Molecular typing of the Legionella pneumophila population isolated from several locations in a contaminated water network

The RAPD (random amplified polymorphic DNA) technique has been developed for the molecular typing of Legionella in order to characterise the populations of hot water systems. During this study, 22 primers were tested and the four most informative ones were selected. The optimisation of the PCR conditions allowed the setting up of a powerful discriminative genotyping method. Moreover, the definition of a quality management method allowed definition of the key steps and the number of replicates to ensure reproducibility of the RAPD pattern. The RAPD was used to study the hot water network of a building. Legionella colonies (91) were isolated from seven locations and genotyped. The diversity of the population in one sample could vary from one to seven different strains. The study of the traceability showed that, in most of the cases, different populations could be present at different locations of the same network.

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Transport of escherichia coli through soil to groundwater traced by randomly amplified polymorphic DNA (RAPD)

Water Science & Technology ◽

10.2166/wst.1997.0758 ◽

1997 ◽

Vol 35 (11-12) ◽

pp. 351-357 ◽

Cited By ~ 2

Author(s):

R. Rothmaier ◽

A. Weidenmann ◽

K. Botzenhart

Keyword(s):

Negative Impact ◽

Random Amplified Polymorphic Dna ◽

Coli Strain ◽

Soil Samples ◽

Test Field ◽

Liquid Manure ◽

E Coli ◽

Rapd Pattern ◽

Geological Situation ◽

Different Strains

Isolates (50) of E. coli obtained from liquid manure (20 bovine, 20 porcine) were genotyped using random amplified polymorphic DNA (RAPD). Typing revealed 9 and 14 different strains in bovine and porcine liquid manure respectively with no strains in common. One porcine strain, showing a simple RAPD pattern, was subcultured and spread on a test field (1.5l/m2 at 1010 cfu/l) in a drinking water protection zone with loamy to sandy sediments in the Donauried area, Baden-Wurttemberg. Soil samples and groundwaters were collected at monthly intervals October 1994 – June 1995 during which 114 E. coli isolates were recovered. The first occurrence and maximum concentration of E. coli in soil samples taken from more than 20cm depth was in January 1995, declining rapidly with depth and time. All isolates from soil and only one from groundwater showed the RAPD pattern of the spread E. coli strain. The results could not demonstrate a severe negative impact of the spreading of liquid manure on the bacteriological quality of the groundwater in the given geological situation. The distinct strain patterns found in different kinds of liquid manure suggest that genotyping of E. coli by RAPD may be an adequate tool for tracing sources of faecal contamination.

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Rate of Legionella pneumophila colonization in hospital hot water network after time flow taps installation

Journal of Hospital Infection ◽

10.1016/j.jhin.2017.08.021 ◽

2018 ◽

Vol 98 (1) ◽

pp. 60-63 ◽

Cited By ~ 9

Author(s):

M. Totaro ◽

P. Valentini ◽

A.L. Costa ◽

S. Giorgi ◽

B. Casini ◽

...

Keyword(s):

Legionella Pneumophila ◽

Hot Water ◽

Water Network ◽

Time Flow

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How Molecular Typing Can Support Legionella Environmental Surveillance in Hot Water Distribution Systems: A Hospital Experience

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17228662 ◽

2020 ◽

Vol 17 (22) ◽

pp. 8662

Author(s):

Luna Girolamini ◽

Silvano Salaris ◽

Jessica Lizzadro ◽

Marta Mazzotta ◽

Maria Rosaria Pascale ◽

...

Keyword(s):

Legionella Pneumophila ◽

Molecular Typing ◽

Distribution Systems ◽

Water Distribution ◽

Water Distribution Systems ◽

Gene Sequencing ◽

Culture Technique ◽

Hot Water ◽

Environmental Surveillance ◽

Risk Map

In this study, we aimed to associate the molecular typing of Legionella isolates with a culture technique during routine Legionella hospital environmental surveillance in hot water distribution systems (HWDSs) to develop a risk map able to be used to prevent nosocomial infections and formulate appropriate preventive measures. Hot water samples were cultured according to ISO 11731:2017. The isolates were serotyped using an agglutination test and genotyped by sequence-based typing (SBT) for Legionella pneumophila or macrophage infectivity potentiator (mip) gene sequencing for non-pneumophila Legionella species. The isolates’ relationship was phylogenetically analyzed. The Legionella distribution and level of contamination were studied in relation to temperature and disinfectant residues. The culture technique detected 62.21% of Legionella positive samples, characterized by L. pneumophila serogroup 1, Legionella non-pneumophila, or both simultaneously. The SBT assigned two sequence types (STs): ST1, the most prevalent in Italy, and ST104, which had never been isolated before. The mip gene sequencing detected L. anisa and L. rubrilucens. The phylogenetic analysis showed distinct clusters for each species. The distribution of Legionella isolates showed significant differences between buildings, with a negative correlation between the measured level of contamination, disinfectant, and temperature. The Legionella molecular approach introduced in HWDSs environmental surveillance permits (i) a risk map to be outlined that can help formulate appropriate disinfection strategies and (ii) rapid epidemiological investigations to quickly identify the source of Legionella infections.

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The Effect of Hydrogen Peroxide on Reducing the Colonization of Legionella spp. in a Hospital Water Network

10.20944/preprints201702.0063.v1 ◽

2017 ◽

Author(s):

Beatrice Casini ◽

Francesco Aquino ◽

Michele Totaro ◽

Mario Miccoli ◽

Irio Galli ◽

...

Keyword(s):

Hydrogen Peroxide ◽

General Hospital ◽

Legionella Pneumophila ◽

Hot Water ◽

Water Network ◽

Progressive Reduction ◽

Complete Control ◽

Food Grade ◽

Legionella Species ◽

The Mean

Objectives: To evaluate the effectiveness of hydrogen peroxide (HP) use in the hospital water network disinfection to control Legionella spp. colonization. Methods: Following the detection of high levels of Legionella contamination in a 136-bed general hospital water network, an HP treatment of the hot water (25 mg/L) was adopted. During a period of 34 months, the effectiveness of HP on Legionella colonization was assessed. Legionella was isolated in accordance with ISO-11731 and identification was carried out by sequencing of the mip gene. Results: Before HP treatment L.pneumophila sg 2-15 was isolated in all sites with a mean count of 9950±8279 CFU/L. After one month of HP-treatment, we observed the disappearance of L. pneumophila 2-15, however other Legionella species never cultured before appeared; Legionella pneumophila 1 was isolated in 1 out of 4 sampling sites (2,000 CFU/L) and other non-pneumophila species in all sites (mean load 3,000 ±2887 CFU/L). Starting from September 2013, HP-treatment was modified adding food-grade polyphosphates and in the following months we observed a progressive reduction of the mean load of all species (p<0.05), until to a substantial disappearing of Legionella colonization. Conclusion: Hydrogen peroxide demonstrated a good efficacy in controlling Legionella. Although in the initial phases of treatment it seemed unable to eliminate all the species, by keeping HP levels to 25 mg/L and adding food-grade polyphosphates, a progressive and complete control of colonisation was obtained.

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Evaluation of Legionella pneumophila Decrease in Hot Water Network of Four Hospital Buildings after Installation of Electron Time Flow Taps

Water ◽

10.3390/w12010210 ◽

2020 ◽

Vol 12 (1) ◽

pp. 210 ◽

Cited By ~ 2

Author(s):

Michele Totaro ◽

Tommaso Mariotti ◽

Costanza Bisordi ◽

Erica De Vita ◽

Paola Valentini ◽

...

Keyword(s):

Legionella Pneumophila ◽

Cold Water ◽

Control Measure ◽

Critical Issue ◽

Hot Water ◽

Low Flow ◽

Water Networks ◽

Water Network ◽

Time Flow ◽

Chemical Disinfection

Legionella spp. control is a critical issue in hospital with old water networks. Chemical disinfection methods are applied as a control measure over prolonged time periods, but Legionella may be resistant to chemical agents in pipeworks with low flow and frequent water stagnation. We evaluated Legionella spp. colonization in the hot water network of Italian hospitals after the installation of time flow taps (TFTs). In the period between 2017 and 2019, TFTs were installed in four hospital water networks. They were programmed in order to obtain a hot water flow of 192 L/day from each TFTs. A continuous chlorination system (chlorine dioxide) and a cold water pre-filtration device were applied in all the buildings. Before and after TFT installation, Legionella spp. was investigated at scheduled times. Before TFT installation, Legionella pneumophila was detected in all the hospitals with counts ranging from 2 × 102 to 1.4 × 105 CFU/L. After TFT installation, a loss in Legionella pneumophila culturability was always achieved in the period between 24 h and 15 days. Total chlorine concentration (Cl2) was detected in the range between 0.23 and 0.36 mg/L while temperature values were from 44.8 to 53.2 °C. TFTs together with chemical disinfection represent a method which improve water quality and disinfectant efficacy, reducing Legionella colonization in dead-end sections.

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Molecular Characterization ofStreptococcus PyogenesIsolates to investigate an outbreak of Puerperal Sepsis

Infection Control and Hospital Epidemiology ◽

10.1086/502567 ◽

2005 ◽

Vol 26 (5) ◽

pp. 455-461 ◽

Cited By ~ 21

Author(s):

Josette Raymond ◽

Laurent Schlegel ◽

Fabien Garnier ◽

Anne Bouvet

Keyword(s):

Molecular Characterization ◽

Rapd Analysis ◽

Random Amplified Polymorphic Dna ◽

Index Patient ◽

Restriction Pattern ◽

Microbiological Characteristics ◽

Rapd Pattern ◽

Close Relationship ◽

Different Strains ◽

Predominant Strain

AbstractObjective:To describe microbiological characteristics and epidemiologic features of an outbreak of postpartum endometritis.Methods:Various markers were investigated in five patients and three throat carriage isolates ofStreptococcus pyogenesobtained during an outbreak of endometritis occurring in a 13-week period. Molecular characterization included biotyping, T-serotyping,emmgene sequence and restriction, pulsed-field gel electrophoresis (PFGE), and random amplified polymorphic DNA (RAPD) analysis.Results:Biotype, T-serotype, and genotypic data (emmanalysis, PFGE, and RAPD analysis) revealed a close relationship among the isolates from three patients, suggesting that cross-contamination had occurred. These isolates were biotype 1, T type 28, andemmtype 28. The isolates from one patient and one carrier differed from those of the index patient by minor variations of theemmamplicon restriction pattern, PFGE pattern, or RAPD pattern. The remaining isolates were phenotypically and genetically different.Conclusion:Identification of different isolates demonstrated that different strains may circulate simultaneously during a true outbreak and that the predominant strain might persist for several months.

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Random amplified polymorphic DNA (RAPD) finger prints evidencing high genetic variability among marine angiosperms of India

Journal of the Marine Biological Association of the United Kingdom ◽

10.1017/s0025315416000631 ◽

2016 ◽

Vol 97 (6) ◽

pp. 1307-1315 ◽

Cited By ~ 3

Author(s):

Elangovan Dilipan ◽

Jutta Papenbrock ◽

Thirunavakkarasu Thangaradjou

Keyword(s):

Genetic Variability ◽

Rapd Analysis ◽

Random Amplified Polymorphic Dna ◽

Rapd Marker ◽

Seagrass Species ◽

Complex Species ◽

Marine Angiosperms ◽

Different Populations ◽

High Degree ◽

Identification Tool

In India 14 seagrass species can be found with monospecific genera (Enhalus, ThalassiaandSyringodium),Cymodoceawith two species andHalophilaandHalodulerepresented by more than two taxonomically complex species. Considering this, the present study was made to understand the level and pattern of genetic variability among these species collected from Tamilnadu coast, India. Random amplified polymorphic DNA (RAPD) analysis was used to evaluate the level of polymorphism existing between the species. Out of the 12 primers tested, 10 primers amplified 415 DNA fragments with an average of 41.5 fragments per primer. Of the total 415 amplified fragments only 123 (29.7%) were monomorphic and the remaining 292 (70.3%) were polymorphic for Indian seagrass species. Among the 10 primers used four are identified as the key primers capable of distinguishing all the Indian seagrasses with a high degree of polymorphism and bringing representative polymorphic alleles in all the tested seagrasses. From the present investigation, this study shows that the RAPD marker technique can be used not only as a tool to analyse genetic diversity but also to resolve the taxonomic uncertainties existing in the Indian seagrasses. The efficiency of these primers in bringing out the genetic polymorphism or homogeneity among different populations of theHalophilaandHalodulecomplex still has to be tested before recommending these primers as an identification tool for Indian seagrasses.

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