scholarly journals Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

2009 ◽  
Vol 75 (11) ◽  
pp. 3502-3512 ◽  
Author(s):  
Pilar Delgado-Viscogliosi ◽  
Lydie Solignac ◽  
Jean-Marie Delattre
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Environmental Water ◽  
Hot Water ◽  
Culture Methods ◽  
Culture Techniques ◽  
Viability Assay ◽  
Viable Cells ◽  
Wide Range

ABSTRACT PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.

scholarly journals Total and Viable Legionella pneumophila Cells in Hot and Natural Waters as Measured by Immunofluorescence-Based Assays and Solid-Phase Cytometry

2011 ◽  
Vol 77 (17) ◽  
pp. 6225-6232 ◽  
Author(s):  
N. Parthuisot ◽  
M. Binet ◽  
A. Touron-Bodilis ◽  
C. Pougnard ◽  
P. Lebaron ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Natural Waters ◽  
Solid Phase ◽  
Environmental Water ◽  
Thermal Spring ◽  
Hot Water ◽  
Content Type ◽  
Thermal Spring Water ◽  
Plate Counts

ABSTRACTA new method was developed for the rapid and sensitive detection of viableLegionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viableL. pneumophilacells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter−1, and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viableL. pneumophilacells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 × 103viable but nonculturableL. pneumophilacells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viableL. pneumophilain both man-made water systems and environmental water samples.

scholarly journals Comparison of Updated Methods for Legionella Detection in Environmental Water Samples

2021 ◽  
Vol 18 (10) ◽  
pp. 5436
Author(s):  
Daniela Toplitsch ◽  
Sabine Platzer ◽  
Romana Zehner ◽  
Stephanie Maitz ◽  
Franz Mascher ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Limit Of Detection ◽  
Culture Method ◽  
Environmental Water ◽  
Microbial Flora ◽  
Culture Methods ◽  
Outbreak Investigations ◽  
Standard Culture ◽  
Selection Of

The difficulty of cultivation of Legionella spp. from water samples remains a strenuous task even for experienced laboratories. The long incubation periods for Legionellae make isolation difficult. In addition, the water samples themselves are often contaminated with accompanying microbial flora, and therefore require complex cultivation methods from diagnostic laboratories. In addition to the recent update of the standard culture method ISO 11731:2017, new strategies such as quantitative PCR (qPCR) are often discussed as alternatives or additions to conventional Legionella culture approaches. In this study, we compared ISO 11731:2017 with qPCR assays targeting Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1. In samples with a high burden of accompanying microbial flora, qPCR shows an excellent negative predictive value for Legionella pneumophila, thus making qPCR an excellent tool for pre-selection of negative samples prior to work-intensive culture methods. This and its low limit of detection make qPCR a diagnostic asset in Legionellosis outbreak investigations, where quick-risk assessments are essential, and are a useful method for monitoring risk sites.

scholarly journals Organogenic Nodule Formation in Hop: A Tool to Study Morphogenesis in Plants with Biotechnological and Medicinal Applications

2010 ◽  
Vol 2010 ◽  
pp. 1-16 ◽  
Author(s):  
Ana M. Fortes ◽  
Filipa Santos ◽  
Maria S. Pais
Keyword(s):  
Tissue Culture ◽  
Large Scale ◽  
Root Nodules ◽  
Molecular Networks ◽  
Nodule Formation ◽  
Culture Methods ◽  
Culture Techniques ◽  
Wide Range ◽  
Morphogenic Process

The usage ofHumulus lupulusfor brewing increased the demand for high-quality plant material. Simultaneously, hop has been used in traditional medicine and recently recognized with anticancer and anti-infective properties. Tissue culture techniques have been reported for a wide range of species, and open the prospect for propagation of disease-free, genetically uniform and massive amounts of plantsin vitro.Moreover, the development of large-scale culture methods using bioreactors enables the industrial production of secondary metabolites. Reliable and efficient tissue culture protocol for shoot regeneration through organogenic nodule formation was established for hop. The present review describes the histological, and biochemical changes occurring during this morphogenic process, together with an analysis of transcriptional and metabolic profiles. We also discuss the existence of common molecular factors among three different morphogenic processes: organogenic nodules and somatic embryogenesis, which strictly speaking depend exclusively on intrinsic developmental reprogramming, and legume nitrogen-fixing root nodules, which arises in response to symbiosis. The review of the key factors that participate in hop nodule organogenesis and the comparison with other morphogenic processes may have merit as a study presenting recent advances in complex molecular networks occurring during morphogenesis and together, these provide a rich framework for biotechnology applications.

scholarly journals In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

2020 ◽  
Vol 103 (3) ◽  
pp. 455-470
Author(s):  
Leah E Simon ◽  
T Rajendra Kumar ◽  
Francesca E Duncan
Keyword(s):  
Wildlife Conservation ◽  
Culture Media ◽  
Ovarian Follicle ◽  
Three Dimensional ◽  
Ovarian Follicles ◽  
Culture Methods ◽  
Growth And Survival ◽  
Follicle Culture ◽  
Culture Techniques

Abstract Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980–2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

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 Legionella Surveillance in a VA Hospital Water System: Cold Water Sampling Is More Sensitive, But Hot Water Samples are More Likely to Yield Legionella pneumophila Serotype 1 (Lp 1)

2015 ◽  
Vol 2 (suppl_1) ◽  
Author(s):  
Monique Boudreaux-Kelly ◽  
Patricia Harris ◽  
Joseph Mikolic ◽  
Ali Sonel ◽  
Cornelius Clancy ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Water Samples ◽  
Cold Water ◽  
Water System ◽  
Hot Water ◽  
Water Sampling ◽  
Serotype 1

scholarly journals Quantitative Real-Time Legionella PCR for Environmental Water Samples: Data Interpretation

2006 ◽  
Vol 72 (4) ◽  
pp. 2801-2808 ◽  
Author(s):  
Philippe Joly ◽  
Pierre-Alain Falconnet ◽  
Janine André ◽  
Nicole Weill ◽  
Monique Reyrolle ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Legionella Pneumophila ◽  
Cooling Tower ◽  
Water System ◽  
Data Interpretation ◽  
Environmental Water ◽  
Hot Water ◽  
Rrna Gene ◽  
Conventional Culture

ABSTRACT Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >103 CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.

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

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.

scholarly journals Study of Seaweed Kappaphycus alvarezii Explants Growth in the Different Salinity Concentrations

2021 ◽  
Vol 13 (1) ◽  
pp. 97
Author(s):  
Muhammad Aris ◽  
Fatma Muchdar ◽  
Rusmawati Labenua
Keyword(s):  
Tissue Culture ◽  
Kappaphycus Alvarezii ◽  
Environmental Parameters ◽  
Culture Method ◽  
Salinity Treatment ◽  
Culture Methods ◽  
Culture Techniques ◽  
Randomized Design ◽  
Salinity Levels

HighlightThe best salinity for the thallus growth of K. alvarezii is 32 mg/LThe salinity indicates the osmotic balance of K. alvarezii seaweedThis osmoregulation process affects the nutrient absorption of K. alvarezii seaweedThis study aims to develop the availibility of superior seeds of K. alvareziiAbstract Kappaphycus alvarezii is one of the leading commodities in Indonesian waters. Demand for this commodity is quite high, as reflected in the increasing volume of exports each year. Fulfillment of these demands is obtained from the production of cultivation. Generally farmers get natural seaweed seedlings, namely cuttings from existing seaweed. The continuous use of seeds from nature can cause deterioration in the quality and quantity. Handling the problem of quality deterioration from seaweed seeds originating from nature, can be overcome by multiplying the seeds through tissue culture methods in vitro. In term of tissue culture techniques method, the most important thing to note is environmental parameters. An environment parameter that changes suddenly such as salinity can inhibit the growth of seaweed. Thus, this work is attampting the different salinity treatment on seaweed explants K. alvarezii. This study aims at determining (weight) the explants of K. alvarezii with a comparison of the different salinity levels in the in vitro tissue culture method. The method used in this study was a completely randomized design (CRD) with the different salinity treatments namely 30, 31, 32, 33, and 34 ppt. The results showed that the different salinities influenced the growth rate of K. alvarezii seaweed explants with the best explant growth at the salinity of 31 ppt, while the lowest growth value was obtained at 34 ppt

scholarly journals Laboratory and field evaluation of selective media for isolation of group B streptococci

1979 ◽  
Vol 9 (4) ◽  
pp. 466-470
Author(s):  
B M Gray ◽  
M A Pass ◽  
H C Dillon
Keyword(s):  
Nalidixic Acid ◽  
Field Evaluation ◽  
Group B Streptococci ◽  
Culture Methods ◽  
Negative Spectrum ◽  
Selective Media ◽  
Culture Techniques ◽  
Inhibitory Agents ◽  
Group B

Problems encountered with currently recommended selective media for group B streptococci (GBS) (selective broth medium and CNA agar) prompted a searach for alternative culture methods in ongoing epidemiological studies. Previously recommended inhibitory agents were tested in vitro. Gentamicin, alone or in combination with nalidixic acid, proved inhibitory for many GBS strains. Among other agents tested, polymyxin was most complementary to the gram-negative spectrum of nalidixic acid, without compromising GBS growth. Crystal violet provided the simplest, most economical staphylococcal inhibitor. Broth and agar media, constituted with these three agents and designated NPC, were evaluated in vitro and in field studies. This investigation represents the first direct comparison of broth media containing inhibitory agents for the preferential isolation of GBS. In maternal colonization studies, NPC broth proved superior to Todd-Hewitt broth containing nalidixic acid and gentamicin at concentrations employed in the previously described selective broth medium (95% versus 59% recovery). Our comparisons were done without added sheep blood since GBS grow well in Todd-Hewitt broth. NPC broth proved more sensitive than NPC agar for detecting GBS colonization in newborns. The NPC agar medium was useful for further purification of broth cultures and quantitative culture techniques.

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