DOCSLIB.ORG

The Development of Legionella Pneumophila Reaches Different End Points in Amoebae, Macrophages and Ciliates

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
The Development of Legionella Pneumophila Reaches Different End Points in Amoebae, Macrophages and Ciliates THE DEVELOPMENT OF LEGIONELLA PNEUMOPHILA REACHES DIFFERENT END POINTS IN AMOEBAE, MACROPHAGES AND CILIATES by Hany A. M. Abdelhady Submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy at Dalhousie University Halifax, Nova Scotia December 2013 © Copyright by Hany A. M. Abdelhady, 2013 DEDICATION To the spirit of my father, who I wish was alive to share this moment with me, and to my mother, who always listened to my complaints and granted me her unlimited support over the years. ii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... viii LIST OF FIGURES .......................................................................................................... x ABSTRACT ..................................................................................................................... xii LIST OF ABBREVIATIONS USED ............................................................................ xiii ACKNOWLEDGEMENTS .......................................................................................... xvi CHAPTER 1: INTRODUCTION .................................................................................... 1 1.1. Legionella pneumophila is an environmental human pathogen ................................ 1 1.2. Legionellosis ............................................................................................................. 1 1.3. L. pneumophila, protozoa and human transmission .................................................. 4 1.4. Developmental cycle and differentiation of L. pneumophila .................................... 5 1.4.1. The central role of MIFs in the life cycle of L. pneumophila ............................. 6 1.4.2. Role of Tetrahymena ciliates in the life cycle of L. pneumophila. ..................... 9 1.5. Overview of the regulatory network of L. pneumophila differentiation ................. 10 1.5.1. Signals that trigger L. pneumophila differentiation .......................................... 10 1.5.2. LetA/LetS two-component system ................................................................... 12 1.5.3. Regulation by small non-coding RNA ............................................................. 14 1.5.4. Alternative sigma factors .................................................................................. 15 1.6. L. pneumophila intracellular trafficking and replication ......................................... 17 1.6.1. L. pneumophila internalization ......................................................................... 18 1.6.2. Inhibition of lysosome fusion with the LCV .................................................... 19 1.6.3. Recruitment of mitochondria to the LCV ......................................................... 19 1.6.4. Remodelling of the LCV into an ER-derived replicative organelle ................. 20 1.6.5. Bacterial egress from host cells ........................................................................ 20 1.7. The Dot/Icm system ................................................................................................ 21 1.8. Transcriptome analysis of L. pneumophila inside different hosts ........................... 23 1.8.1. The control of differentiation in L. pneumophila is not yet fully understood. ...................................................................................................... 24 1.8.2. Changes in the gene expression of L. pneumophila inside A. castellanii ......... 25 1.8.3. Changes in the gene expression of L. pneumophila inside human macrophages.................................................................................................... 28 iii 1.8.4. Changes in the gene expression of L. pneumophila inside Tetrahymena ciliates ............................................................................................................. 29 1.8.5. Comparison of L. pneumophila gene expression in amoebae and macrophages.................................................................................................... 30 1.9. Metabolism of L. pneumophila ............................................................................... 30 1.9.1. Amino acids ...................................................................................................... 31 1.9.2. Iron ................................................................................................................... 32 1.9.3. Carbohydrate metabolism ................................................................................. 35 1.10. Study hypotheses and objectives ........................................................................... 37 CHAPTER 2: MATERIALS AND METHODS .......................................................... 39 2.1. Bacterial strains and growth conditions .................................................................. 39 2.1.1. L. pneumophila ................................................................................................. 39 2.1.2. Escherichia coli ................................................................................................ 39 2.2. Hosts Infected with L. pneumophila ....................................................................... 41 2.2.1. Culture and infection of Acanthamoeba castellanii ......................................... 41 2.2.2. Culture of Tetrahymena tropicalis ciliates and feeding experiments ............... 42 2.2.3. Culture and infection of mammalian cell lines ................................................. 43 2.2.3.1. Human U937 and THP-1 cells .................................................................. 43 2.2.3.2. Mouse L929 cells ...................................................................................... 44 2.2.3.3. Human HeLa cells ..................................................................................... 44 2.3. Purification of L. pneumophila progenies from infected host cells ........................ 44 2.3.1. Purification of L. pneumophila progenies from infected cells ......................... 45 2.3.2. Isolation and mechanical disruption of legionellae-laden pellets ..................... 45 2.4. Characterization of L. pneumophila progenies from infected cells ........................ 46 2.4.1. Transmission electron microscopy (TEM) ....................................................... 46 2.4.2. Stress-resistance assays .................................................................................... 46 2.4.3. Intracellular growth experiments ...................................................................... 47 2.4.4. Attachment experiments ................................................................................... 48 2.4.5. Competition assays ........................................................................................... 48 2.4.6. Plaque assays .................................................................................................... 49 2.4.7. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS- PAGE) ............................................................................................................. 50 2.5. Molecular techniques .............................................................................................. 51 iv 2.5.1. Isolation of L. pneumophila genomic DNA ..................................................... 51 2.5.2. Isolation of L. pneumophila RNA .................................................................... 52 2.5.3. Polymerase chain reaction (PCR) ..................................................................... 53 2.5.4. Agarose gel electrophoresis .............................................................................. 54 2.5.5. Restriction endonuclease digestion .................................................................. 54 2.5.6. Plasmid and DNA purification ......................................................................... 54 2.5.7. DNA ligation .................................................................................................... 56 2.5.8. Preparation of electrocompetent E. coli DH5α cells ........................................ 56 2.5.9. Preparation of electrocompetent L. pneumophila cells .................................... 56 2.5.10. Bacterial transformation by electroporation ................................................... 56 2.6. Transcriptome analysis of L. pneumophila inside different hosts ........................... 57 2.6.1. Analysis of the genetic expression of L. pneumophila in different hosts ......... 57 2.6.2. Confirmation of microarray data by qRT-PCR ................................................ 58 2.6.2.1. RNA isolation and concentration calculation ........................................... 58 2.6.2.2. Deoxyribonuclease (DNase) I treatment of RNA ..................................... 59 2.6.2.3. Reverse transcription and cDNA synthesis ............................................... 59 2.6.2.4. Primer design ............................................................................................ 60 2.6.2.5. qRT-PCR reaction conditions ................................................................... 60 2.6.2.6. qRT-PCR data analysis ............................................................................. 61 2.7. Investigation of the role of lpg1669 in the differentiation of L. pneumophila ........ 61 2.7.1. Construction of an L. pneumophila JR32 ∆lpg1669 (amyA) mutant ................ 61 2.7.2. Complementation
Load more

Recommended publications
  • Legionnaires' Disease, Pontiac Fever, Legionellosis and Legionella
    Legionnaires’ Disease, Pontiac Fever, Legionellosis and Legionella Q: What is Legionellosis and who is at risk? Legionellosis is an infection caused by Legionella bacteria. Legionellosis can present as two distinct illnesses: Pontiac fever (a self-limited flu-like mild respiratory illness), and Legionnaires’ Disease (a more severe illness involving pneumonia). People of any age can get Legionellosis, but the disease occurs most frequently in persons over 50 years of age. The disease most often affects those who smoke heavily, have chronic lung disease, or have underlying medical conditions that lower their immune system, such as diabetes, cancer, or renal dysfunction. Persons taking certain drugs that lower their immune system, such as steroids, have an increased risk of being affected by Legionellosis. Many people may be infected with Legionella bacteria without developing any symptoms, and others may be treated without having to be hospitalized. Q: What is Legionella? Legionella bacteria are found naturally in freshwater environments such as creeks, ponds and lakes, as well as manmade structures such as plumbing systems and cooling towers. Legionella can multiply in warm water (77°F to 113°F). Legionella pneumophila is responsible for over 90 percent of Legionnaires’ Disease cases and several different species of Legionella are responsible for Pontiac Fever. Q: How is Legionella spread and how does someone acquire Legionellosis (Legionnaires’ Disease/Pontiac Fever)? Legionella bacteria become a health concern when they grow and spread in manmade structures such as plumbing systems, hot water tanks, cooling towers, hot tubs, decorative fountains, showers and faucets. Legionellosis is acquired after inhaling mists from a contaminated water source containing Legionella bacteria.
    [Show full text]
  • Management Weaknesses Delayed Response to Flint Water Crisis
    U.S. ENVIRONMENTAL PROTECTION AGENCY OFFICE OF INSPECTOR GENERAL Ensuring clean and safe water Compliance with the law Management Weaknesses Delayed Response to Flint Water Crisis Report No. 18-P-0221 July 19, 2018 Report Contributors: Stacey Banks Charles Brunton Kathlene Butler Allison Dutton Tiffine Johnson-Davis Fred Light Jayne Lilienfeld-Jones Tim Roach Luke Stolz Danielle Tesch Khadija Walker Abbreviations CCT Corrosion Control Treatment CFR Code of Federal Regulations EPA U.S. Environmental Protection Agency FY Fiscal Year GAO U.S. Government Accountability Office LCR Lead and Copper Rule MDEQ Michigan Department of Environmental Quality OECA Office of Enforcement and Compliance Assurance OIG Office of Inspector General OW Office of Water ppb parts per billion PQL Practical Quantitation Limit PWSS Public Water System Supervision SDWA Safe Drinking Water Act Cover Photo: EPA Region 5 emergency response vehicle in Flint, Michigan. (EPA photo) Are you aware of fraud, waste or abuse in an EPA Office of Inspector General EPA program? 1200 Pennsylvania Avenue, NW (2410T) Washington, DC 20460 EPA Inspector General Hotline (202) 566-2391 1200 Pennsylvania Avenue, NW (2431T) www.epa.gov/oig Washington, DC 20460 (888) 546-8740 (202) 566-2599 (fax) [email protected] Subscribe to our Email Updates Follow us on Twitter @EPAoig Learn more about our OIG Hotline. Send us your Project Suggestions U.S. Environmental Protection Agency 18-P-0221 Office of Inspector General July 19, 2018 At a Glance Why We Did This Project Management Weaknesses
    [Show full text]
  • Host-Adaptation in Legionellales Is 2.4 Ga, Coincident with Eukaryogenesis
    bioRxiv preprint doi: https://doi.org/10.1101/852004; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 Host-adaptation in Legionellales is 2.4 Ga, 2 coincident with eukaryogenesis 3 4 5 Eric Hugoson1,2, Tea Ammunét1 †, and Lionel Guy1* 6 7 1 Department of Medical Biochemistry and Microbiology, Science for Life Laboratories, 8 Uppsala University, Box 582, 75123 Uppsala, Sweden 9 2 Department of Microbial Population Biology, Max Planck Institute for Evolutionary 10 Biology, D-24306 Plön, Germany 11 † current address: Medical Bioinformatics Centre, Turku Bioscience, University of Turku, 12 Tykistökatu 6A, 20520 Turku, Finland 13 * corresponding author 14 1 bioRxiv preprint doi: https://doi.org/10.1101/852004; this version posted February 27, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 15 Abstract 16 Bacteria adapting to living in a host cell caused the most salient events in the evolution of 17 eukaryotes, namely the seminal fusion with an archaeon 1, and the emergence of both the 18 mitochondrion and the chloroplast 2. A bacterial clade that may hold the key to understanding 19 these events is the deep-branching gammaproteobacterial order Legionellales – containing 20 among others Coxiella and Legionella – of which all known members grow inside eukaryotic 21 cells 3.
    [Show full text]
  • Legionella Information for Community Public Water Systems, Health Care
    Legionella Information FOR COMMUNITY PUBLIC WATER SYSTEMS (CPWSs), HEALTH CARE FACILITIES, AND ALL TYPES OF BUILDINGS What is Legionella? Legionella is a bacterium commonly found in natural and man-made aquatic environments. Legionella can be found at low concentrations in any public water system. Legionella only poses a health risk when growth occurs in warm stagnant water, the water is aerosolized, and the small droplets are inhaled. Legionella generally does not pose a health risk if a person drinks the water. Those that are infected may develop legionellosis, a type of pneumonia called Legionnaires’ disease, or a flu-like illness called Pontiac fever. There has been an increase in Legionnaires’ disease cases nationwide and in Minnesota, with 17 confirmed cases in 2004 and 115 cases in 2016. The risk to people depends on many factors, including: 1. Amplification or growth of Legionella. ▪ Conditions such as water temperatures between 77oF and 108oF, stagnation, and the presence of scale, sediment, biofilm, and protozoa promote amplification of Legionella. 2. Aerosolization of colonized water. ▪ Common modes of aerosolization include showers and faucets, cooling towers, hot tubs, and decorative fountains and water features. 3. Size of the water droplets. 4. Strain of Legionella. 5. Susceptibility of exposed individual. ▪ Those who are over 50 years of age, are male, smoke, have chronic lung disease, and/or are immunocompromised or immunosuppressed are more susceptible. According to the Centers for Disease Control and Prevention (CDC), about 5,000 cases of Legionnaires’ disease are reported each year in the United States, and one out of every 10 people who get Legionnaires’ disease will die.
    [Show full text]
  • Legionella Monitoring in NYC's Distribution System
    Legionella Monitoring in NYC’s Distribution System ENOMA OMOREGIE NEW YORK CITY DEPARTMENT OF HEALTH AND MENTAL HYGIENE PUBLIC HEALTH LABORATORY NATIONAL ENVIRONMENTAL MONITORING CONFERENCE 2019 Legionella spp. Monitoring in the New York City Drinking Water Distribution System (LMINDS) Joint study between the NYC Department of Environmental Protection and Department of Health and Mental Hygiene. Four primary study questions: What is the prevalence and geographic distribution of Legionella in NYC’s water distribution system? What Legionella species inhabit the NYC distribution system, and how do they relate to species described in clinical and environmental studies? How do environmental factors such as physiochemical water parameters, water age, disinfectant levels etc., and utility management practices correlate with the presence of Legionella? Can utility management practices that reduce Legionella levels be identified? What is Legionella? Gram negative bacterium that is part of the Gammaproteobacteria. Naturally occurring and often found in aquatic systems: rivers, streams, cooling towers, hot and cold domestic water systems etc. Grows optimally at around 35°C. Causative agent of legionellosis. Source: CDC.gov; Chemology.com.au Legionellosis is two distinct syndromes Legionnaires’ disease, which is a serious type of pneumonia. ~6,000 reported cases per year in the USA and NYC has about 200 ‐ 440 cases per year. 9% mortality rate. Risk factors include smoking, chronic lung disease, age, and a compromised immune system.
    [Show full text]
  • Effect of Common Oxidative Water Treatments on Acanthamoeba Internalized Legionella
    UNLV Theses, Dissertations, Professional Papers, and Capstones May 2019 Effect of Common Oxidative Water Treatments on Acanthamoeba Internalized Legionella James Park Follow this and additional works at: https://digitalscholarship.unlv.edu/thesesdissertations Part of the Microbiology Commons Repository Citation Park, James, "Effect of Common Oxidative Water Treatments on Acanthamoeba Internalized Legionella" (2019). UNLV Theses, Dissertations, Professional Papers, and Capstones. 3659. http://dx.doi.org/10.34917/15778517 This Thesis is protected by copyright and/or related rights. It has been brought to you by Digital Scholarship@UNLV with permission from the rights-holder(s). You are free to use this Thesis in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Thesis has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. EFFECT OF COMMON OXIDATIVE WATER TREATMENTS ON ACANTHAMOEBA INTERNALIZED LEGIONELLA By James Bodeen Park Bachelor of Science- Life Sciences University of Nevada, Las Vegas 2013 A thesis submitted in partial fulfillment of the requirements for the Master of Public Health Department of Environmental and Occupational Health School of Public Health The Graduate College University of Nevada, Las Vegas May 2019 Thesis Approval The Graduate College The University of Nevada, Las Vegas April 4, 2019 This thesis prepared by James Bodeen Park entitled Effect of Common Oxidative Water Treatments on Acanthamoeba Internalized Legionella is approved in partial fulfillment of the requirements for the degree of Master of Public Health Department of Environmental and Occupational Health Patricia Cruz, Ph.D.
    [Show full text]
  • Monitoring Distribution Systems for Legionella Pneumophila Using Legiolert
    Received: 21 September 2018 Revised: 26 November 2018 Accepted: 17 December 2018 DOI: 10.1002/aws2.1122 ORIGINAL RESEARCH Monitoring distribution systems for Legionella pneumophila using Legiolert Mark W. LeChevallier Dr. Water Consulting, Morrison, Colorado This study implemented the Legiolert test (a culture-based assay for L. pneumo- Correspondence Mark W. LeChevallier, Dr. Water Consulting, phila based on the most probable number [MPN]) at 12 utilities to assess their Morrison, Colorado. experiences and to develop a baseline of Legionella pneumophila occurrence in Email: [email protected] drinking water distribution systems. A total of 679 samples were analyzed during Funding information the study: 53 source water, 50 from the plant effluent, and 576 from the distribution IDEXX Laboratories, Westbrook, ME system. L. pneumophila was detected in three of five source water samples at one utility but was not detected in any of the treated plant effluent samples. L. pneumophila was detected in only one distribution sample (0.17%) at a concen- tration of 1 MPN/100 mL in a sample that contained 0.72 mg/L free chlorine and was serotyped as belonging to the 2–14 serogroup. Four (0.7%) distribution sam- ples could not be confirmed by serotyping. Overall, the utilities found the test easy to learn and apply in their systems. This study provides a precedent for future mon- itoring of drinking water systems. KEYWORDS distribution, L. pneumophila, Legiolert, Legionella, microbiology 1 | INTRODUCTION L. pneumophila has become the most commonly identi- fied drinking water pathogen, responsible for about two-thirds Legionellosis is a respiratory infection caused by bacteria in of all potable water outbreaks and nearly all the fatalities asso- the genus Legionella.
    [Show full text]
  • Flint Water Crisis
    Case Examples FLINT WATER CRISIS Flint Water Crisis The Challenge The safety of our nation’s drinking water systems has recently been called into question. The water crisis that began in Flint, Michigan, in April 2014 sheds light on the interplay between weak governmental policy, the physical environmental health infrastructure, and the lack of oversight and accountability in a post-industrial region suffering from years of under-employment and industry divestment. Over the course of many months, a population of nearly 100,000 was stripped of one of the most basic human needs: clean drinking water. Flint’s aged water system—similar to that in many US urban areas—contains a large percentage of lead pipe sand plumbing.1 In April 2014, the city switched its water source from treated Lake Huron/Detroit River water to improperly treated Flint River water, which corroded pipes and caused lead to leach into the water. Lead can enter the body through drinking water, through foods cooked with contaminated water, or through infant formulas mixed with lead-tainted water. While lead occurs in the environment naturally, both EPA and CDC maintain no safe blood lead level in children.2, 3 In fact, lead exposure is linked to developmental and neurological damage in children,4 whose bodies absorb ingested lead at the alarming rate of 40 to 50 percent, compared with 3 to 10 percent in adults.1 Groups suffering disproportionate lead exposure include children, pregnant women, and low-income individuals.1 Most water system damage, as in Flint, is preventable with enforcement of proper regulatory safeguards.
    [Show full text]
  • Evidence That Bacteria Packaging by Tetrahymena Is a Widespread Phenomenon
    microorganisms Article Evidence that Bacteria Packaging by Tetrahymena Is a Widespread Phenomenon Alicia F. Durocher 1,2,3, Alix M. Denoncourt 1,2,3, Valérie E. Paquet 1,2,3 and Steve J. Charette 1,2,3,* 1 Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC G1V 0A6, Canada; [email protected] (A.F.D.); [email protected] (A.M.D.); [email protected] (V.E.P.) 2 Centre de Recherche de l’Institut Universitaire de Cardiologie et de Pneumologie de Québec, Hôpital Laval, Quebec City, QC G1V 4G5, Canada 3 Département de Biochimie, de Microbiologie et de Bio-Informatique, Faculté des Sciences et de Génie, Université Laval, Quebec City, QC G1V 0A6, Canada * Correspondence: [email protected] Received: 19 September 2020; Accepted: 4 October 2020; Published: 7 October 2020 Abstract: Protozoa are natural predators of bacteria, but some bacteria can evade digestion once phagocytosed. Some of these resistant bacteria can be packaged in the fecal pellets produced by protozoa, protecting them from physical stresses and biocides. Depending on the bacteria and protozoa involved in the packaging process, pellets can have different morphologies. In the present descriptive study, we evaluated the packaging process with 20 bacteria that have never been tested before for packaging by ciliates. These bacteria have various characteristics (shape, size, Gram staining). All of them appear to be included in pellets produced by the ciliates Tetrahymena pyriformis and/or T. thermophila in at least one condition tested. We then focused on the packaging morphology of four of these bacteria.
    [Show full text]
  • The Structural Complexity of the Gammaproteobacteria Flagellar Motor Is Related to the 2 Type of Its Torque-Generating Stators 3
    bioRxiv preprint doi: https://doi.org/10.1101/369397; this version posted July 14, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The structural complexity of the Gammaproteobacteria flagellar motor is related to the 2 type of its torque-generating stators 3 4 Mohammed Kaplan1, Debnath Ghosal1, Poorna Subramanian1, Catherine M. Oikonomou1, Andreas Kjær1*, Sahand 5 Pirbadian2, Davi R. Ortega1, Mohamed Y. El-Naggar2 and Grant J. Jensen1,3,4 6 1Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125. 7 2Department of Physics and Astronomy, Biological Sciences, and Chemistry, University of Southern California, Los 8 Angeles, CA 90089. 9 3Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125. 10 *Present address: Rex Richards Building, South Parks Road, Oxford OX1 3QU 11 4Corresponding author: [email protected]. 12 Abstract: 13 14 The bacterial flagellar motor is a cell-envelope-embedded macromolecular machine that functions as a propeller to 15 move the cell. Rather than being an invariant machine, the flagellar motor exhibits significant variability between 16 species, allowing bacteria to adapt to, and thrive in, a wide range of environments. For instance, different torque- 17 generating stator modules allow motors to operate in conditions with different pH and sodium concentrations and 18 some motors are adapted to drive motility in high-viscosity environments. How such diversity evolved is unknown.
    [Show full text]
  • Biological Pollutants in the Indoor Environment
    CHAPTER 3 Biological Pollutants in the Indoor Environment Peter W.H. Binnie ABSTRACT Studies carried out by Healthy Buildings International (HBI) found that in over one third of the more than 400 buildings studied, the major pollutants included allergenic fungi, and in greater than !Wo thirds, air supply systems wore contaminated with dust, dirt, and microbes. This paper discusses the recognition of sick building syndrome (SBS) as an accepted malady and lhe possible association or microbial contaminants with SBS. The different types of microbes are described along with the problems they can produce. Sources and spread of microbes within buildings is discussed along with descriptions of methods for sampling from surfaces, water, and indoor air. Special mention is made of sampling for and identification of Legione/la pneumophi/a, using a new rapid assay technique, and the importance of correct interpretation of microbial findings against available standards. INTRODUCTION In 1982 the World Health Organization' made an attempt to define sick build­ ing syndrome and, at least since then, it has been recognized as a malady affecting a proportion of people in certain problem buildings. By its nature, SBS is difficult to define, and despite an ever-increasing number of publications on the subject, no single cause has been identified.2 The indicators that a building may be sick usually are increased staff complaints of minor health symptoms and of stuffy air, intermittent odors, and visible increases in dust levels. Other factors may be uneven temperature zones, noticeable smoke accumulation, and dirt coming out of air supply diffusers. Management may also be aware of increased staff absentee­ ism and reduced productivity.
    [Show full text]
  • Detection of Legionella Pneumophila in Water and Biofilm Samples by Culture and Molecular Methods from Man-Made Systems in São Paulo - Brazil
    Brazilian Journal of Microbiology (2007) 38:743-751 ISSN 1517-8382 DETECTION OF LEGIONELLA PNEUMOPHILA IN WATER AND BIOFILM SAMPLES BY CULTURE AND MOLECULAR METHODS FROM MAN-MADE SYSTEMS IN SÃO PAULO - BRAZIL Fábio R. S. Carvalho; Annette S. Foronda; Vivian H. Pellizari* Laboratório de Microbiologia Ambiental, Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil. Submitted: January 09, 2007; Returned to authors for corrections: March 30, 2007; Approved: September 20, 2007. ABSTRACT Legionella pneumophila is a pathogenic bacteria associated to aquatic habitat of natural and artificial environments. Clinical cases of legionellosis have been reported in Brazil but there is a lack of information about the incidence and concentration of this bacterium in environmental sources. Thus, the present study was designed to evaluate the occurrence of legionellae in São Paulo city, Brazil, using different methods of detection and identification. Sixty-seven water and biofilm samples from natural reservoirs and man-made systems were collected and analyzed for the presence of Legionella spp by culturing onto a selective medium, coculture in axenic free-living amoebae and direct fluorescent antibody (DFA) assay. Results showed that freshwater of reservoirs did not contain legionellae, Legionella pneumophila was isolated from man-made systems, with predominance of Legionella pneumophila serogroup 1 strains. Although there was no statistical difference among the proposed detection methods, the plate culture method yielded a higher number of L. pneumophila positive samples, followed by amoebic coculture procedure and direct fluorescent antibody assay. Results of PCR and sequencing reactions revealed that application of macrophage infectivity potentiator gene as a molecular marker was an important tool for the identification of environmental isolates of L.
    [Show full text]