Biorisk An Engineering Safety Module Prepared by Valentin

Biorisk An Engineering Safety Module Prepared by Valentin Malenkov Reviewed by Prof. Marc Aucoin Sponsored by: MINERVA (www. safetymanagementeducation. com/) and MITACS Chapter 2: Microorganisms, Pathogens, and Toxins

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Module Outline 2 Chapter 1: Introduction to Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Chapter 3: Regulation of Biohazardous Materials and Risk Management Systems Chapter 4: Risk Assessments, Risk Groups, and Containment Levels Chapter 5: Biohazardous Material Containment

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Learning Objectives 3 1. Know the basic characteristics which define pathogenic microorganisms 2. Be able to differentiate between a pathogen and a toxin 3. Understand the factors affecting a pathogen’s handling requirements 4. Know the factors considered when determining the risk a microorganism poses 5. Understand the basics of recombinant creation and related risk assessments

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Introduction to Microorganisms 4 • Microorganisms are all around us! – – • Method of replication, physical and chemical properties, etc all vary – • covered in detail later for pathogenic agents Most are benign, some are beneficial, a few are dangerous – • Invisible to naked eye Coat most surfaces on Earth Human digestive tract contains over 128 types of bacteria aiding in digestion[1] When are microorganisms pathogenic? Image [1]

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Pathogens: Introduction 5 Pathogen: biological agent that causes disease or infection to its host [2] – Not necessarily microorganism – Must be infectious • Main pathogen categories: – – – Bacteria Viruses Parasites Fungi Prions Fun Fact: There are more bacterial cells in your body than there are human cells and more microorganisms in your body than there are people on earth!

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Pathogens: Knowledge is Power 6 q Pathogen characteristics inform handling requirements q Know your pathogens! q q Biological classification? Storage and replication method? Threat to life and environment? Treatment and decontamination?

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Pathogenicity 7 • Pathogenicity: threat level of a pathogen • Measures of pathogenicity – Incidence: incidents from pathogen/population/time – Attack rate: individuals infected/exposed – Death Rate: individuals killed/infected – Population Specific Mortality: death rate for specific population • Overarching properties defined by pathogen characteristics

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Pathogen Communicability 8 Communicability: the ability of a pathogen to spread from one host to another – – Often major factor in pathogenicity Defined by 5 factors 1. Transmission type – Direct Contact • • – Casual (ex. shaking hands) Intimate (ex. sexual intercourse, kissing) Indirect Contact • Fomites (inanimate object/substance carrying pathogen) – • Clothing, skin cells, bed sheets Vectors (living organism carrying pathogen) – – Mosquitoes, ticks, fleas Must infect host directly, not simply carry pathogen

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Pathogen Communicability (cont’d) 9 2. Route of Infection – – – Inhalation: airborne or aerosol inhaled Mucus Membrane or Skin Contact Ingestion Injection: direct infection into tissue (needle, bite) Zoonoses: animal hosts and carriers 3. Infectious Dose – – Amount of initial inoculum required for infection Expressed in amount of pathogen causing infection in 50% of those exposed (Cl 50)

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Pathogen Communicability (cont’d) 10 4. Host Range – Variety of hosts more difficult to control – More possible transmission events 5. Natural Distribution – Presence or lack of pathogen/hosts in natural environment – May provide information on pathogenicity

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Incidence of Disease 11 Image [2] [1] http: //www. health. gov. on. ca/en/publications/disease/listeria_2008. aspx

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Incidence of Disease 12 • Defines level of pathogen-associated disease in the environment – Above-normal incidence of disease – Defined by size of area involved • Outbreak – Small area or single population (single city, school system) – Ex: 2008 Listeriosis in Canada from processed meat[3]

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Incidence of Disease (cont’d) 13 • Epidemic – Larger area (whole country) – Ex. 1974 Smallpox outbreak in India[4], yearly flu epidemics • Pandemic – Globe-spanning (continent, hemisphere, or whole world) – Ex. Worldwide HIV/AIDS pandemic, 2009 H 1 N 1 pandemic[5]

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Chain of Infection 14 • Alternate look at pathogen properties – Describes conditions required for transmission • Focus on breaking the chain; prevention 6 links, each with possible infection control 1. Causative agent: The microorganism itself • Eliminate organisms/replace with non-pathogenic 2. Reservoir: Original host/source of microorganism • Early detection of infection, remove possible reservoirs

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Chain of Infection (cont’d) 15 3. Portal of Exit: path for leaving original reservoir • Prevent exit (face mask), decontaminate by-products (waste, tissues, etc) 4. Mode of transmission: method of transportation to new reservoir • Containment in place, filter carriers of pathogens (air, water) 5. Portal of entry: method for entering new host • Protect with PPE and SOPs 6. Susceptible host: availability of new reservoir • Separate viable hosts, provide vaccines, or use nonhuman diseases

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Basics of Pathogen Biology

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Prokaryotic vs. Eukaryotic[6] 17 • Basic classification of cell structure • Prokaryotes – – – Small (typically ~2µm diameter) No membrane-bound organelles DNA circular, not nucleus-enclosed Peptidoglycan cell walls* Most wide-spread life-forms on Earth • • Chemoautotrophic bacteria can be found in deepsea geysers [7] Bacteria and archea can be found in Antarctic ice[8] *with some exceptions such as mycoplasma

Biorisk 18 Chapter 2: Microorganisms, Pathogens, and Toxins Prokaryotic vs Eukaryotic (cont’d) • Eukaryotes – – 5 -20µm in diameter (on average) Membrane-bound organelles Linear DNA in multiple chromosomes Non-peptidoglycan cell walls, or none • Added complexity in multi-cellular organisms – Specialized cells – Tissues and organs Image [4] Image [3]

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Bacteria 19 • Present in every habitat on earth – Free-living or obligate intracellular – Sporulation to survive extreme conditions • • Very durable (80 o. C, radiation, drying, chemicals) Saves DNA to replicate when conditions improve • Basic properties – – Prokaryotic cells (unicellular) 0. 2 -2µm diameter, 2 -8µm length Stationary or motile Reproduce by binary fission • Genetic info sharing possible - Conjugation

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Bacteria (cont’d) 20 • Bacteria as pathogens – Transmission by ingestion, contact, through abrasion, and via animal vectors Can survive a long time outside body – • – Symptoms result from bacterial lifecycle • – Toxic excretions (Ex. Botulinum toxin) Treated with antibiotics* • • Spores survive almost indefinitely Resistant strains from mutation/conjugation Bacterial diseases include Anthrax, Lyme, Typhus, Syphilis, Plague – – Severe but fairly rare Treatable at early stages *organism not toxin

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Aside: Mycoplasmas 21 • Small bacteria lacking cell walls – – – • Cause diseases like Pneumonia and linked to cancer[9] – – – • Smallest known self-replicating cells (~100 nm in diameter) Variable shape Form filaments Attaches to host cell membrane Alters cell structure and genetics[10] Causes cell detachment Unaffected by common antibiotics

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Viruses 22 • Non-living biological entities – Acellular entities which cannot self-replicate – ~30 -400 nm in size – Typically parasitic • Can infect all living organisms – Plants, animals, fungi, bacteria – Adept at bypassing immune response • • Silence host cell signaling Prevent “cell suicide”

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Viruses (cont’d) 23 • • Viral structure varied Nucleic acids in protein core – – • RNA or DNA Structural protein “capsid” Additional structures for entry/attachment – – • Tail fibers (bacteriophages) Membranes with glycoproteins (animal viruses) Host cell organelles and even DNA altered – – Hijack host cell mechanisms Latent infections

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Viruses (cont’d 2) 24 • Wide range of transmission pathways – Small enough to become airborne – Direct contact of mucous membranes – Indirect transmission by vectors • Treated with anti-virals – – Target viral protein Specific to viral lifecycle step Inhibits lifecycle progression Latent infections can still occur • Immunization as preventative measure

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Parasites 25 • Covers wide range of organisms – – – • Single-celled to visible to naked eye Pathogenic or pathogen vector Only includes living organisms (viruses separate) General Characteristics: – – Draw nutrients from host to survive Provides no benefits to host Able to self-replicate Mostly spread by direct and indirect contact • • • Fecal-oral route Fomites (water, soil) Vectors (mosquitoes, ticks)

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Protozoa 26 • Single-celled eukaryotic organisms – Motile with help of microstructures • • – – • Cilia Flagella Mostly free-living (if not parasitic) Sexual or asexual reproduction Inhabits aquatic environments – – – Required for motility Found inside hosts (blood stream, organs) Form dormant “cysts” in harsh environments • • Resistant to high temperature and chemicals Not a modern classification – Image [5] Genetic and biochemical variability

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Protozoa (cont’d) 27 • • Spread by direct/indirect contact and vectors Diseases caused by Protozoa: – – – • Malaria Toxoplasmosis Amoebiasis Chagas Disease Sleeping Sickness Treated with specialized compounds – – – Low similarity between protozoa Resistance build-up Use of single compound strongly discouraged

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Helminthes[11] 28 • Multi-cellular worm-like parasites – – Visible to the naked eye Disrupt host nutrient absorption Many do not replicate in humans Varied characteristics • Spread by contact with eggs – – Protective shells Remain viable for months Found in soil, water, faeces Indirect contact through vectors

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Helminthes (cont’d) 29 • High prevalence in affected populations – Developing regions of Africa, Asia, Latin America – >800 million cases globally[11] – Exceeds Malaria and Tuberculosis[11] • Diseases caused by Helminthes – Onchocerciasis, “river blindness” – Schictosomiasis, “snail fever” – Filariasis, can cause Elephantiasis

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Helminthes (cont’d 2) 30 • Treatment with invertebrate-targeting drugs – Anthelmintics • Inhibition of helminth enzymes – Avermectin • Blocks transmittance in nerve and muscle cells of parasite • No human vaccines available[12, 13] – Successful livestock vaccination – Helminthes highly immunomodulatory • Influences immune system to survive

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Ectoparasites 31 • Parasites which live on or in the skin – Feed on blood of host – Can stay attached for weeks • Generally arthropods – Invertebrates with exoskeletons – Visible to the naked eye – Mites, ticks, lice • Act as vectors for pathogens – Difficult to control indirect contact

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Fungi 32 • Simple eukaryotes – Many form filaments, “hyphae” (not yeasts) • • – – Cell walls of cellulose of chitin Sexual and asexual reproduction • • Form networks “mushrooms” and other external features for reproduction Spread by spores Many saprophoric – – – Gain nutrients from dead organic matter External digestion with enzymes Only require light for spore production Image [6]

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Fungi (cont’d) 33 • Common crop pathogen – – • Cause of severe crop damage (potato famine[14]) Monocultures vulnerable Can infect animals (infections called Mycoses) – Mostly opportunistic pathogens • • • – Cuts and abrasions Inhalation Immunocompromised individuals Levels of mycoses • • • Cutaneous: skin’s surface, nails, hair Subcutaneous: under skin and adjacent tissue (through cuts) Systemic: internal organs affected

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Fungi (cont’d 2) 34 • Treatment with antifungal medication – Polyenes: changes membrane structure, killing cells – Azoles: inhibit essential enzyme – Echinocandins: inhibit synthesis of cell walls • Over 300 known pathogenic fungi[15] – Ringworm (common skin infection) – Many infect lungs

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Prions 35 • Infectious protein agents – No nucleic acid – Misfolded version of host proteins – Resistant to inactivation (heating and enzyme) • Cause neurodegenerative diseases – Propagate misfolding of proteins – Can be acquired or genetic – Scrapie, Mad Cow Disease, fatal familial insomnia

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Prions (cont’d) 36 • No known treatment – Symptom relief only – Cannot replace protein • Transmission routes not well understood – Likely through contact – Pathogenicity not well understood • Very recently discovered – Discovered in 1980’s – 1997 Nobel Prize, Stanley B Prusiner[11]

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Viroids and Virusoids 37 • Two very small virus-like pathogen groups – – • Both consist of single circular strand of RNA Effects unique to each member Viroids – – • Infect plants causing disease Replicated in nucleus No proteins coded Potato spindle viroid Virusoids – Require helper virus for replication • – – Coated with helper virus protein Code for one or more gene product Hepatitis D virusoid (uses Hepatitis B)

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Cell Culture 38 • • Can harbour infectious agents Grown for research and industrial applications – – – • Maintained for experiments Used in bio-manufacturing Mammalian, insect, fungal (yeast) Factors affecting risk – – Source of cell line Source tissue Cell type Source population Image [7]

Biorisk Toxins Chapter 2: Microorganisms, Pathogens, and Toxins

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins What is a Toxin? 40 • Substance produced by a microorganism harmful to other organisms – – – • Biological molecules Non-replicating and non-infectious Most not volatile Common effects and symptoms – – Acute: fever, chills, rashes, aches, vomiting, shock, death, weakness Long-term: organ damage/failure, chronic arthritis, brain damage, nerve damage, musculoskeletal effects

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Microbial Toxins: Endotoxin vs Exotoxins 41 • Endotoxins: non-secreted cell-associated toxin – Almost all from Gram-negative bacteria • – – Released on lysis No specific target of toxicity • • Found in outer layer of cell wall Damage near site of colony Exotoxins – – – Secreted by microorganism Wider range of pathogens Targets specific cell types/tissue away from colony

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Factors for Toxin Containment 42 • Routes of exposure – • Concentration/Volume – • Facility and application dependant Toxicity – • LD 50, or lethal dose for 50% of the population Rate of action – • Compare toxin action time to response time in emergency Severity of symptoms – • Pathogen or application-specific Acute and long term effects Availability of anti-sera

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Toxin Production 43 • Main reason for pathogenicity of certain infectious agents – Bacteria biggest producer • Most potent toxins known – Fungal toxins, “Mycotoxins” • Exposure from contaminated crops – Parasites’ byproducts toxic • Natural waste excreted • Viruses, prions, virouds, and virusoids produce no toxins

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Recombinant Organisms and Biosafety

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Recombinants 45 • Organism or gene vector modified by genetic recombination – New gene introduced or old gene modified – Joining nucleic acid molecules • Pathogens modification may increase risk – Changes in behaviour • • • Host range expanded Resistance to treatment Increased virulence – Changes can be unpredictable

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Genetic Engineering 46 • Controlled/purposeful manipulation of nucleic acid – – Applicable to all cells Alters cell metabolism (reactions occurring in the cell) • • • Increase/decrease of native metabolites Addition of novel metabolite(s) Modified cells carefully selected – Contain required cellular machinery • • – – Gene translation Metabolite production/modification Robust and easily propagated Manipulation can create strain

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Genetic Engineering (cont’d) 47 • • Direct injection possible Gene vector: Particle for introducing genetic material into cells – – • Required for gene delivery Bypasses cell barriers Plasmids: circular DNA sequence inserted directly – – • Cell DNA unaltered Non-permanent modification Viral Vectors: Modified virus with gene of interest – – Can integrate gene into cell DNA Can be more permanent

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Working with Recombinants 48 • Start with unmodified organisms’ risk factors – – • Organism modified Source of genetic information Additional Considerations – Source of virulence modified? • – Pharmacological activity change? • – Drug resistance Adverse genetic information added? • – – Toxin, infectivity, motility, etc Gene coding for toxin Is a novel mechanism added? Can modified cells replicate?

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins References [1] livescience (2006) Bacteria Thrive in Hostile Human Bellies. Accessed at http: //www. livescience. com/515 bacteria-thrive-hostile-human-bellies. html [2] Science Daily, Pathogens. Accessed at http: //www. sciencedaily. com/articles/p/pathogen. htm [3] http: //www. health. gov. on. ca/en/publications/disease/listeria_2008. aspx [4] http: //www. nmji. in/archives/Volume_19_1_january_february 2006/SPECIAL_SERIES/Special_Series. htm [5] http: //www. flu. gov/about_the_flu/h 1 n 1/ [6] Sharma, Dushyant Kumar (2013) Microbiology. Alpha Science International, Oxford [7] Extreme. Science (2013) Deep Sea Hydrothermal Vents. http: //www. extremescience. com/deep-sea-vents. htm [8] Brent C. Christner et al (2014) A microbial ecosystem beneath the West Antarctic ice sheet. Nature, Volume 512, accessed at http: //www. nature. com/nature/journal/v 512/n 7514/full/nature 13667. html [9] Tsai S et al (1995). Mycoplasmas and oncogenesis: Persistent infection and multistage malignant transformation. Proc Nat Acad Sci USA 92 (22) [10] Sinkovics JG (2012) Molecular biology of oncogenic inflammatory processes. I. Non-oncogenic and oncogenic pathogens, intrinsic inflammatory reactions without pathogens, and micro. RNA/DNA interactions (Review)". International Journal of Oncology 40 (2): 305– 349 [11] Hotez, Peter et al. (2008) Helminth infections: the great neglected tropical diseases. J Clin Invest. v. 118, pg 1311 -1321 [12] Hewitson, James; Maizels, Rick (2014) Vaccination against helminth parasite infections. Expert Reviews of Vaccines online, pg 1 -15

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins References [13] Maizels, RM et al. (1999) Vaccination against helminth parasites--the ultimate challenge for vaccinologists? . Immunology Review, v. 171 pg 125 -147 [14] The History Place (2000) Irish Potatoe Famine – The Blight Begins. http: //www. historyplace. com/worldhistory/famine/begins. htm [15] Garcia-Solache MA, Casadevall A. Global warming will bring new fungal diseases for mammals. m. Bio 2010; 1.

Biorisk Chapter 2: Microorganisms, Pathogens, and Toxins Attribution for images used 51 1. 2. 3. 4. 5. 6. 7. “This 2005 scanning electron micrograph (SEM) depicted numerous clumps of methicillin-resistant Staphylococcus aureus bacteria, commonly referred to by the acronym, MRSA; Magnified 2381 x. ” Authors: Jeff Hageman, Janice Haney Carr. This image is a work of the Centers for Disease Control and Prevention, part of the United States Department of Health and Human Services, taken or made as part of an employee's official duties. As a work of the U. S. federal government, the image is in the public domain. Original source: his media comes from the Centers for Disease Control and Prevention's Public Health Image Library (PHIL), with identification number #10048 “A Board of Health quarantine poster warning that the premises are contaminated by smallpox”. Author: Buckley & Curtin. This work is in the public domain in the United States because it is a work prepared by an officer or employee of the United States Government as part of that person’s official duties under the terms of Title 17, Chapter 1, Section 105 of the US Code. Original source: Courtesy of the National Library of Medicine. Images from the History of Medicine (IHM), http: //ihm. nlm. nih. gov/images/A 21137. Also showcased in the book: Hidden Treasure (New York, NY: Blast Books, 2012), p. 178. HMD Prints & Photos call number: WA 234 C 25 no. 14 sol “Acanthamoeba spp. ”. Author and source: http: //enfo. agt. bme. hu/drupal/en/node/11277. This file is licensed under the Creative Commons Attribution-Share Alike 4. 0 International license. “White whales performing at the Moscow Zoo dolphinarium”. Authors: Iliya Pitalev and Илья Питалев. This image was provided to Wikimedia Commons by Russian International News Agency (RIA Novosti) (rian. ru and visualrian. ru) as part of a cooperation project. Original source: RIA Novosti archive, image #374827, http: //visualrian. ru/ru/site/gallery/#374827 “Dileptus ciliate, collected from a natural pond in Wakefield, Quebec”. Author: Deuterostome. This file is licensed under the Creative Commons Attribution-Share Alike 3. 0 Unported license. Original source: https: //commons. wikimedia. org/wiki/File: Dileptus. jpg “Flora Batava. Afbeelding en beschrijving der Nederlandsche Gewassen. (1898)”. This work is in the public domain in its country of origin and other countries and areas where the copyright term is the author's life plus 100 years or less. Permission is granted to copy, distribute and/or modify this document under the terms of the GNU Free Documentation License, Version 1. 2 or any later version published by the Free Software Foundation; with no Invariant Sections, no Front -Cover Texts, and no Back-Cover Texts. A copy of the license is included in the section entitled GNU Free Documentation License “Cho cells adherend 2”. Author: Wikimedia. com User: Alcibiades. “I, the copyright holder of this work, release this work into the public domain. This applies worldwide” (https: //commons. wikimedia. org/wiki/File: Cho_cells_adherend 2. jpg)

Biorisk An Engineering Safety Module Prepared by Valentin Malenkov Reviewed by Prof. Marc Aucoin Sponsored by: MINERVA (www. safetymanagementeducation. com/) and MITACS Chapter 2: Quiz

Biorisk Chapter 2 1. True of false? All pathogens are infectious living organisms. 2. What are the four main measures of pathogenicity? 3. If you catch an infection from sleeping in a bed in which a sick person slept, what form of contact was involved? 4. True or False: If a plant pathogen is released in an area rich with the plants it infects, the host range was the main issue. 5. True or false: bacteria are mostly harmless. 6. Can a bacteria infect a virus? 7. Can a virus be caught via inhalation in a perfectly dry room? 53

Biorisk Chapter 2 8. Can protozoa be found only in the bodies of other organisms? 9. True or false: Helminth infections are found only in developing countries 10. What is the technical term for a fungal filament structure 11. What is the main method of reproduction used by fungi? 12. What treatment is used for infections caused by prions? 13. True or false: Insect and mammalian cell lines are not pathogenic to humans. 14. What type of toxin is excreted by a pathogen rather than being cell-associated? 15. Of the pathogen groups discussed, which are known to produce toxins? 16. A bacterial culture has been barraged by antibiotics and a resistant mutant strain isolated. Is this new strain a recombinant organism?