scholarly journals Intracellular Proliferation of Legionella pneumophila in Hartmannella vermiformis in Aquatic Biofilms Grown on Plasticized Polyvinyl Chloride

2004 ◽  
Vol 70 (11) ◽  
pp. 6826-6833 ◽  
Author(s):  
Melanie W. Kuiper ◽  
Bart A. Wullings ◽  
Antoon D. L. Akkermans ◽  
Rijkelt R. Beumer ◽  
Dick van der Kooij
Keyword(s):  
Polyvinyl Chloride ◽  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
Tap Water ◽  
Water System ◽  
Intracellular Growth ◽  
Mixed Microbial Community ◽  
Hartmannella Vermiformis ◽  
High Concentrations

ABSTRACT The need for protozoa for the proliferation of Legionella pneumophila in aquatic habitats is still not fully understood and is even questioned by some investigators. This study shows the in vivo growth of L. pneumophila in protozoa in aquatic biofilms developing at high concentrations on plasticized polyvinyl chloride in a batch system with autoclaved tap water. The inoculum, a mixed microbial community including indigenous L. pneumophila originating from a tap water system, was added in an unfiltered as well as filtered (cellulose nitrate, 3.0-μm pore size) state. Both the attached and suspended biomasses were examined for their total amounts of ATP, for culturable L. pneumophila, and for their concentrations of protozoa. L. pneumophila grew to high numbers (6.3 log CFU/cm2) only in flasks with an unfiltered inoculum. Filtration obviously removed the growth-supporting factor, but it did not affect biofilm formation, as determined by measuring ATP. Cultivation, direct counting, and 18S ribosomal DNA-targeted PCR with subsequent sequencing revealed the presence of Hartmannella vermiformis in all flasks in which L. pneumophila multiplied and also when cycloheximide had been added. Fluorescent in situ hybridization clearly demonstrated the intracellular growth of L. pneumophila in trophozoites of H. vermiformis, with 25.9% � 10.5% of the trophozoites containing L. pneumophila on day 10 and >90% containing L. pneumophila on day 14. Calculations confirmed that intracellular growth was most likely the only way for L. pneumophila to proliferate within the biofilm. Higher biofilm concentrations, measured as amounts of ATP, gave higher L. pneumophila concentrations, and therefore the growth of L. pneumophila within engineered water systems can be limited by controlling biofilm formation.

scholarly journals Detection of Protozoan Hosts for Legionella pneumophila in Engineered Water Systems by Using a Biofilm Batch Test

2010 ◽  
Vol 76 (21) ◽  
pp. 7144-7153 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Dick van der Kooij
Keyword(s):  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Batch Test ◽  
Free Living ◽  
Water Types ◽  
Operational Taxonomic Units ◽  
Hartmannella Vermiformis

ABSTRACT Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.

scholarly journals Copper Supplementation in Watering Solution Reaches the Xylem But Does Not Protect Tobacco Plants Against Xylella fastidiosa Infection

Plant Disease ◽  
2020 ◽  
Vol 104 (3) ◽  
pp. 724-730 ◽  
Author(s):  
Qing Ge ◽  
Paul A. Cobine ◽  
Leonardo De La Fuente
Keyword(s):  
Biofilm Formation ◽  
Inductively Coupled Plasma ◽  
Tap Water ◽  
Xylella Fastidiosa ◽  
In Vitro Study ◽  
Optical Emission ◽  
In Planta ◽  
Inductively Coupled ◽  
High Concentrations

Xylella fastidiosa is a xylem-limited plant pathogenic bacterium that causes disease in many crops worldwide. Copper (Cu) is an antimicrobial agent widely used on X. fastidiosa hosts to control other diseases. Although the effects of Cu for control of foliar pathogens are well known, it is less studied on xylem-colonizing pathogens. Previous results from our group showed that low concentrations of CuSO4 increased biofilm formation, whereas high concentrations inhibited biofilm formation and growth in vitro. In this study, we conducted in planta experiments to determine the influence of Cu in X. fastidiosa infection using tobacco as a model. X. fastidiosa-infected and noninfected plants were watered with tap water or with water supplemented with 4 mM or 8 mM of CuSO4. Symptom progression was assessed, and sap and leaf ionome analysis was performed by inductively coupled plasma with optical emission spectroscopy. Cu uptake was confirmed by increased concentrations of Cu in the sap of plants treated with CuSO4-amended water. Leaf scorch symptoms in Cu-supplemented plants showed a trend toward more severe at later time points. Quantification of total and viable X. fastidiosa in planta indicated that CuSO4-amended treatments did not inhibit but slightly increased the growth of X. fastidiosa. Cu in sap was in the range of concentrations that promote X. fastidiosa biofilm formation according to our previous in vitro study. Based on these results, we proposed that the plant Cu homeostasis machinery controls the level of Cu in the xylem, preventing it from becoming elevated to a level that would lead to bacterial inhibition.

Investigation of Virulence Genes and Biofilm Formation Among Legionella Pneumophila Isolated from Hospital Water Sources

2020 ◽  
Author(s):  
shiva mirkalantari ◽  
Sara Hayatimehr ◽  
Noor Amirmozafari ◽  
Faramarz Masjedian
Keyword(s):  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
Virulence Genes ◽  
Tap Water ◽  
Severe Pneumonia ◽  
Water Sources ◽  
Hospital Environment ◽  
Contamination Rate ◽  
Elevated Concentration ◽  
Contributing Factors

Abstract Background: Legionella pneumophila as a ubiquitous bacterium is inherently resistant to chlorine in tap water. It can easily enter water piping systems and get transmitted to immunocompromised populations and cause severe pneumonia. Owing to the fact that its presence in water sources doesn’t necessarily lead to onset of disease; therefore, several factors such as inhaled bacteria dose, virulence factors and diversity of serogroups can be considered as contributing factors. The main aim of current project was to investigate the contamination rate of hospital water systems with Legionella by culture and evaluate presence of major virulence factor genes as well as the ability to form biofilms among the Legionella isolates. Results: Twelve (12%) of the 100 water samples produced positive results in culture method. Additional confirmation was performed by PCR method with specific primers for Legionella genus (16SrRNA) and pneumophila species (mip). Fifty (5%) samples of 12 with positive culture have a colony forming unit higher than 1000cfu/100 ml. Legionella were isolated with a rate of 8%, 3% and 1% from shower heads, oxygen humidifier bottle and water bath, respectively. PCR assay for the virulence genes showed that all 12 (100%) isolates were positive for mip genes, 9 (75%) were positive for dot gene, 8 (66.66%) were positive for hsp, 6(50%) were positive for lvh and 4(33.33%) for rtx. Two of the isolates displayed higher ability to form biofilm in reference to the standard strain.Conclusion: Although the presence of Legionella pneumophila in hospital environment does not necessarily confer a threat to public health; continuous monitoring of water sources should be conducted in order to avoid elevated concentration of this bacterium and visible biofilm formation.

scholarly journals csrR, a Paralog and Direct Target of CsrA, Promotes Legionella pneumophila Resilience in Water

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Zachary D. Abbott ◽  
Helen Yakhnin ◽  
Paul Babitzke ◽  
Michele S. Swanson
Keyword(s):  
Legionella Pneumophila ◽  
Tap Water ◽  
Bacterial Species ◽  
Regulatory Protein ◽  
Opportunistic Pathogen ◽  
Water Systems ◽  
Phenotypic Analysis ◽  
Term Survival ◽  
Content Type

ABSTRACTCritical to microbial versatility is the capacity to express the cohort of genes that increase fitness in different environments.Legionella pneumophilaoccupies extensive ecological space that includes diverse protists, pond water, engineered water systems, and mammalian lung macrophages. One mechanism that equips this opportunistic pathogen to adapt to fluctuating conditions is a switch between replicative and transmissive cell types that is controlled by the broadly conserved regulatory protein CsrA. A striking feature of the legionellae surveyed is that each of 14 strains encodes 4 to 7csrA-like genes, candidate regulators of distinct fitness traits. Here we focus on the onecsrAparalog (lpg1593) that, like the canonicalcsrA, is conserved in all 14 strains surveyed. Phenotypic analysis revealed that long-term survival in tap water is promoted by thelpg1593locus, which we namecsrR(for “CsrA-similar protein forresilience”). As predicted by its GGA motif,csrRmRNA was bound directly by the canonical CsrA protein, as judged by electromobility shift and RNA-footprinting assays. Furthermore, CsrA repressed translation ofcsrRmRNAin vivo, as determined by analysis ofcsrR-gfpreporters,csrRmRNA stability in the presence and absence ofcsrAexpression, and mutation of the CsrA binding site identified on thecsrRmRNA. Thus, CsrA not only governs the transition from replication to transmission but also represses translation of its paralogcsrRwhen nutrients are available. We propose that, during prolonged starvation, relief of CsrA repression permits CsrR protein to coordinateL. pneumophila's switch to a cell type that is resilient in water supplies.IMPORTANCEPersistence ofL. pneumophilain water systems is a public health risk, and yet there is little understanding of the genetic determinants that equip this opportunistic pathogen to adapt to and survive in natural or engineered water systems. A potent regulator of this pathogen's intracellular life cycle is CsrA, a protein widely distributed among bacterial species that is understood quite well. Our finding that every sequencedL. pneumophilastrain carries severalcsrAparalogs—including two common to all isolates—indicates that the legionellae exploit CsrA regulatory switches for multiple purposes. Our discovery that one paralog, CsrR, is a target of CsrA that enhances survival in water is an important step toward understanding colonization of the engineered environment by pathogenicL. pneumophila.

scholarly journals Legionella pneumophila OxyR Is a Redundant Transcriptional Regulator That Contributes to Expression Control of the Two-Component CpxRA System

2016 ◽  
Vol 199 (5) ◽  
Author(s):  
Jennifer R. Tanner ◽  
Palak G. Patel ◽  
Jacqueline R. Hellinga ◽  
Lynda J. Donald ◽  
Celine Jimenez ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Mutational Analysis ◽  
Transcriptional Regulator ◽  
Intracellular Growth ◽  
Content Type ◽  
Expression Control ◽  
Two Component ◽  
Legionnaires Disease

ABSTRACT Nominally an environmental organism, Legionella pneumophila is an intracellular parasite of protozoa but is also the causative agent of the pneumonia termed Legionnaires' disease, which results from inhalation of aerosolized bacteria by susceptible humans. Coordination of gene expression by a number of identified regulatory factors, including OxyR, assists L. pneumophila in adapting to the stresses of changing environments. L. pneumophila OxyR (OxyRLp) is an ortholog of Escherichia coli OxyR; however, OxyRLp was shown elsewhere to be functionally divergent, such that it acts as a transcription regulator independently of the oxidative stress response. In this study, the use of improved gene deletion methods has enabled us to generate an unmarked in-frame deletion of oxyR in L. pneumophila. Lack of OxyRLp did not affect in vitro growth or intracellular growth in Acanthamoeba castellanii protozoa and U937-derived macrophages. The expression of OxyRLp does not appear to be regulated by CpxR, even though purified recombinant CpxR bound a DNA sequence similar to that reported for CpxR elsewhere. Surprisingly, a lack of OxyRLp resulted in elevated activity of the promoters located upstream of icmR and the lpg1441-cpxA operon, and OxyRLp directly bound to these promoter regions, suggesting that OxyRLp is a direct repressor. Interestingly, a strain overexpressing OxyRLp demonstrated reduced intracellular growth in A. castellanii but not in U937-derived macrophages, suggesting that balanced expression control of the two-component CpxRA system is necessary for survival in protozoa. Taken together, this study suggests that OxyRLp is a functionally redundant transcriptional regulator in L. pneumophila under the conditions evaluated herein. IMPORTANCE Legionella pneumophila is an environmental pathogen, with its transmission to the human host dependent upon its ability to replicate in protozoa and survive within its aquatic niche. Understanding the genetic factors that contribute to L. pneumophila survival within each of these unique environments will be key to limiting future point-source outbreaks of Legionnaires' disease. The transcriptional regulator L. pneumophila OxyR (OxyRLp) has been previously identified as a potential regulator of virulence traits warranting further investigation. This study demonstrated that oxyR is nonessential for L. pneumophila survival in vitro and in vivo via mutational analysis. While the mechanisms of how OxyRLp expression is regulated remain elusive, this study shows that OxyRLp negatively regulates the expression of the cpxRA two-component system necessary for intracellular survival in protozoa.

scholarly journals Optimizing production efficiencies of hot water units using building energy simulations - Trade-off between Legionella pneumophila contamination risk and energy efficiency

2019 ◽  
Vol 111 ◽  
pp. 04053
Author(s):  
Elisa Van Kenhove ◽  
Lien De Backer ◽  
Jelle Laverge
Keyword(s):  
Simulation Model ◽  
Legionella Pneumophila ◽  
Energy Demand ◽  
Energy Use ◽  
Tap Water ◽  
Water System ◽  
High Energy ◽  
Hot Water ◽  
Domestic Hot Water ◽  
Production Efficiencies

The energy needed for domestic hot water represents an important share in the total energy use of well-insulated and airtight buildings. One of the main reasons for this high energy demand is that hot water is produced at temperatures above 60°C to mitigate the risk of contaminating the hot water system with Legionella pneumophila. However, this elevated temperature is not necessary for most domestic hot water applications, and has a negative effect on the efficiency of hot water production units. A simulation model has been developed which proposes an alternative to this constant 60°C by predicting the Legionella pneumophila concentration dynamically throughout the hot water system. Based on this knowledge, a hot water controller is added to the simulation model that sets a lower hot water comfort temperature in combination with heat shocks. In this paper, the simulation model is used to estimate the energy saving potential in a case study building, at the level of the heat production system by reaching higher production efficiencies. Three different production units, namely an electric boiler, heat pump and solar collector have been investigated. The controller is expected to become an alternative for the current, energy intensive, high temperature tap water heating systems.

Corroding copper and steel exposed to intermittently flowing tap water promote biofilm formation and growth of Legionella pneumophila

2020 ◽  
Vol 183 ◽  
pp. 115951 ◽  
Author(s):  
Dick van der Kooij ◽  
Harm R. Veenendaal ◽  
Ronald Italiaander
Keyword(s):  
Legionella Pneumophila ◽  
Biofilm Formation ◽  
Tap Water

A simple method for the eradication ofLegionella pneumophilafrom potable water systems

1997 ◽  
Vol 43 (12) ◽  
pp. 1189-1196 ◽  
Author(s):  
Carmen Moreno ◽  
Isabel de Blas ◽  
Francisca Miralles ◽  
David Apraiz ◽  
Vicente Catalan
Keyword(s):  
Legionella Pneumophila ◽  
Potable Water ◽  
Cold Water ◽  
Tap Water ◽  
Water System ◽  
Water Systems ◽  
Hot Water ◽  
Residual Chlorine ◽  
Water Heater ◽  
Simple Method

In this paper we describe a simple method, noncorrosive to pipes, for the eradication of Legionella pneumophila from potable water systems. This method is based on the systematic purging of the pipe networks with cold water containing 1 – 1.5 mg residual chlorine/L. In the hot water system, a new pipe bypassing the water heater was installed, whereas in the air conditioning system, the circuit is purged with water from the tap water system. The feasibility of this method was studied in two hotels in which the presence of Legionella was detected despite treatment of the water by the hyperchlorination method. The evolution of the presence of Legionella was studied by culture and polymerase chain reaction. Eighty samples from hotel A and sixty-seven samples from hotel B were analyzed during the time that the eradication method was applied. Our results showed that this method permitted the effective elimination of L. pneumophila after 5 months in hotel A and 7 months in hotel B.Key words: Legionella pneumophila, eradication.

Multiplication of Legionella pneumophila Philadelphia-1 in cultured peritoneal macrophages and its correlation to susceptibility of animals

1986 ◽  
Vol 32 (5) ◽  
pp. 438-442 ◽  
Author(s):  
Shin-Ichi Yoshida ◽  
Yasuo Mizuguchi
Keyword(s):  
Growth Rate ◽  
Guinea Pig ◽  
Legionella Pneumophila ◽  
Lethal Dose ◽  
Peritoneal Macrophages ◽  
The Other ◽  
Natural Resistance ◽  
Intracellular Growth ◽  
Golden Hamsters

Intracellular growth of Legionella pneumophila Philadelphia-1 strain in peritoneal macrophages (PMP) from various rodents was measured and its correlation to the level of susceptibility of the animal was examined. In guinea pig PMP, the organism grew well and the guinea pig was very susceptible to it (50% lethal dose, LD50 = 7.6 × 104). On the other hand, the bacteria hardly multiplied in mouse PMP and the animal was resistant to infection (LD50 = 6.7 × 107). Intracellular growth rate correlated well with susceptibility in these animals. In golden hamsters, a discrepancy between intracellular growth and susceptibility was found. The organism grew intracellularly as rapid as in guinea pig PMP, but the golden hamster was very resistant to infection (LD50 = 2.2 × 108). In rat PMP, the organism did not grow intracellularly during a 24-h period of infection, but started to grow after that and the growth rate thereafter was as rapid as in guinea pig PMP. WKA rats were resistant and the LD50 in the animal was 1.9 × 107. In vivo natural resistance of rats and golden hamsters to the organism was considered to be a result of other factors than macrophages.

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