Characterization and cloning of a 37.6-kb plasmid carried byLegionella pneumophilarecovered from patients and hospital water over a 12-year period

1997 ◽  
Vol 43 (2) ◽  
pp. 193-197 ◽  
Author(s):  
Shawn D. Mansfield ◽  
Greg. S. Bezanson ◽  
Thomas J. Marrie
Keyword(s):  
Legionella Pneumophila ◽  
Plasmid Dna ◽  
Potable Water ◽  
Pattern Analysis ◽  
Plasmid Stability ◽  
Physical Map ◽  
Fragmentation Pattern ◽  
Single Plasmid ◽  
Double Digestion ◽  
Victoria General Hospital

For 12 years, strains of Legionella pneumophila serogroup 1 harbouring a 37.6-kb (23 MDa) plasmid have predominated among patient and potable water isolates at the Victoria General Hospital, Halifax, N.S. Plasmid DNA recovered from 24 strains isolated between 1983 and 1995 was digested with the restriction endonucleases EcoRI, HindIII, KpnI, PvuII, XbaI, and BamHI. The distribution of cutting sites indicated that the 23-MDa size group had remained essentially unchanged during this period, suggesting the persistence of a single plasmid type. Further fragmentation pattern analysis permitted the construction of a physical map of the prototype 23-MDA plasmid, pLp4269. Double digestion with BamHI–HindIII enabled the cloning of 94.4% of pLp4269 into pBluescript vector. A 2.1-kb fragment was not clonable. Plasmid pLp4269 is the first of the smaller Legionella extrachromosomal DNAs to be characterized in this way.Key words: Legionella, plasmid, stability, map, cloning.

scholarly journals Lessons From an Outbreak of Legionnaires’ Disease on a Hematology-Oncology Unit

2016 ◽  
Vol 38 (3) ◽  
pp. 306-313 ◽  
Author(s):  
Louise K. Francois Watkins ◽  
Karrie-Ann E. Toews ◽  
Aaron M. Harris ◽  
Sherri Davidson ◽  
Stephanie Ayers-Millsap ◽  
...  
Keyword(s):  
Legionella Pneumophila ◽  
Potable Water ◽  
Single Case ◽  
Healthcare Providers ◽  
Water System ◽  
Positive Sequence ◽  
Environmental Isolates ◽  
Legionnaires Disease ◽  
Oncology Unit ◽  
Healthcare Associated

OBJECTIVESTo define the scope of an outbreak of Legionnaires’ disease (LD), to identify the source, and to stop transmission.DESIGN AND SETTINGEpidemiologic investigation of an LD outbreak among patients and a visitor exposed to a newly constructed hematology-oncology unit.METHODSAn LD case was defined as radiographically confirmed pneumonia in a person with positive urinary antigen testing and/or respiratory culture forLegionellaand exposure to the hematology-oncology unit after February 20, 2014. Cases were classified as definitely or probably healthcare-associated based on whether they were exposed to the unit for all or part of the incubation period (2–10 days). We conducted an environmental assessment and collected water samples for culture. Clinical and environmental isolates were compared by monoclonal antibody (MAb) and sequence-based typing.RESULTSOver a 12-week period, 10 cases were identified, including 6 definite and 4 probable cases. Environmental sampling revealedLegionella pneumophilaserogroup 1 (Lp1) in the potable water at 9 of 10 unit sites (90%), including all patient rooms tested. The 3 clinical isolates were identical to environmental isolates from the unit (MAb2-positive, sequence type ST36). No cases occurred with exposure after the implementation of water restrictions followed by point-of-use filters.CONCLUSIONSContamination of the unit’s potable water system with Lp1 strain ST36 was the likely source of this outbreak. Healthcare providers should routinely test patients who develop pneumonia at least 2 days after hospital admission for LD. A single case of LD that is definitely healthcare associated should prompt a full investigation.Infect Control Hosp Epidemiol2017;38:306–313

scholarly journals Evaluation of Legiolert for Quantification of Legionella pneumophila from Non-potable Water

2018 ◽  
Vol 75 (10) ◽  
pp. 1282-1289 ◽  
Author(s):  
Melanie M. Rech ◽  
Brian M. Swalla ◽  
Jason K. Dobranic
Keyword(s):  
Legionella Pneumophila ◽  
Potable Water

scholarly journals Cre/LoxP-HBV plasmids generating recombinant covalently closed circular DNA genome upon transfection

2020 ◽  
Author(s):  
Robert L. Kruse ◽  
Xavier Legras ◽  
Mercedes Barzi
Keyword(s):  
Plasmid Dna ◽  
Plasmid Stability ◽  
Intron Insertion ◽  
Circular Dna ◽  
Immunodeficient Mice ◽  
New Therapies ◽  
Covalently Closed Circular Dna ◽  
Dna Silencing

AbstractNew therapies against hepatitis B virus (HBV) require the elimination of covalently closed circular DNA (cccDNA), the episomal HBV genome. HBV plasmids containing an overlength 1.3-mer genome and bacterial backbone (pHBV1.3) are used in many different models, but do not replicate the unique features of cccDNA. Since the stable cccDNA pool is a barrier to HBV eradication in patients, we developed a recombinant circular HBV genome (rcccDNA) to mimic the cccDNA using Cre/LoxP technology. We validated four LoxP insertion sites into the HBV genome using hydrodynamic tail vein injection into murine liver, demonstrating high levels of HBV surface antigen (HBsAg) and HBV DNA expression with rcccDNA formation. HBsAg expression from rcccDNA was >30,000 ng/mL over 78 days, while HBsAg-expression from pHBV1.3 plasmid DNA declined from 2,753 ng/mL to 131 ng/mL over that time in immunodeficient mice (P<0.001), reflective of plasmid DNA silencing. We then cloned Cre-recombinase in cis on the LoxP-HBV plasmids, achieving plasmid stability in bacteria with intron insertion into Cre and demonstrating rcccDNA formation after transfection in vitro and in vivo. These cis-Cre/LoxP-HBV plasmids were then used to create HBx-mutant and GFP reporter plasmids to further probe cccDNA biology and antiviral strategies against cccDNA. Overall, we believe these auto-generating rcccDNA plasmids will be of great value to model cccDNA for testing new therapies against HBV infection.

scholarly journals Performance of Legiolert Test vs. ISO 11731 to Confirm Legionella pneumophila Contamination in Potable Water Samples

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 690 ◽  
Author(s):  
Maria Scaturro ◽  
Matteo Buffoni ◽  
Antonietta Girolamo ◽  
Sandra Cristino ◽  
Luna Girolamini ◽  
...  
Keyword(s):  
Risk Assessment ◽  
Legionella Pneumophila ◽  
Water Samples ◽  
Gold Standard ◽  
Liquid Culture ◽  
Potable Water ◽  
Culture Method ◽  
Alternative Methods ◽  
Significant Difference

Detection and enumeration of Legionella in water samples is of great importance for risk assessment analysis. The plate culture method is the gold standard, but has received several well-known criticisms, which have induced researchers to develop alternative methods. The purpose of this study was to compare Legionella counts obtained by the analysis of potable water samples through the plate culture method and through the IDEXX liquid culture Legiolert method. Legionella plate culture, according to ISO 11731:1998, was performed using 1 L of water. Legiolert was performed using both the 10 mL and 100 mL Legiolert protocols. Overall, 123 potable water samples were analyzed. Thirty-seven (30%) of them, positive for L. pneumophila, serogroups 1 or 2–14 by plate culture, were used for comparison with the Legiolert results. The Legiolert 10 mL test detected 34 positive samples (27.6%) and the Legiolert 100 mL test detected 37 positive samples, 27.6% and 30% respectively, out of the total samples analyzed. No significant difference was found between either the Legiolert 10 mL and Legiolert 100 mL vs. the plate culture (p = 0.9 and p = 0.3, respectively) or between the Legiolert 10 mL and Legiolert 100 mL tests (p = 0.83). This study confirms the reliability of the IDEXX Legiolert test for Legionella pneumophila detection and enumeration, as already shown in similar studies. Like the plate culture method, the Legiolert assay is also suitable for obtaining isolates for typing purposes, relevant for epidemiological investigations.

scholarly journals Legionella pneumophila and Protozoan Hosts: Implications for the Control of Hospital and Potable Water Systems

Pathogens ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 286 ◽  
Author(s):  
Muhammad Atif Nisar ◽  
Kirstin E. Ross ◽  
Melissa H. Brown ◽  
Richard Bentham ◽  
Harriet Whiley
Keyword(s):  
Legionella Pneumophila ◽  
Distribution Systems ◽  
Water Distribution ◽  
Potable Water ◽  
Water Systems ◽  
Water Disinfection ◽  
Health Concern ◽  
Waterborne Pathogen ◽  
Microbial Biofilms ◽  
Pontiac Fever

Legionella pneumophila is an opportunistic waterborne pathogen of public health concern. It is the causative agent of Legionnaires’ disease (LD) and Pontiac fever and is ubiquitous in manufactured water systems, where protozoan hosts and complex microbial communities provide protection from disinfection procedures. This review collates the literature describing interactions between L. pneumophila and protozoan hosts in hospital and municipal potable water distribution systems. The effectiveness of currently available water disinfection protocols to control L. pneumophila and its protozoan hosts is explored. The studies identified in this systematic literature review demonstrated the failure of common disinfection procedures to achieve long term elimination of L. pneumophila and protozoan hosts from potable water. It has been demonstrated that protozoan hosts facilitate the intracellular replication and packaging of viable L. pneumophila in infectious vesicles; whereas, cyst-forming protozoans provide protection from prolonged environmental stress. Disinfection procedures and protozoan hosts also facilitate biogenesis of viable but non-culturable (VBNC) L. pneumophila which have been shown to be highly resistant to many water disinfection protocols. In conclusion, a better understanding of L. pneumophila-protozoan interactions and the structure of complex microbial biofilms is required for the improved management of L. pneumophila and the prevention of LD.

Fatal NosocomialLegionella pneumophilaInfection Due to Exposure to Contaminated Water From a Washbasin in a Hematology Unit

2008 ◽  
Vol 29 (11) ◽  
pp. 1091-1093 ◽  
Author(s):  
Alexandre Brûlet ◽  
Marie-Christine Nicolle ◽  
Marine Giard ◽  
Franck-Emmanuel Nicolini ◽  
Mauricette Michallet ◽  
...  
Keyword(s):  
Nosocomial Infection ◽  
Water Supply ◽  
Legionella Pneumophila ◽  
Potable Water ◽  
Contaminated Water ◽  
Genomic Profile ◽  
Source Of Infection ◽  
Potable Water Supply

A fatal nosocomial infection withLegionella pneumophilaserogroup 5 occurred in a patient with leukemia. Isolates recovered from both the potable water supply and the patient showed an identical genomic profile. With no other exposure identified, the water from the washbasin was evidently the source of infection.

Comparison of two molecular methods used for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply

2008 ◽  
Vol 58 (3) ◽  
pp. 683-688 ◽  
Author(s):  
B. Casini ◽  
P. Valentini ◽  
A. Baggiani ◽  
F. Torracca ◽  
C. Lorenzini ◽  
...  
Keyword(s):  
Water Supply ◽  
Legionella Pneumophila ◽  
Potable Water ◽  
Water System ◽  
Epidemiological Studies ◽  
Molecular Methods ◽  
Pfge Analysis ◽  
Typing Method ◽  
Electrophoretic Patterns ◽  
Low Degree

The results of the pulsed-field gel electrophoresis and the sequence-based typing (using the loci flaA, pilE, asd, mip, mompS and proA) were compared for subtyping of Legionella pneumophila 1 strains isolated from a hospital water supply. Molecular typing was carried out on 61 isolates (38% of the positive samples) selected on space and temporal criteria in order to follow the evolution of the water-system colonization. For all the 61 isolates, the sequence of the amplified mip gene fragment identified Legionella pneumophila strain Wadsworth. Genotype testing by PFGE analysis showed three different patterns, correspondent to three SBT types according to the allelic profiles. Both PFGE and SBT indicated the circulation and the persistence in the hospital potable water-system of three types randomly distributed in space and time. The two molecular methods adopted showed a 100% concordance, although a low degree of genetic heterogeneity characterized the isolates. The electrophoretic patterns were sufficiently unambiguous to consider PFGE a highly discriminatory typing method, but the SBT technique besides accurately characterizing isolates, was able to identify Legionella strains through analysis of the mip gene. A typing method with this level of discriminatory power has great potential for assisting in epidemiological studies.

scholarly journals Intracellular Tracking of Single-plasmid DNA Particles After Delivery by Electroporation

2013 ◽  
Vol 21 (12) ◽  
pp. 2217-2226 ◽  
Author(s):  
Christelle Rosazza ◽  
Annette Buntz ◽  
Thorsten Rieß ◽  
Dominik Wöll ◽  
Andreas Zumbusch ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Single Plasmid
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