Highlighting the Potency of Biosurfactants Produced by Pseudomonas Strains as Anti-Legionella Agents

Legionella pneumophila, the causative agent of Legionnaires’ disease, is a waterborne bacterium mainly found in man-made water systems in close association with free-living amoebae and multispecies biofilms. Pseudomonas strains, originating from various environments including freshwater systems or isolated from hospitalized patients, were tested for their antagonistic activity towards L. pneumophila. A high amount of tested strains was thus found to be active. This antibacterial activity was correlated to the presence of tensioactive agents in culture supernatants. As Pseudomonas strains were known to produce biosurfactants, these compounds were specifically extracted and purified from active strains and further characterized using reverse-phase HPLC and mass spectrometry methods. Finally, all biosurfactants tested (lipopeptides and rhamnolipids) were found active and this activity was shown to be higher towards Legionella strains compared to various other bacteria. Therefore, described biosurfactants are potent anti-Legionella agents that could be used in the water treatment industry although tests are needed to evaluate how effective they would be under field conditions.

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Computational framework for evaluating risk trade-offs in costs associated with Legionnaires’ Disease risk, energy, and scalding risk for hospital hot water systems

Environmental Science Water Research & Technology ◽

10.1039/d1ew00397f ◽

2021 ◽

Author(s):

Ashley Heida ◽

Alexis Mraz ◽

Mark Hamilton ◽

Mark Weir ◽

Kerry A Hamilton

Keyword(s):

Legionella Pneumophila ◽

Disease Risk ◽

Disease Outbreaks ◽

Febrile Illness ◽

Water Systems ◽

Hot Water ◽

Computational Framework ◽

Trade Offs ◽

Pontiac Fever ◽

Legionnaires Disease

Legionella pneumophila are bacteria that when inhaled cause Legionnaires’ Disease (LD) and febrile illness Pontiac Fever. As of 2014, LD is the most frequent cause of waterborne disease outbreaks due...

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Legionellosis and legionnaires’ disease

Oxford Textbook of Medicine ◽

10.1093/med/9780198746690.003.0143 ◽

2020 ◽

pp. 1226-1229

Author(s):

Diego Viasus ◽

Jordi Carratalà

Keyword(s):

Legionella Pneumophila ◽

Case Fatality ◽

Water Systems ◽

Natural Habitats ◽

Cooling Systems ◽

Freshwater Streams ◽

First Choice ◽

Legionnaires Disease ◽

Failure Prognosis ◽

Human Infections

Legionellaceae are Gram-negative bacilli, of which Legionella pneumophila is the principal cause of human infections. Their natural habitats are freshwater streams, lakes, thermal springs, moist soil, and mud, but the principal source for large outbreaks of legionellosis is cooling systems used for air conditioning and other cooling equipment. Legionella spp. are principally transmitted to humans through contaminated water aerosols. Middle-aged men, smokers, regular alcohol drinkers, and those with comorbidity are most at risk. Aside from supportive care, the first-choice antibiotics are macrolides (mainly azithromycin) and/or fluoroquinolones (especially levofloxacin). Case fatality is 5–15% in previously well adults, but much higher in those who are immunocompromised or develop respiratory failure. Prognosis is improved by early administration of effective anti-legionella antibiotic therapy. Prevention is by the correct design, maintenance, and monitoring of water systems.

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Legionella Contamination in Hot Water of Italian Hotels

Applied and Environmental Microbiology ◽

10.1128/aem.71.10.5805-5813.2005 ◽

2005 ◽

Vol 71 (10) ◽

pp. 5805-5813 ◽

Cited By ~ 82

Author(s):

Paola Borella ◽

Maria Teresa Montagna ◽

Serena Stampi ◽

Giovanna Stancanelli ◽

Vincenzo Romano-Spica ◽

...

Keyword(s):

Risk Factors ◽

Legionella Pneumophila ◽

Water Systems ◽

Hot Water ◽

Cross Sectional ◽

Multicenter Survey ◽

Multivariate Logistic Model ◽

Legionnaires Disease ◽

Building Characteristics

ABSTRACT A cross-sectional multicenter survey of Italian hotels was conducted to investigate Legionella spp. contamination of hot water. Chemical parameters (hardness, free chlorine concentration, and trace element concentrations), water systems, and building characteristics were evaluated to study risk factors for colonization. The hot water systems of Italian hotels were strongly colonized by Legionella; 75% of the buildings examined and 60% of the water samples were contaminated, mainly at levels of ≥103 CFU liter−1, and Legionella pneumophila was the most frequently isolated species (87%). L. pneumophila serogroup 1 was isolated from 45.8% of the contaminated sites and from 32.5% of the hotels examined. When a multivariate logistic model was used, only hotel age was associated with contamination, but the risk factors differed depending on the contaminating species and serogroup. Soft water with higher chlorine levels and higher temperatures were associated with L. pneumophila serogroup 1 colonization, whereas the opposite was observed for serogroups 2 to 14. In conclusion, Italian hotels, particularly those located in old buildings, represent a major source of risk for Legionnaires' disease due to the high frequency of Legionella contamination, high germ concentration, and major L. pneumophila serogroup 1 colonization. The possible role of chlorine in favoring the survival of Legionella species is discussed.

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Reduction of Legionella spp. in Water and in Soil by a Citrus Plant Extract Vapor

Applied and Environmental Microbiology ◽

10.1128/aem.01275-14 ◽

2014 ◽

Vol 80 (19) ◽

pp. 6031-6036 ◽

Cited By ~ 2

Author(s):

Katie Laird ◽

Elena Kurzbach ◽

Jodie Score ◽

Jyoti Tejpal ◽

George Chi Tangyie ◽

...

Keyword(s):

Legionella Pneumophila ◽

Water System ◽

Severe Form ◽

Gas Chromatography Mass Spectrometry ◽

Free Living ◽

Content Type ◽

Soil Systems ◽

Viable Cells ◽

Legionnaires Disease ◽

Soil And Water

ABSTRACTLegionnaires' disease is a severe form of pneumonia caused byLegionellaspp., organisms often isolated from environmental sources, including soil and water.Legionellaspp. are capable of replicating intracellularly within free-living protozoa, and once this has occurred,Legionellais particularly resistant to disinfectants. Citrus essential oil (EO) vapors are effective antimicrobials against a range of microorganisms, with reductions of 5 log cells ml−1on a variety of surfaces. The aim of this investigation was to assess the efficacy of a citrus EO vapor againstLegionellaspp. in water and in soil systems. Reductions of viable cells ofLegionella pneumophila,Legionella longbeachae,Legionella bozemanii, and an intra-amoebal culture ofLegionella pneumophila(water system only) were assessed in soil and in water after exposure to a citrus EO vapor at concentrations ranging from 3.75 mg/liter air to 15g/liter air. Antimicrobial efficacy via different delivery systems (passive and active sintering of the vapor) was determined in water, and gas chromatography-mass spectrometry (GC-MS) analysis of the antimicrobial components (linalool, citral, and β-pinene) was conducted. There was up to a 5-log cells ml−1reduction inLegionellaspp. in soil after exposure to the citrus EO vapors (15 mg/liter air). The most susceptible strain in water wasL. pneumophila, with a 4-log cells ml−1reduction after 24 h via sintering (15 g/liter air). Sintering the vapor through water increased the presence of the antimicrobial components, with a 61% increase of linalool. Therefore, the appropriate method of delivery of an antimicrobial citrus EO vapor may go some way in controllingLegionellaspp. from environmental sources.

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Balamuthia mandrillaris, Free-Living Ameba and Opportunistic Agent of Encephalitis, Is a Potential Host for Legionella pneumophila Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.71.5.2244-2249.2005 ◽

2005 ◽

Vol 71 (5) ◽

pp. 2244-2249 ◽

Cited By ~ 29

Author(s):

Winlet Sheba Shadrach ◽

Kerstin Rydzewski ◽

Ulrike Laube ◽

Gudrun Holland ◽

Muhsin Özel ◽

...

Keyword(s):

Legionella Pneumophila ◽

Mammalian Cells ◽

Mammalian Species ◽

Acanthamoeba Castellanii ◽

Electron Microscopic ◽

Free Living ◽

Balamuthia Mandrillaris ◽

Microscopic Studies ◽

Legionnaires Disease ◽

Free Living Ameba

ABSTRACT Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.

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Legionella pneumophila CRISPR-Cas suggests recurrent encounters with Gokushovirinae

10.1101/2021.03.08.434514 ◽

2021 ◽

Author(s):

Shayna R. Deecker ◽

Malene L. Urbanus ◽

Beth Nicholson ◽

Alexander W. Ensminger

Keyword(s):

Causative Agent ◽

Legionella Pneumophila ◽

Molecular Mechanisms ◽

Human Lung ◽

Mobile Element ◽

Water Systems ◽

Current Data ◽

Remediation Strategies ◽

Legionnaires Disease ◽

Outstanding Question

ABSTRACTLegionella pneumophila is a ubiquitous freshwater pathogen and the causative agent of Legionnaires’ disease. This pathogen and its ability to cause disease is closely tied to its environmental encounters. From phagocytic protists, L. pneumophila has “learned” how to avoid predation and exploit conserved eukaryotic processes to establish an intracellular replicative niche. Legionnaires’ disease is a product of these evolutionary pressures as L. pneumophila uses the same molecular mechanisms to replicate in grazing protists and in macrophages of the human lung. L. pneumophila growth within protists also provides a refuge from desiccation, disinfection, and other remediation strategies. One outstanding question has been whether this protection extends to phages. L. pneumophila isolates are remarkably devoid of prophages and to date no Legionella phages have been identified. Nevertheless, many L. pneumophila isolates maintain active CRISPR-Cas defenses. So far, the only known target of these systems has been an episomal element that we previously named Legionella Mobile Element-1 (LME-1). In this study, we have identified over 150 CRISPR-Cas systems across 600 isolates, to establish the clearest picture yet of L. pneumophila’s adaptive defenses. By leveraging the sequence of 1,500 unique spacers, we can make two main conclusions: current data argue against CRISPR-Cas targeted integrative elements beyond LME-1 and the heretofore “missing” L. pneumophila phages are most likely lytic gokushoviruses.IMPORTANCEThe causative agent of Legionnaires’ disease, an often-fatal pneumonia, is an intracellular bacterium, Legionella pneumophila, that normally grows inside amoebae and other freshwater protists. Unfortunately for us, this has two major consequences: the bacterium can take what it has learned in amoebae and use similar strategies to grow inside our lungs; and these amoebae can protect Legionella from various forms of chemical and physical disinfection regimes. Legionella are ubiquitous in the environment and frequently found in man-made water systems. Understanding the challenges to Legionella survival before it reaches the human lung is critical to preventing disease.We have leveraged our earlier discovery that L. pneumophila CRISPR-Cas systems are active and adaptive – meaning that they respond to contemporary threats encountered in the environment. In this way, CRISPR arrays can be considered genomic diaries of past encounters, with spacer sequences used to identify elements that may impinge on the pathogen’s survival. One outstanding question in the field is whether L. pneumophila is susceptible to phage, given the presumptive protection provided by intracellular replication within its eukaryotic hosts. In this work, we use CRISPR spacer sequences to suggest that the heretofore “missing” L. pneumophila phage are most likely lytic gokushoviruses. Such information is critical to the long-term goal of developing of new strategies for preventing colonization of our water systems by Legionella and subsequent human exposure to the pathogen.

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Differential Bacterial Predation by Free-Living Amoebae May Result in Blooms of Legionella in Drinking Water Systems

Microorganisms ◽

10.3390/microorganisms9010174 ◽

2021 ◽

Vol 9 (1) ◽

pp. 174 ◽

Cited By ~ 1

Author(s):

Mohamed Shaheen ◽

Nicholas J. Ashbolt

Keyword(s):

Legionella Pneumophila ◽

Fluorescent Microscopy ◽

Feeding Preference ◽

Water Systems ◽

Food Sources ◽

Resistant Bacteria ◽

Initial Growth ◽

Intracellular Growth ◽

Free Living ◽

E Coli

Intracellular growth of pathogenic Legionella in free-living amoebae (FLA) results in the critical concentrations that are problematic in engineered water systems (EWS). However, being amoeba-resistant bacteria (ARB), how Legionella spp. becomes internalized within FLA is still poorly understood. Using fluorescent microscopy, we investigated in real-time the preferential feeding behavior of three water-related FLA species, Willaertia magna, Acanthamoeba polyphaga, and Vermamoeba vermiformis regarding Legionella pneumophila and two Escherichia coli strains. Although all the studied FLA species supported intracellular growth of L. pneumophila, they avoided this bacterium to a certain degree in the presence of E. coli and mostly fed on it when the preferred bacterial food-sources were limited. Moreover, once L. pneumophila were intracellular, it inhibited digestion of co-occurring E. coli within the same trophozoites. Altogether, based on FLA–bacteria interactions and the shifts in microbial population dynamics, we propose that FLA’s feeding preference leads to an initial growth of FLA and depletion of prey bacteria, thus increases the relative abundance of Legionella and creates a “forced-feeding” condition facilitating the internalization of Legionella into FLA to initiate the cycles of intracellular multiplication. These findings imply that monitoring of FLA levels in EWS could be useful in predicting possible imminent high occurrence of Legionella.

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Cytotoxicity, Intracellular Replication, and Contact-Dependent Pore Formation of Genotyped Environmental Legionella pneumophila Isolates from Hospital Water Systems in the West Bank, Palestine

Pathogens ◽

10.3390/pathogens10040417 ◽

2021 ◽

Vol 10 (4) ◽

pp. 417

Author(s):

Ashraf R. Zayed ◽

Marina Pecellin ◽

Lina Jaber ◽

Suha Butmeh ◽

Shereen A. Bahader ◽

...

Keyword(s):

Legionella Pneumophila ◽

West Bank ◽

Water Systems ◽

Host Cells ◽

Mammalian Host ◽

Atypical Pneumonia ◽

Risk Area ◽

The West ◽

High Risk Area ◽

Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ disease. Due to the hot climate and intermittent water supply, the West Bank, Palestine, can be considered a high-risk area for this often fatal atypical pneumonia. L. pneumophila occurs in biofilms of natural and man-made freshwater environments, where it infects and replicates intracellularly within protozoa. To correlate the genetic diversity of the bacteria in the environment with their virulence properties for protozoan and mammalian host cells, 60 genotyped isolates from hospital water systems in the West Bank were analyzed. The L. pneumophila isolates were previously genotyped by high resolution Multi Locus Variable Number of Tandem Repeat Analysis (MLVA-8(12)) and sorted according to their relationship in clonal complexes (VACC). Strains of relevant genotypes and VACCs were compared according to their capacity to infect Acanthamoeba castellanii and THP-1 macrophages, and to mediate pore-forming cytotoxicity in sheep red blood cells (sRBCs). Based on a previous detailed analysis of the biogeographic distribution and abundance of the MLVA-8(12)-genotypes, the focus of the study was on the most abundant L. pneumophila- genotypes Gt4(17), Gt6 (18) and Gt10(93) and the four relevant clonal complexes [VACC1, VACC2, VACC5 and VACC11]. The highly abundant genotypes Gt4(17) and Gt6(18) are affiliated with VACC1 and sequence type (ST)1 (comprising L. pneumophila str. Paris), and displayed seroroup (Sg)1. Isolates of these two genotypes exhibited significantly higher virulence potentials compared to other genotypes and clonal complexes in the West Bank. Endemic for the West Bank was the clonal complex VACC11 (affiliated with ST461) represented by three relevant genotypes that all displayed Sg6. These genotypes unique for the West Bank showed a lower infectivity and cytotoxicity compared to all other clonal complexes and their affiliated genotypes. Interestingly, the L. pneumophila serotypes ST1 and ST461 were previously identified by in situ-sequence based typing (SBT) as main causative agents of Legionnaires’ disease (LD) in the West Bank at a comparable level. Overall, this study demonstrates the site-specific regional diversity of L. pneumophila genotypes in the West Bank and suggests that a combination of MLVA, cellular infection assays and hierarchical agglomerative cluster analysis allows an improved genotype-based risk assessment.

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The Impact of Storms on Legionella pneumophila in Cooling Tower Water, Implications for Human Health

Frontiers in Microbiology ◽

10.3389/fmicb.2020.543589 ◽

2020 ◽

Vol 11 ◽

Author(s):

Robin L. Brigmon ◽

Charles E. Turick ◽

Anna S. Knox ◽

Courtney E. Burckhalter

Keyword(s):

Human Health ◽

Legionella Pneumophila ◽

Count Data ◽

Distribution Systems ◽

Cooling Tower ◽

Water Systems ◽

The Other ◽

Cooling Towers ◽

Legionnaires Disease ◽

The Impact

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 108 cells/L in cooling tower water systems. Extreme weather conditions contributed to elevations in L. pneumophila to 107–108 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 108 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.

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Distribution of Sequence-Based Types of Legionella pneumophila Serogroup 1 Strains Isolated from Cooling Towers, Hot Springs, and Potable Water Systems in China

Applied and Environmental Microbiology ◽

10.1128/aem.03844-13 ◽

2014 ◽

Vol 80 (7) ◽

pp. 2150-2157 ◽

Cited By ~ 25

Author(s):

Tian Qin ◽

Haijian Zhou ◽

Hongyu Ren ◽

Hong Guan ◽

Machao Li ◽

...

Keyword(s):

South Korea ◽

Legionella Pneumophila ◽

Potable Water ◽

Hot Springs ◽

Cooling Tower ◽

Hot Spring ◽

Water Systems ◽

Cooling Towers ◽

Content Type ◽

Legionnaires Disease

ABSTRACTLegionella pneumophilaserogroup 1 causes Legionnaires' disease. Water systems contaminated withLegionellaare the implicated sources of Legionnaires' disease. This study analyzedL. pneumophilaserogroup 1 strains in China using sequence-based typing. Strains were isolated from cooling towers (n= 96), hot springs (n= 42), and potable water systems (n= 26). Isolates from cooling towers, hot springs, and potable water systems were divided into 25 sequence types (STs; index of discrimination [IOD], 0.711), 19 STs (IOD, 0.934), and 3 STs (IOD, 0.151), respectively. The genetic variation among the potable water isolates was lower than that among cooling tower and hot spring isolates. ST1 was the predominant type, accounting for 49.4% of analyzed strains (n= 81), followed by ST154. With the exception of two strains, all potable water isolates (92.3%) belonged to ST1. In contrast, 53.1% (51/96) and only 14.3% (6/42) of cooling tower and hot spring, respectively, isolates belonged to ST1. There were differences in the distributions of clone groups among the water sources. The comparisons amongL. pneumophilastrains isolated in China, Japan, and South Korea revealed that similar clones (ST1 complex and ST154 complex) exist in these countries. In conclusion, in China, STs had several unique allelic profiles, and ST1 was the most prevalent sequence type of environmentalL. pneumophilaserogroup 1 isolates, similar to its prevalence in Japan and South Korea.

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