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Chapter Outlines

Chapter 21      The Best for Last: Bacteriophages

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21.1 Bacteriophage Research History Bacteriophages - viruses that infect bacteria Discovered after recognition of bacterial hosts in the 1880's (the golden age of microbiology) Frederick W. Twort observed glassy transformation Used a Chamberland filter to discover the filterable "bacteriophages" that lysed cultures of micrococci His work was not acknowledged for 5 years Felix d'Herelle (1873-1849) Credited as the sole-discoverer of bacteriophages 1917 report by d'Herelle describes the lysis of dysentery-causing bacteria grown in liquid medium Tworts paper was not cited in d'Herrelle's publication Felix d'Herelle's Research 2 directions >Determining the biological nature of bacteriophages Exploring the use of bacteriophages as therapy to treat bacterial infections in a preantibiotic era d'Herelle and Bacteriophage Therapy 1919 field trials to control an epidemic of chicken typhoid caused by the bacterium Salmonella gallinarum Inoculated chickens either orally or by injection with bacteriophages Flocks treated with bacteriophages suffered fewer deaths and shorter epidemics that did not reoccur Positive results motivated d'Herelle to conduct human trials in the 1920's d'Herelle's Human Trials To prove that the bacteriophage preparations were safe: d'Herelle injected himself Family members Coworkers No harmful effects observed 1925 report by d'Herelle regarding experiments at the League of Nations quarantine station in Alexandria, Egypt Injected 4 patients suffering from laboratory confirmed bubonic plague with bacteriophages into the bubos present in their lymph nodes All 4 patients recovered rapidly After this success, d'Herelle traveled the world continuing bacteriophage therapy as a means of controlling cholera outbreaks d'Herelle's Career Accepted an appointment as a professor of protobiology at Yale University in 1928 Played a role in establishing a bacteriophage institute in Tbilisi, Soviet Georgia in 1934 This institute exists today as the Georgia Eliava Institute of Bacteriophage, Microbiology and Virology Bacteriophage Therapy Abandoned in the 1930's Published reports were not consistent The therapy appeared to be "hit or miss" The main reason for the abandonment of bacteriophage therapy was the development of antibiotics Today the Western world has a renewed interest in bacteriophage therapy to combat antibiotic-resistant strains of pathogenic bacteria The History of the Georgia Eliava Institute of Bacteriophage, Microbiology and Virology Founded in 1923 by Professor Giorgyi Eliava Felix d'Herelle visited Eliava at the Institute during 1934-1935 via invites by Josef Stalin Stalin was interested in bacteriophage therapy for military use Eliava was executed in 1937 d'Herelle did not return to the Institute afer his death Activities of the Georgia Eliava Institute Continued after Eliava's death Research focus centered around bacteriological and bacteriophage research The Institute change names in 1952 and was reorganized in 1988 The Institute manufactured bacteriophage sprays, salves, ointments and pills The Institute was damaged during the Georgian civil war. Thousands of bacteriophage samples were lost The Institute was revitalized by energetic entrepreneurs after a 1997 BBC Broadcast entitled The Virus That Cures The American Phage Group Formed in the 1940's Consisted of scientists from universities throughout the U.S. that were studying bacteria and bacteriophages Max Delbruck Salvador Luria Alfred Hershey The Phage Group spent summers doing research experiments at Cold Spring Harbor (Long Island, NY) Optimized experiments to study the biology of bacteriophages such as: One-step growth experiments Plaque assays Focused on a selected group of "authorized" bacteriophages such as the Type (T) bacteriophages 21.2 Bacteriophage Ecology Scientific community estimates that aquatic communities contain 4-6 X 1030 bacteria and 1 X 1031 bacteriophages Bacteriophages recycle bacterial carbon in the marine environment Marine microbial ecology is a rapidly developing field 21.3 The Biology of Bacteriophages: Composition and Structure Over 5,100 bacteriophages have been analyzed using the transmission electron microscope ICTV recognizes 1 order 13 families 31 genera Bacteriophage Structure 4 basic shapes or symmetries Binary (head and tail structure) Icosahedral (also called cubic) Helical (filamentous) Pleomorphic The majority of bacteriophages contain a head and tail structure Representatives of the 13 Bacteriophage Families. See Figure 21-3. Bacteriophage Structure and Genomes 3 families of bacteriophages are enveloped 96% of all bacteriophages contain dsDNA genomes The remaining 4% contain ssDNA, ssRNA or dsRNA genomes Genomes may code for as few as 3-5 genes to as many as 100 The genomes of bacteriophages contain unusual or modified bases that protect them from degradation by host nucleases during phage infection Most famous group of bacteriophages T-Type 21.4 Overview of Bacteriophage Infection: Bacteriophages Possess Alternative Lifestyles: Lytic vs Temperate Phages Bacteriophages adsorb to the surface receptor molecules of bacteria during the first step of infection Receptors may be: Pili Proteins Oligosaccharides Lipopolysaccharides (LPS) Adsorption and Penetration T4 bacteriophages anchors its tail fibers in the LPS layer of its bacterial host This adsorption step causes a conformational change, resulting in contraction of the tail sheath and penetration of the cell membrane and the bacteriophage The bacteriophage DNA genome is subsequently injected into the host cell through the tail tube Other Bacteriophages May bind to other receptors Bacteriophages that do not have tails penetrate the host by producing polysaccharide-degrading enzymes that digest components of the bacterial envelope or cell wall Resistance to Bacteriophage Infection Bacteria become resistant to phage infection when their host cell receptors are altered by mutation There is interest in engineering new receptor-recognition elements into the tail fibers of well-characterized bacteriophages so they can infect genetically distant hosts Genome Penetration Not an injection process Bacteriophages may package lysozyme in the base of its tail and uses the enzyme to degrade a portion of the peptidoglycan of the bacterial cell wall The DNA is drawn into the cell by a process that is not well understood for most bacteriophages After the DNA enters the cell, it circularizes rapidly by sticky ends or termini or the linear ends are modified and protected from bacterial nucleases Transcription and Translation are Coupled Bacterial host RNA polymerase recognizes the viral DNA promoters and begins transcription of early genes Translation of the early genes is coupled with transcription Late genes are transcribed and translated Assembly and Release Process: Lytic Infection After all bacteriophage "parts" are produced, new bacteriophages are assembled A copy of the genomic DNA is "reeled into" a preassembled icosahedral head A few molecules of lysozyme are packaged into the tail plate Phage lysin, endolysins, muramidases or virolysins hydrolyze bonds in the murein or peptidoglycan of the cell wall, allowing the viruses to escape or be released Holin is used to create pores in the inner membrane of the host, allwing the lysin or other enzymes to facilitate bacteriophage release Lysogenic (Temperate) Infections These bacteriophages infect their hosts but do not kill them. Instead their genome becomes integrated into a specific region of the host chromosome The integrated bacteriophage genome is called a prophage The viral DNA replicates every time the cell copies its chromosomal DNA during cell division Some temperate phages encode transposase which allows the bacteriophage to insert randomly into the chromosome Other bacteriophages integrate into site specific locations within the chromosome All bacteriophage gene expression is repressed by a repressor protein Derepression or Induction If the repressor loses function (becomes inactivated), viral DNA is excised from the bacterial chromosome and the excised DNA acts like a lytic virus Spontaneous derepression or induction happens about 1 in every 10,000 cell divisions Temperate bacteriophages can carry host genes from one bacterial cell to another in a process called transduction 21.5 Bacteriophages Create Pathogenic Bacteria in Nature Lysogenic conversion - occurs when a bacteriophage alters the phenotype of a lysogenic bacterium e.g. Corynebacterium diphtheriae produces a toxin responsible for diphtheria only if it carries the temperate bacteriophage called b The tox gene is located near one end of the phage genome Other Examples of Lysogenic Conversion If Streptococcus pyogenes contains a temperate bacteriophage T2, the bacterium changes to a pyrogenic or erythrogenic exotoxin producing bacterium This exotoxin causes the rash of scarlet fever Examples of Virulence Factors Carried on Bacteriophages. See Table 21.1 21.6 Control of Bacteriophages in Industrial Fermentations Bacteria are used in a variety of food fermentation processes: Yogurt Cheese Sauerkraut Soy sauce Pharmaceutical and biotechnology industries use bacteria on a large scale to produce products such as: Alcohols Vitamins Amino acids Enzymes Hormones Biopolymers Antibiotics Other therapeutics Bacteriophages are a Threat to Fermentation and Pharmaceutical Industries Attacks by bacteriophages can results in considerable economic losses About 1-10% of dairy product fermentation batches are lost to bacteriophage infection Often happens because the raw starting materials are contaminated with undetectable numbers of bacteriophages Laboratory staff are trained to expect and respond to bacteriophage outbreaks Bacteriophages are ubiquitous Bacteriophage "seasons" are January to March and October to November 21.7 Biofilms and Bacteriophages Bacteria form complex communities called biofilms on solid wet surfaces in natures such as River rocks Walls of limestone caves Pipes Industrial equipment Hulls of ships Teeth Lining of the colon Sutures Lungs of Cystic Fibrosis Patients Medical devices such as catheters, heart valves Surrounding tissues of the heart Orthopedic devices Facial implants Biofilms may consist of a single bacterial species to hundreds or thousands of bacterial species The bacteria produce extracellular polysaccharide polymers that surround and encase the microbes, facilitating their adhesion to surfaces Biofilms can cause environmental problems Biofilms Can Cause Chronic Bacterial Infections in Humans Biofilms are a challenge to medical care Bacteria commonly isolated from medical devices: Enterococcus faecalis S. aureus S. epidermidis Streptococcus viridans E. coli Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosa These bacteria originate from the skin of the patient or the healthcare workers, entry ports of catheters etc. Preventative strategies to treat biofilms on medical devices: Application of silver-impregnated catheters Coating devices with antibiotics Disinfection at the surface Adding inline filters into the ports of catheters Bacteriophages studies to pretreat catheter surfaces to control biofilms are promising 21.8 FDA-Approved Listeria-Specific Bacteriophage Preparation on Ready-to-Eat Meats Listeriosis is a foodborne illness caused by the bacterium Listeria monocytogenes Listeriosis occurs in pregnant women (can cause a miscarriage, stillbirth or premature delivery), newborns and people with weakened immune systems 2006 - FDA approved LISTEX P100 which is a cocktail of 6 bacteriophages in the form of an application spray LISTEX P100 LISTEX P100 will be used in clean rooms of cheese, meat, and poultry processing plants to inhibit the growth of Listeria on products such as lunch meat and hot dogs LISTEX P100 will not be declared as an ingredient on the label of a treated product