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 Free-Living Protozoa in Two Unchlorinated Drinking Water Supplies, Identified by Phylogenic Analysis of 18S rRNA Gene Sequences

2009 ◽  
Vol 75 (14) ◽  
pp. 4736-4746 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Geo Bakker ◽  
Hauke Smidt ◽  
Dick van der Kooij
Keyword(s):  
Drinking Water ◽  
Legionella Pneumophila ◽  
Distribution System ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Free Living ◽  
Water Supplies ◽  
Molecular Approaches ◽  
High Level

ABSTRACT Free-living protozoan communities in water supplies may include hosts for Legionella pneumophila and other undesired bacteria, as well as pathogens. This study aimed at identifying free-living protozoa in two unchlorinated groundwater supplies, using cultivation-independent molecular approaches. For this purpose, samples (<20°C) of treated water, distributed water, and distribution system biofilms were collected from supply A, with a low concentration of natural organic matter (NOM) (<0.5 ppm of C), and from supply B, with a high NOM concentration (7.9 ppm of C). Eukaryotic communities were studied using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). In both supplies, highly diverse eukaryotic communities were observed, including free-living protozoa, fungi, and metazoa. Sequences of protozoa clustered with Amoebozoa (10 operational taxonomic units [OTUs]), Cercozoa (39 OTUs), Choanozoa (26 OTUs), Ciliophora (29 OTUs), Euglenozoa (13 OTUs), Myzozoa (5 OTUs), and Stramenopiles (5 OTUs). A large variety of protozoa were present in both supplies, but the estimated values for protozoan richness did not differ significantly. H. vermiformis was observed in both supplies but was not a predominant protozoan. One OTU with the highest similarity to Acanthamoeba polyphaga, an opportunistic human pathogen and a host for undesired bacteria, was observed in supply A. The high level of NOM in supply B corresponded with an elevated level of active biomass and with elevated concentrations of H. vermiformis in distributed water. Hence, the application of qPCR may be promising in elucidating the relationship between drinking water quality and the presence of specific protozoa.

scholarly journals Quantitative Detection of the Free-Living Amoeba Hartmannella vermiformis in Surface Water by Using Real-Time PCR

2006 ◽  
Vol 72 (9) ◽  
pp. 5750-5756 ◽  
Author(s):  
Melanie W. Kuiper ◽  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Harry Boonstra ◽  
Hauke Smidt ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
18S Rrna ◽  
18S Rrna Gene ◽  
Quantitative Detection ◽  
Rrna Gene ◽  
Free Living ◽  
Copy Numbers ◽  
Free Living Amoeba ◽  
Hartmannella Vermiformis

ABSTRACT A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 � 10−1 and 1.14 � 104 cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 � 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (≥98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.

scholarly journals Relationships between Free-Living Protozoa, Cultivable Legionella spp., and Water Quality Characteristics in Three Drinking Water Supplies in the Caribbean

2011 ◽  
Vol 77 (20) ◽  
pp. 7321-7328 ◽  
Author(s):  
Rinske M. Valster ◽  
Bart A. Wullings ◽  
Riemsdijk van den Berg ◽  
Dick van der Kooij
Keyword(s):  
Drinking Water ◽  
Legionella Pneumophila ◽  
18S Rrna Gene ◽  
Molecular Techniques ◽  
Tropical Region ◽  
Rrna Gene ◽  
Free Living ◽  
Water Supplies ◽  
Content Type ◽  
Protozoan Community

ABSTRACTThe study whose results are presented here aimed at identifying free-living protozoa (FLP) and conditions favoring the growth of these organisms and cultivableLegionellaspp. in drinking water supplies in a tropical region. Treated and distributed water (±30°C) of the water supplies of three Caribbean islands were sampled and investigated with molecular techniques, based on the 18S rRNA gene. The protozoan hostHartmannella vermiformisand cultivableLegionella pneumophilawere observed in all three supplies. Operational taxonomic units (OTUs) with the highest similarity to the potential or candidate hostsAcanthamoebaspp.,Echinamoeba exundans,E. thermarum, and anNeoparamoebasp. were detected as well. In total, 59 OTUs of FLP were identified. The estimated protozoan richness did not differ significantly between the three supplies. In supply CA-1, the concentration ofH. vermiformiscorrelated with the concentration ofLegionellaspp. and clones related to Amoebozoa predominated (82%) in the protozoan community. These observations, the low turbidity (<0.2 nephelometric turbidity units [NTU]), and the varying ATP concentrations (1 to 12 ng liter−1) suggest that biofilms promoted protozoan growth in this supply. Ciliophora represented 25% of the protozoan OTUs in supply CA-2 with elevated ATP concentrations (maximum, 55 ng liter−1) correlating with turbidity (maximum, 62 NTU) caused by corroding iron pipes. Cercozoan types represented 70% of the protozoan clones in supply CA-3 with ATP concentrations of <1 ng liter−1and turbidity of <0.5 NTU in most samples of distributed water. The absence ofH. vermiformisin most samples from supply CA-3 suggests that growth of this protozoan is limited at ATP concentrations of <1 ng liter−1.

scholarly journals Sensitivity of Vermamoeba (Hartmannella) vermiformis cysts to conventional disinfectants and protease

2014 ◽  
Vol 13 (2) ◽  
pp. 302-310 ◽  
Author(s):  
Emilie Fouque ◽  
Yann Héchard ◽  
Philippe Hartemann ◽  
Philippe Humeau ◽  
Marie-Cécile Trouilhé
Keyword(s):  
Legionella Pneumophila ◽  
Pathogenic Bacteria ◽  
Hot Water ◽  
Water Networks ◽  
Important Health ◽  
Hartmannella Vermiformis ◽  
Important Health Issue ◽  
Few Data ◽  
First Time

Vermamoeba vermiformis is a free-living amoeba (FLA) widely distributed in the environment, known to colonize hot water networks and to be the reservoir of pathogenic bacteria such as Legionella pneumophila. FLA are partly resistant to biocides, especially in their cyst form. The control of V. vermiformis in hot water networks represents an important health issue, but there are very few data on their resistance to disinfection treatments. The sensitivity of cysts of two strains of V. vermiformis to three disinfectants frequently used in hot water networks (chlorine, heat shock, peracetic acid (PAA) mixed with hydrogen peroxide (H2O2)) was investigated. In vitro, several concentrations of biocides, temperatures and exposure times according to the French regulation were tested. Cysts were fully inactivated by the following conditions: 15 mg/L of chlorine for 10 min; 60 °C for 30 min; and 0.5 g/L equivalent H2O2 of PAA mixed with H2O2 for 30 min. For the first time, the strong efficacy of subtilisin (0.625 U/mL for 24 h), a protease, to inactivate the V. vermiformis cysts has been demonstrated. It suggests that novel approaches may be efficient for disinfection processes. Finally, V. vermifomis cysts were sensitive to all the tested treatments and appeared to be more sensitive than Acanthamoeba cysts.

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.

Identification of Acanthamoeba genotype T4 and Paravahlkampfia sp. from two clinical samples

2008 ◽  
Vol 57 (3) ◽  
pp. 392-396 ◽  
Author(s):  
Soykan Ozkoc ◽  
Sema Tuncay ◽  
Songul Bayram Delibas ◽  
Ciler Akisu ◽  
Zeynep Ozbek ◽  
...  
Keyword(s):  
18S Rrna ◽  
Single Agent ◽  
18S Rrna Gene ◽  
Maximum Temperature ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Free Living ◽  
History Of ◽  
Simplex Virus ◽  
Corneal Trauma

In this study, two free-living amoebae strains, Acanthamoeba genotype T4 and Paravahlkampfia sp., which were isolated from keratitis cases are presented. While the Acanthamoeba strain was isolated as a single agent, the Paravahlkampfia strain was found together with herpes simplex virus. Neither of the patients were contact lens wearers, but they did have a history of minor corneal trauma. Amoebae were detected on non-nutrient agar covered with Escherichia coli. Based on PCR-amplified 18S rRNA-gene analysis the first isolate was identified as Acanthamoeba genotype T4 and the second as Paravahlkampfia sp. In thermotolerance tests, the maximum temperature at which trophozoites continued to divide was determined as 37 °C for this Acanthamoeba strain and 35 °C for the Paravahlkampfia strain. To the best of our knowledge, the Acanthamoeba strain described herein is the second molecularly identified Acanthamoeba strain in an Acanthamoeba keratitis patient in Turkey. However, the Paravahlkampfia isolate is believed to be the first strain that has been isolated from a keratitis patient and has been molecularly differentiated from Vahlkampfia.

scholarly journals MixedTheileriainfections in free-ranging buffalo herds: implications for diagnosingTheileria parvainfections in Cape buffalo (Syncerus caffer)

Parasitology ◽  
2011 ◽  
Vol 138 (7) ◽  
pp. 884-895 ◽  
Author(s):  
RONEL PIENAAR ◽  
FRED T. POTGIETER ◽  
ABDALLA A. LATIF ◽  
ORIEL M. M. THEKISOE ◽  
BEN J. MANS
Keyword(s):  
South Africa ◽  
Hypervariable Region ◽  
18S Rrna Gene ◽  
Control Measures ◽  
Locked Nucleic Acid ◽  
Rrna Gene ◽  
Mixed Infections ◽  
Endemic Region ◽  
Signal Suppression

SUMMARYBuffalo-adaptedTheileria parvacauses Corridor disease in cattle. Strict control measures therefore apply to the movement of buffalo in South Africa and include mandatory testing of buffalo for the presence ofT. parva. The official test is a real-time hybridization PCR assay that amplifies the V4 hypervariable region of the 18S rRNA gene ofT. parva, T.sp. (buffalo) andT.sp. (bougasvlei). The effect that mixedT. parvaandT.sp. (buffalo)-like infections have on accurateT. parvadiagnosis was investigated in this study.In vitromixed infection simulations indicated PCR signal suppression at 100 to 1000-foldT.sp. (buffalo) excess at lowT. parvaparasitaemia. Suppression of PCR signal was found in field buffalo with mixed infections. TheT. parva-positive status of these cases was confirmed by selective suppression ofT.sp. (buffalo) amplification using a locked nucleic acid clamp and independent assays based on the p67, p104 andTprgenes. The incidence of mixed infections in the Corridor disease endemic region of South Africa is significant, while the prevalence in buffalo outside the endemic area is currently low. A predicted increase ofT.sp. (buffalo)-like infections can affect future diagnoses where mixed infections occur, prompting the need for improvements in current diagnostics.

scholarly journals Synergism between feremycorrhizal symbiosis and free-living diazotrophs leads to improved growth and nutrition of wheat under nitrogen deficiency conditions

2022 ◽  
Author(s):  
Khalil Kariman ◽  
Benjamin Moreira-Grez ◽  
Craig Scanlan ◽  
Saleh Rahimlou ◽  
Gustavo Boitt ◽  
...  
Keyword(s):  
Mycorrhizal Fungi ◽  
Nitrogen Deficiency ◽  
Soil Microbiome ◽  
Control Treatment ◽  
Shoot Biomass ◽  
Rrna Gene ◽  
Free Living ◽  
Total N ◽  
Synergistic Interactions

AbstractA controlled-environment study was conducted to explore possible synergistic interactions between the feremycorrhizal (FM) fungus Austroboletus occidentalis and soil free-living N2-fixing bacteria (diazotrophs). Wheat (Triticum aestivum) plants were grown under N deficiency conditions in a field soil without adding microbial inoculum (control: only containing soil indigenous microbes), or inoculated with a consortium containing four free-living diazotroph isolates (diazotrophs treatment), A. occidentalis inoculum (FM treatment), or both diazotrophs and A. occidentalis inoculums (dual treatment). After 7 weeks of growth, significantly greater shoot biomass was observed in plants inoculated with diazotrophs (by 25%), A. occidentalis (by 101%), and combined inoculums (by 106%), compared to the non-inoculated control treatment. All inoculated plants also had higher shoot nutrient contents (including N, P, K, Mg, Zn, Cu, and Mn) than the control treatment. Compared to the control and diazotrophs treatments, significantly greater shoot N content was observed in the FM treatment (i.e., synergism between the FM fungus and soil indigenous diazotrophs). Dually inoculated plants had the highest content of nutrients in shoots (e.g., N, P, K, S, Mg, Zn, Cu, and Mn) and soil total N (13–24% higher than the other treatments), i.e., synergism between the FM fungus and added diazotrophs. Root colonization by soil indigenous arbuscular mycorrhizal fungi declined in all inoculated plants compared to control. Non-metric multidimensional scaling (NMDS) analysis of the bacterial 16S rRNA gene amplicons revealed that the FM fungus modified the soil microbiome. Our in vitro study indicated that A. occidentalis could not grow on substrates containing lignocellulosic materials or sucrose, but grew on media supplemented with hexoses such as glucose and fructose, indicating that the FM fungus has limited saprotrophic capacity similar to ectomycorrhizal fungi. The results revealed synergistic interactions between A. occidentalis and soil free-living diazotrophs, indicating a potential to boost microbial N2 fixation for non-legume crops.

scholarly journals Fungi found in Mediterranean and North Sea sponges: how specific are they?

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3722 ◽  
Author(s):  
Mohd Azrul Naim ◽  
Hauke Smidt ◽  
Detmer Sipkema
Keyword(s):  
North Sea ◽  
Ribosomal Rna ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Anaerobic Digesters ◽  
Operational Taxonomic Units ◽  
The North ◽  
Hypervariable Regions ◽  
The North Sea ◽  
Biological Sources

Fungi and other eukaryotes represent one of the last frontiers of microbial diversity in the sponge holobiont. In this study we employed pyrosequencing of 18S ribosomal RNA gene amplicons containing the V7 and V8 hypervariable regions to explore the fungal diversity of seven sponge species from the North Sea and the Mediterranean Sea. For most sponges, fungi were present at a low relative abundance averaging 0.75% of the 18S rRNA gene reads. In total, 44 fungal OTUs (operational taxonomic units) were detected in sponges, and 28 of these OTUs were also found in seawater. Twenty-two of the sponge-associated OTUs were identified as yeasts (mainly Malasseziales), representing 84% of the fungal reads. Several OTUs were related to fungal sequences previously retrieved from other sponges, but all OTUs were also related to fungi from other biological sources, such as seawater, sediments, lakes and anaerobic digesters. Therefore our data, supported by currently available data, point in the direction of mostly accidental presence of fungi in sponges and do not support the existence of a sponge-specific fungal community.

Bench-scale experiments for the development of a unified loop-mediated isothermal amplification (LAMP) assay for the in vitro diagnosis of Leishmania species' promastigotes

2013 ◽  
Vol 142 (8) ◽  
pp. 1671-1677 ◽  
Author(s):  
M. KARANI ◽  
I. SOTIRIADOU ◽  
J. PLUTZER ◽  
P. KARANIS
Keyword(s):  
Lamp Assay ◽  
High Sensitivity ◽  
Leishmania Species ◽  
18S Rrna Gene ◽  
Isothermal Amplification ◽  
Rrna Gene ◽  
Bench Scale ◽  
Loop Mediated Isothermal Amplification ◽  
Wide Range

SUMMARYWe developed, in bench-scale experiments, a unified loop-mediated isothermal amplification (LAMP) assay for the detection of cutaneous, mucocutaneous and visceral leishmaniasis using DNA of cultivated promastigotes. Two primer sets for the LAMP assay were designed based on the 18S rRNA gene, and their sensitivity and specificity were tested and compared. Both of them were specific for Leishmania as the DNA of all ten Leishmania species tested was amplified, whereas the DNA of other parasites, including that of Trypanosoma, was not. The detection limit for primer set 1 ranged between 30 pg and 3·6 fg, depending on which Leishmania species tested. Primer set 2 showed high sensitivity, but was less sensitive than primer set 1. Our findings lead to the conclusion that the LAMP assay with primer set 1 is a promising and effective assay for the successful detection of a wide range of Leishmania infections using only a unified multiplex LAMP test.

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