distribution of the bacterium in nature.
Article
The amino acids required for growth and as energy sources by 10 strains of Legionella pneumophila were determined by using a chemically defined medium. All strains required arginine, cysteine, isoleucine, leucine, threonine, valine, methionine, and phenylalanine or tyrosine. Most strains (7 of 10) required serine, and two strains had to be supplied proline before growth could be established. All 10 strains used serine and, to a lesser extent, threonine as the sole sources of carbon and energy. The Y serine calculated was 94.9 +/- 8.5 g (dry weight) of cells/mol of serine. Assuming that the value of Y adenosine 5'-triphosphate is 10.5, these results indicate that oxidative catabolism of 1 mol of serine yielded approximately 9 mol of adenosine 5'-triphosphate. This high yield suggests that although serine was the major source of carbon, other amino acids may also be metabolized.
Article
Bacteria were concentrated 500-fold from 20-liter water samples collected from 67 different lakes and rivers in the United States. The data suggest that Legionella pneumophila is part of the natural aquatic environment and that the bacterium is capable of surviving extreme ranges of environmental conditions. The data further demonstrate the effectiveness of the direct fluorescent-antibody technique for detecting L. pneumophila in natural aquatic systems. Smears of the concentrated samples were screened microscopically for serogroups of L. pneumophila by the direct fluorescent-antibody technique. Virtually all of the 793 samples were found to be positive by this method. The 318 samples containing the largest numbers of positive bacteria which were morphologically consistent with L. pneumophila were injected into guinea pigs for attempted isolations. Isolates were obtained from habitats with a wide range of physical, chemical, and biological parameters. Samples collected monthly from a thermally altered lake and injected into guinea pigs demonstrated a seasonality of infection, with the highest frequency of infection occurring during the summer months.
Article
A total of 100 water samples, 95% of which were taken from hospital potable water fixtures, were cultured on three different media used for the isolation of Legionella pneumophila. The media used were buffered charcoal-yeast extract medium (BCYE alpha medium), BCYE alpha medium with antimicrobial agents (BMPA alpha medium), and BCYE alpha medium with antimicrobial agents, glycine, and differential dyes (MWY medium). An acid wash procedure was also used for specimens plated on BCYE alpha and BMPA alpha media. A total of 24 samples were culture positive for L. pneumophila by one or more techniques. MWY medium detected 92% of positive cultures, BCYE alpha medium with the acid wash detected 83% of positive cultures, BMPA alpha medium detected 79% of positive cultures. BCYE alpha medium detected 71% of positive cultures, and BMPA alpha medium with the acid wash detected 62% of positive cultures. MWY medium was the best medium for isolating L. pneumophila from potable water specimens and can probably be depended upon as the sole medium for this type of testing.
Article
Legionella pneumophila, the causative organism of Legionnaires' disease, is pathogenic for free living, ubiquitous, freshwater, and soil amoebae of the genera Acanthamoeba and Naegleria. Some species support the growth of strains from serogroups 1 to 6, others only strains from certain serogroups. Initial studies with seeded material indicate that amoebal enrichment could be utilised for the isolation of legionellae from clinical specimens and natural habitats. It is suggested that a vacuole, or amoeba, full of legionellae, rather than free legionellae, could be the infective particle for man.