Impact of temperature on Legionella pneumophila, its protozoan host cells, and the microbial diversity of the biofilm community of a pilot cooling tower

ABSTRACTLegionella 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.

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Unravelling the importance of the eukaryotic and bacterial communities and their relationship with Legionella spp. ecology in cooling towers: a complex network

Microbiome ◽

10.1186/s40168-020-00926-6 ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Kiran Paranjape ◽

Émilie Bédard ◽

Deeksha Shetty ◽

Mengqi Hu ◽

Fiona Chan Pak Choon ◽

...

Keyword(s):

Legionella Pneumophila ◽

Cooling Tower ◽

Severe Pneumonia ◽

Amplicon Sequencing ◽

Host Community ◽

Cooling Towers ◽

Eukaryotic Community ◽

Protozoan Community ◽

Survival And Growth

Abstract Background Cooling towers are a major source of large community-associated outbreaks of Legionnaires’ disease, a severe pneumonia. This disease is contracted when inhaling aerosols that are contaminated with bacteria from the genus Legionella, most importantly Legionella pneumophila. How cooling towers support the growth of this bacterium is still not well understood. As Legionella species are intracellular parasites of protozoa, it is assumed that protozoan community in cooling towers play an important role in Legionella ecology and outbreaks. However, the exact mechanism of how the eukaryotic community contributes to Legionella ecology is still unclear. Therefore, we used 18S rRNA gene amplicon sequencing to characterize the eukaryotic communities of 18 different cooling towers. The data from the eukaryotic community was then analysed with the bacterial community of the same towers in order to understand how each community could affect Legionella spp. ecology in cooling towers. Results We identified several microbial groups in the cooling tower ecosystem associated with Legionella spp. that suggest the presence of a microbial loop in these systems. Dissolved organic carbon was shown to be a major factor in shaping the eukaryotic community and may be an important factor for Legionella ecology. Network analysis, based on co-occurrence, revealed that Legionella was correlated with a number of different organisms. Out of these, the bacterial genus Brevundimonas and the ciliate class Oligohymenophorea were shown, through in vitro experiments, to stimulate the growth of L. pneumophila through direct and indirect mechanisms. Conclusion Our results suggest that Legionella ecology depends on the host community, including ciliates and on several groups of organisms that contribute to its survival and growth in the cooling tower ecosystem. These findings further support the idea that some cooling tower microbiomes may promote the survival and growth of Legionella better than others.

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Presence ofLegionellaspp. in cooling towers: the role of microbial diversity,Pseudomonas, and continuous chlorine application

10.1101/540302 ◽

2019 ◽

Cited By ~ 3

Author(s):

Kiran Paranjape ◽

Émilie Bédard ◽

Lyle G. Whyte ◽

Jennifer Ronholm ◽

Michèle Prévost ◽

...

Keyword(s):

Bacterial Community ◽

Microbial Diversity ◽

Legionella Pneumophila ◽

Cooling Tower ◽

Severe Pneumonia ◽

Water Source ◽

Amplicon Sequencing ◽

Physical Parameters ◽

Cooling Towers ◽

Strong Negative Correlation

ABSTRACTLegionnaire’s Disease (LD) is a severe pneumonia caused byLegionella pneumophila. Cooling towers are the main source ofL. pneumophiladuring large outbreaks. Colonization, survival, and proliferation ofL. pneumophilain cooling towers are necessary for outbreaks to occur. These steps are affected by chemical and physical parameters of the cooling tower environment. We hypothesize that the bacterial community residing in the cooling tower could also affect the presence ofL. pneumophila. A16S rRNAtargeted amplicon sequencing approach was used to study the bacterial community of cooling towers and its relationship with theLegionella spp.andL. pneumophilacommunities. The results indicated that the water source shaped the bacterial community of cooling towers. Several taxa were enriched and positively correlated withLegionella spp.andL. pneumophila. In contrast,Pseudomonasshowed a strong negative correlation withLegionella spp.and several other genera. Most importantly, continuous chlorine application reduced microbial diversity and promoted the presence ofPseudomonascreating a non-permissive environment forLegionella spp. This suggests that disinfection strategies as well as the resident microbial population influences the ability ofLegionella spp.to colonize cooling towers.

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Toxoflavin secreted by Pseudomonas alcaliphila inhibits growth of Legionella pneumophila and its host Vermamoeba vermiformis

10.1101/2022.01.08.475489 ◽

2022 ◽

Author(s):

Sebastien P. Faucher ◽

Sara Matthews ◽

Arvin Nickzad ◽

Passoret Vounba ◽

Deeksha Shetty ◽

...

Keyword(s):

Legionella Pneumophila ◽

Severe Pneumonia ◽

Amplicon Sequencing ◽

Inhibitory Effect ◽

Water Systems ◽

Bulk Water ◽

Cooling Towers ◽

High Concentration ◽

16S Rrna Amplicon Sequencing

Legionella pneumophila is a natural inhabitant of water systems. From there, it can be transmitted to humans by aerosolization resulting in severe pneumonia. Most large outbreaks are caused by cooling towers contaminated with L. pneumophila. The resident microbiota of the cooling tower is a key determinant for the colonization and growth of L. pneumophila. The genus Pseudomonas correlates negatively with the presence of L. pneumophila, but it is not clear which species is responsible. Therefore, we identified the Pseudomonas species inhabiting 14 cooling towers using a Pseudomonas-specific 16S rRNA amplicon sequencing strategy. Cooling towers free of L. pneumophila contained a high relative abundance of members from the Pseudomonas alcaliphila/oleovorans phylogenetic cluster. In vitro, P. alcaliphila JCM 10630 inhibited the growth of L. pneumophila on agar plates. Analysis of the P. alcaliphila genome revealed the presence of a genes cluster predicted to produce toxoflavin. L. pneumophila growth was inhibited by pure toxoflavin and by extract from P. alcaliphila culture found to contain toxoflavin by LC-ESI-MS. In addition, toxoflavin inhibits growth of Vermameoba vermiformis, a host cell of L. pneumophila. Our study indicates that P. alcaliphila may be important to restrict growth of L. pneumophila in water systems through the production of toxoflavin. A sufficiently high concentration is likely not achieved in the bulk water but might have a local inhibitory effect such as in biofilm.

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MultipleLegionella pneumophilaeffector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1708553114 ◽

2017 ◽

Vol 114 (48) ◽

pp. E10446-E10454 ◽

Cited By ~ 31

Author(s):

Stephanie R. Shames ◽

Luying Liu ◽

James C. Havey ◽

Whitman B. Schofield ◽

Andrew L. Goodman ◽

...

Keyword(s):

Legionella Pneumophila ◽

Severe Pneumonia ◽

Effector Proteins ◽

Host Cells ◽

Limiting Factor ◽

Effector Protein ◽

Host Immune System ◽

Loss Of Function ◽

High Throughput Analysis ◽

Single Strain

Legionella pneumophilais the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain ofL. pneumophilaencodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number ofL. pneumophilaeffectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhancedL. pneumophilain the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute toL. pneumophilavirulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis.

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The Legionella longbeachae Icm/Dot Substrate SidC Selectively Binds Phosphatidylinositol 4-Phosphate with Nanomolar Affinity and Promotes Pathogen Vacuole-Endoplasmic Reticulum Interactions

Infection and Immunity ◽

10.1128/iai.01685-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4021-4033 ◽

Cited By ~ 33

Author(s):

Stephanie Dolinsky ◽

Ina Haneburger ◽

Adam Cichy ◽

Mandy Hannemann ◽

Aymelt Itzen ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Legionella Pneumophila ◽

Severe Pneumonia ◽

Biological Data ◽

Effector Proteins ◽

Host Cells ◽

Titration Calorimetry ◽

Dependent Manner ◽

Content Type ◽

Phosphatidylinositol 4

ABSTRACTLegionellaspp. cause the severe pneumonia Legionnaires' disease. The environmental bacteria replicate intracellularly in free-living amoebae and human alveolar macrophages within a distinct, endoplasmic reticulum (ER)-derived compartment termed theLegionella-containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS) that translocates into host cells a plethora of different “effector” proteins, some of which anchor to the pathogen vacuole by binding to phosphoinositide (PI) lipids. Here, we identified by unbiased pulldown assays inLegionella longbeachaelysates a 111-kDa SidC homologue as the major phosphatidylinositol 4-phosphate [PtdIns(4)P]-binding protein. The PI-binding domain was mapped to a 20-kDa P4C [PtdIns(4)Pbinding of SidC] fragment. Isothermal titration calorimetry revealed that SidC ofL. longbeachae(SidCLlo) binds PtdIns(4)Pwith aKd(dissociation constant) of 71 nM, which is 3 to 4 times lower than that of the SidC orthologue ofLegionella pneumophila(SidCLpn). Upon infection of RAW 264.7 macrophages withL. longbeachae, endogenous SidCLloor ectopically produced SidCLpnlocalized in an Icm/Dot-dependent manner to the PtdIns(4)P-positive LCVs. AnL. longbeachaeΔsidCdeletion mutant was impaired for calnexin recruitment to LCVs inDictyostelium discoideumamoebae and outcompeted by wild-type bacteria inAcanthamoeba castellanii. Calnexin recruitment was restored by SidCLloor its orthologues SidCLpnand SdcALpn. Conversely, calnexin recruitment was restored by SidCLloinL. pneumophilalackingsidCandsdcA. Together, biochemical, genetic, and cell biological data indicate that SidCLlois anL. longbeachaeeffector that binds through a P4C domain with high affinity to PtdIns(4)Pon LCVs, promotes ER recruitment to the LCV, and thus plays a role in pathogen-host interactions.

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Investigating Bacterial and Free-Living Protozoa Diversity in Biofilms of Hot Water Pipes of Apartment Buildings in the City of Riga (Latvia)

Frontiers in Water ◽

10.3389/frwa.2021.799840 ◽

2021 ◽

Vol 3 ◽

Author(s):

Baiba Vilne ◽

Lelde Grantiņa-Ieviņa ◽

Juris Ķibilds ◽

Artjoms Mališevs ◽

Genadijs Konvisers ◽

...

Keyword(s):

Water Temperature ◽

Legionella Pneumophila ◽

Amplicon Sequencing ◽

Hot Water ◽

Community Diversity ◽

Strictly Aerobic ◽

Free Living ◽

Water Pipes ◽

Apartment Buildings ◽

Wide Range

Background: Biofilms, when formed on the surfaces of water pipes, can be responsible for a wide range of water quality and operational problems. We sought to assess the bacterial and free-living protozoa (FLP) diversity, in relation to the presence of Legionnaire's disease-causing bacteria Legionella pneumophila (L. pneumophila) in 45 biofilms of hot water distribution system pipes of apartment buildings in Riga, the capital city of Latvia.Results: 16S rRNA amplicon sequencing (metataxonomics) revealed that each biofilm contained 224 rather evenly distributed bacterial genera and that most common and most abundant were two genera, completely opposites in terms of their oxygen requirements: the obligately anaerobic Thermodesulfovibrio and the strictly aerobic Phenylobacterium. Water temperature and north-south axis (i.e., different primary water sources) displayed the most significant effect on the inter-sample variations, allowing us to re-construct three sub-networks (modules) of co-occurring genera, one involving (potentially FLP-derived) Legionella spp. Pangenome-based functional profile predictions suggested that all three may be dominated by pathways related to the development and maintenance of biofilms, including quorum sensing and nutrient transport, as well as the utilization of various energy sources, such as carbon and nitrogen. In our 18S rRNA amplicon sequencing data, potential hosts of L. pneumophila were detected in 11 out of 12 biofilm samples analyzed, however, in many cases, their relative abundance was very low (<1%). By validating our findings using culture-based methods, we detected L. pneumophila (serogroups 2, 3, 6 and 9) in nine (20%) biofilms, whereas FLP (mostly Acanthamoeba, Vahlkampfidae and Vermamoeba spp.) were present in six (~13%) biofilms. In two biofilms, L. pneumophila and its potential hosts were detected simultaneously, using culture-based methods.Conclusions: Overall, our study sheds light on the community diversity of hot water biofilms and predicts how several environmental factors, such as water temperature and source might shape it.

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Comparison of Legiolert and a Conventional Culture Method for Detection of Legionella pneumophila from Cooling Towers in Québec

Journal of AOAC International ◽

10.5740/jaoacint.18-0245 ◽

2019 ◽

Vol 102 (4) ◽

pp. 1235-1240 ◽

Cited By ~ 2

Author(s):

Isabelle Barrette

Keyword(s):

Legionella Pneumophila ◽

Cooling Tower ◽

Test Procedure ◽

Agar Plate ◽

Culture Method ◽

Most Probable Number ◽

Cooling Towers ◽

Plate Method ◽

Probable Number ◽

Compliance Testing

Abstract Background: Legionnaires’ disease is a potentially lethal pneumonia contracted through inhalation of aerosolized water contaminated with Legionella bacteria. Detection and control of L. pneumophila, the primary species responsible for the disease, is critical to public health. In Québec, cooling towers and evaporative condensers are required to follow a maintenance and testing program to ensure L. pneumophila concentrations remain at acceptable levels. Objective: This study compared a new culture method based on the most probable number approach, Legiolert®, with the formal culture method used at EnvironeX for regulatory compliance testing to quantify L. pneumophila from cooling tower waters in Québec. Methods: A split-sample analysis was performed in which 401 samples from cooling towers in Québec were tested with both methods. Results: Results with 74 positive samples showed that Legiolert provided a significant increase in sensitivity for L. pneumophila compared with the agar plate method. Cooling tower samples often contain non-Legionella flora that necessitate multiple treatment and plating conditions to prevent interference with the test. Legiolert showed little to no impact from non-Legionella organisms in this study. Conclusions: Overall, Legiolert showed several advantages over the agar plate method, including increased sensitivity, reduced interference, a simplified test procedure, and an easy-to-read positive signal.

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Alveolar Macrophages and Neutrophils Are the Primary Reservoirs for Legionella pneumophila and Mediate Cytosolic Surveillance of Type IV Secretion

Infection and Immunity ◽

10.1128/iai.01891-14 ◽

2014 ◽

Vol 82 (10) ◽

pp. 4325-4336 ◽

Cited By ~ 31

Author(s):

Alan M. Copenhaver ◽

Cierra N. Casson ◽

Hieu T. Nguyen ◽

Thomas C. Fung ◽

Matthew M. Duda ◽

...

Keyword(s):

Immune Response ◽

Alveolar Macrophages ◽

Legionella Pneumophila ◽

Pulmonary Infection ◽

Severe Pneumonia ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Content Type ◽

Type Iv

ABSTRACTLegionella pneumophila, an intracellular pathogen responsible for the severe pneumonia Legionnaires' disease, uses itsdot/icm-encoded type IV secretion system (T4SS) to translocate effector proteins that promote its survival and replication into the host cell cytosol. However, by introducing bacterial products into the host cytosol,L. pneumophilaalso activates cytosolic immunosurveillance pathways, thereby triggering robust proinflammatory responses that mediate the control of infection. Thus, the pulmonary cell types thatL. pneumophilainfects not only may act as an intracellular niche that facilitates its pathogenesis but also may contribute to the immune response againstL. pneumophila. The identity of these host cells remains poorly understood. Here, we developed a strain ofL. pneumophilaproducing a fusion protein consisting of β-lactamase fused to the T4SS-translocated effector RalF, which allowed us to track cells injected by the T4SS. Our data reveal that alveolar macrophages and neutrophils both are the primary recipients of T4SS-translocated effectors and harbor viableL. pneumophiladuring pulmonary infection of mice. Moreover, both alveolar macrophages and neutrophils from infected mice produced tumor necrosis factor and interleukin-1α in response to T4SS-sufficient, but not T4SS-deficient,L. pneumophila. Collectively, our data suggest that alveolar macrophages and neutrophils are both an intracellular reservoir forL. pneumophilaand a source of proinflammatory cytokines that contribute to the host immune response againstL. pneumophiladuring pulmonary infection.

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Legionella species and serogroups in Malaysian water cooling towers: identification by latex agglutination and PCR-DNA sequencing of isolates

Journal of Water and Health ◽

10.2166/wh.2009.002 ◽

2009 ◽

Vol 8 (1) ◽

pp. 92-100 ◽

Cited By ~ 4

Author(s):

Stacey Foong Yee Yong ◽

Fen-Ning Goh ◽

Yun Fong Ngeow

Keyword(s):

Dna Sequencing ◽

Legionella Pneumophila ◽

Cooling Tower ◽

Latex Agglutination ◽

Cooling Towers ◽

Water Cooling ◽

Public Health Policies ◽

Legionella Species ◽

Repeat Sampling ◽

East West

In this study, we investigated the distribution of Legionella species in water cooling towers located in different parts of Malaysia to obtain information that may inform public health policies for the prevention of legionellosis. A total of 20 water samples were collected from 11 cooling towers located in three different states in east, west and south Malaysia. The samples were concentrated by filtration and treated with an acid buffer before plating on to BCYE agar. Legionella viable counts in these samples ranged from 100 to 2,000 CFU ml−1; 28 isolates from the 24 samples were examined by latex agglutination as well as 16S rRNA and rpoB PCR-DNA sequencing. These isolates were identified as Legionella pneumophila serogroup 1 (35.7%), L. pneumophila serogroup 2–14 (39%), L. pneumophila non-groupable (10.7%), L. busanensis, L. gormanii, L. anisa and L. gresilensis.L. pneumophila was clearly the predominant species at all sampling sites. Repeat sampling from the same cooling tower and testing different colonies from the same water sample showed concurrent colonization by different serogroups and different species of Legionella in some of the cooling towers.

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Community outbreak of Legionnaires’ disease in Vic-Gurb, Spain in October and November 2005

Eurosurveillance ◽

10.2807/esm.12.03.00691-en ◽

2007 ◽

Vol 12 (3) ◽

pp. 5-6 ◽

Cited By ~ 8

Author(s):

M R Sala ◽

C Arias ◽

J M Oliva ◽

A Pedrol ◽

P Roura ◽

...

Keyword(s):

Central Region ◽

Legionella Pneumophila ◽

Cooling Tower ◽

Weather Conditions ◽

High Humidity ◽

Cooling Towers ◽

Large Area ◽

Environmental Investigation ◽

Flat Terrain ◽

Legionnaires Disease

This paper reports the investigation of a community-acquired outbreak of Legionnaires'; disease in the municipalities of Vic and Gurb (Central Region of Catalonia, Spain). There were 55 cases reported in October and November 2005. An epidemiological and environmental investigation was undertaken. Thirty-five case patients (64%) lived in Vic or Gurb, while 36% had visited or worked in Vic or Gurb during the 10 days before onset of symptoms, but no commonly frequented building could be identified. Water probes for culture were obtained from 30 cooling towers. In five cooling towers of two industrial settings in Gurb (plants A and B), Legionella pneumophila (Lp) serogroup 1 was present. Two Lp-1 strains were recovered from cooling towers in plants A and B. The Lp-1 strain from plant A showed a PGFE profile identical with those obtained from three patients. The exposure to Legionella pneumophila apparently occurred in a large area, since 43 of the 55 cases lived, visited or worked within a distance of 1,800 m from plant A, and six cases in a distance between 2,500 and 3,400 m. The inspections of cooling towers in plant A revealed inadequate disinfectant doses of biocide, non-existent maintenance records on weekends and wrong sample points for routine microbial check-ups. Weather conditions in October 2005 template temperature and high humidity (wind conditions are unappreciable) could have been favourable factors in this outbreak together with the flat terrain of Gurb and Vic area, explaining the extensive horizontal airborne dissemination of contaminated aerosols. The outbreak could have been prevented by proper and correct maintenance of the cooling tower at plant A.

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