Microbiology with Diseases by Body System Robert W. Bauman Third Edition

Transcription

1 Microbiology with Diseases by Body System Robert W. Bauman Third Edition

2 Pearson Education Limited Edinburgh Gate Harlow Essex CM20 2JE England and Associated Companies throughout the world Visit us on the World Wide Web at: Pearson Education Limited 2014 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without either the prior written permission of the publisher or a licence permitting restricted copying in the United Kingdom issued by the Copyright Licensing Agency Ltd, Saffron House, 6 10 Kirby Street, London EC1N 8TS. All trademarks used herein are the property of their respective owners. The use of any trademark in this text does not vest in the author or publisher any trademark ownership rights in such trademarks, nor does the use of such trademarks imply any affiliation with or endorsement of this book by such owners. ISBN 10: ISBN 13: British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Printed in the United States of America

3 CRITICAL THINKING Why is liquid water necessary for microbial metabolism? Jonathon Blair/Corbis Figure 8 The use of desiccation as a means of preserving apricots in Pakistan. In this time-honored practice, drying inhibits microbial growth in the food by removing the water that microbes need for metabolism. most pathogens, including the bacteria that cause syphilis, gonorrhea, and the more common forms of bacterial pneumonia and diarrhea. However, most molds can grow on dried raisins and apricots, which have as little as 16% water content. Scientists use lyophilization (lī-of i-li-zā shŭn), a technique combining freezing and drying, to preserve microbes and other cells for many years. In this process, scientists instantly freeze a culture in liquid nitrogen or frozen carbon dioxide (dry ice); then, they subject it to a vacuum that removes frozen water through a process called sublimation, in which the water is transformed directly from a solid to a gas. Lyophilization prevents the formation of large, damaging ice crystals. Although not all cells survive, enough are viable to enable the culture to be reconstituted many years later. Filtration Describe the use of filters for disinfection and sterilization. Filtration is the passage of a fluid (either a liquid or a gas) through a sieve designed to trap particles in this case, cells or viruses and separate them from the fluid. Researchers often use a vacuum to assist the movement of fluid through the filter (Figure 9a). Filtration traps microbes larger than the pore size, allowing smaller microbes to pass through. In the late 1800s, filters were able to trap cells, but their pores were too large to trap the pathogens of such diseases as rabies and measles. These pathogens were thus named filterable viruses, which today has been shortened to viruses. 9 Now, filters with pores small enough to trap even viruses are available, so filtration can be used to sterilize such heat-sensitive materials as ophthalmic solutions, antibiotics, vaccines, liquid vitamins, enzymes, and culture media. Over the years, filters have been constructed from porcelain, glass, cotton, asbestos, and diatomaceous earth, a substance composed of the innumerable glasslike cell walls of single-celled algae called diatoms. Scientists today typically use thin (only 0.1 mm thick), circular membrane filters manufactured of nitrocellulose or plastic and containing specific pore sizes ranging from 25 µm to less than 0.01 µm in diameter (Figure 9b). The pores of the latter filters are small enough to trap small viruses and even some large protein molecules. Microbiologists also use filtration to estimate the number of microbes in a fluid by counting 9 Latin, meaning poisons. Figure 9 Filtration equipment used for microbial control. (a) Assembly for sterilization by vacuum filtration. (b) Membrane filters composed of various substances and with pores of various sizes can be used to trap diverse microbes, here bacteria known as spirochetes. Nonsterile medium Membrane filter To vacuum pump J.B. Woolsey Associates, LLC (a) Sterile medium (b) SEM 1 µm Science Source/Photo Researchers 273

4 Exhaust HEPA filter Blower Supply HEPA filter Light High-velocity air barrier the number deposited on the filter after passing a given volume through the filter. Table 3 lists some pore sizes of membrane filters and the microbes they allow through. Health care and laboratory workers routinely use filtration to prevent airborne contamination by microbes. Medical personnel wear surgical masks to prevent exhaled microbes from contaminating the environment, and cotton plugs are placed in culture vessels to prevent contamination by airborne microbes. Additionally, high-efficiency particulate air (HEPA) filters are crucial parts of biological safety cabinets (Figure 10), and HEPA filters are mounted in the air ducts of some operating rooms, rooms occupied by patients with airborne diseases such as tuberculosis, and rooms of immunocompromised patients such as burn victims and AIDS patients. CRITICAL THINKING Outside Safety glass viewscreen Figure 10 The roles of high-efficiency particulate air (HEPA) filters in biological safety cabinets. HEPA filters protect workers from exposure to microbes (by maintaining a barrier of filtered air across the opening of the cabinet). Hospital also use HEPA filters in air ducts of operating rooms and of the rooms of highly contagious or immunocompromised patients. A virologist needs to remove all bacteria from a solution containing viruses without removing the viruses. What size membrane filter should the scientist use? J.B. Woolsey Associates, LLC/Precision Graphics TABLE 3 Membrane Filters Pore Size (µm) Smallest Microbes That Are Trapped 5 Multicellular algae, animals, and fungi 3 Yeasts and larger unicellular algae 1.2 Protozoa and small unicellular algae 0.45 Largest bacteria 0.22 Largest viruses and most bacteria Larger viruses and pliable bacteria (mycoplasmas, rickettsias, chlamydias, and some spirochetes) 0.01 Smallest viruses Osmotic Pressure Discuss the use of hypertonic solutions in microbial control. Another ancient method of microbial control is the use of high concentrations of salt or sugar in foods to inhibit microbial growth by osmotic pressure. Osmosis is the net movement of water across a semipermeable membrane (such as a cytoplasmic membrane) from an area of higher water concentration to an area of lower water concentration. Cells in a hypertonic solution of salt or sugar lose water, and the cell desiccates. The removal of water inhibits cellular metabolism because enzymes are fully functional only in aqueous environments. Thus, osmosis preserves honey, jerky, jams, jellies, salted fish, and some types of pickles from most microbial attacks. Fungi have a greater ability than bacteria to tolerate hypertonic environments with little moisture, which explains why jelly in your refrigerator may grow a colony of Penicillium (pen-i-sil ē-ŭm) mold but is not likely to grow the bacterium Salmonella. Radiation Differentiate ionizing radiation from nonionizing radiation as they relate to microbial control. Another physical method of microbial control is the use of radiation. There are two types of radiation: particulate radiation and electromagnetic radiation. Particulate radiation consists of high-speed subatomic particles, such as protons, that have been freed from their atoms. Electromagnetic radiation can be defined as energy without mass traveling in waves at the speed of light ( 3 * 10 5 km/sec ). Electromagnetic energy is released from atoms that have undergone internal changes. The wavelength of electromagnetic radiation, defined as the distance between two crests of a wave, ranges from very short gamma rays, through X rays, ultraviolet light, and visible light, to long infrared rays, and finally to very long radio waves. Though they are particles, electrons also have a wave nature, with wavelengths that are even shorter than gamma rays. 274

5 The shorter the wavelength of an electromagnetic wave, the more energy it carries; therefore, shorter-wavelength radiation is more suitable for microbial control than longer-wavelength radiation, which carries less energy and is less penetrating. Scientists describe all types of radiation as either ionizing or nonionizing according to its effects on the chemicals within cells. Ionizing Radiation Electron beams, gamma rays, and X rays, all of which have wavelengths shorter than 1 nm, are ionizing radiation because when they strike molecules, they have sufficient energy to eject electrons from atoms, creating ions. Such ions disrupt hydrogen bonding, oxidize double covalent bonds, and create highly reactive hydroxyl radicals. These ions in turn denature other molecules, particularly DNA, causing fatal mutations and cell death. Electron beams are produced by cathode ray machines. Electron beams are highly energetic and therefore very effective in killing microbes in just a few seconds, but they cannot sterilize thick objects or objects coated with large amounts of organic matter. They are used to sterilize spices, meats, microbiological plastic ware, and dental and medical supplies such as gloves, syringes, and suturing material. Gamma rays, which are emitted by some radioactive elements such as radioactive cobalt, penetrate much farther than electron beams but require hours to kill microbes. The U.S. Food and Drug Administration (FDA) has approved the use of gamma irradiation for microbial control in meats, spices, and fresh fruits and vegetables (Figure 11). Irradiation with gamma rays kills not only microbes but also the larvae and eggs of insects; it also kills the cells of fruits and vegetables, preventing both microbial spoilage and overripening. Consumers have been reluctant to accept irradiated food. A number of reasons have been cited, including fear that radiation makes food radioactive and claims that it changes the taste and nutritive value of foods or produces potentially carcinogenic (cancer-causing) chemicals. Supporters of irradiation reply that gamma radiation passes through food and cannot make it radioactive any more than a dental X ray produces radioactive teeth, and they cite numerous studies that conclude that irradiated foods are tasty, nutritious, and safe. X rays travel the farthest through matter, but they have less energy than gamma rays and require a prohibitive amount of time to make them practical for microbial control. Nonionizing Radiation Electromagnetic radiation with a wavelength greater than 1 nm does not have enough energy to force electrons out of orbit, so it is nonionizing radiation. However, such radiation does contain enough energy to excite electrons and cause them to make new covalent bonds, which can affect the three-dimensional structure of proteins and nucleic acids. Ultraviolet (UV) light, visible light, infrared radiation, and radio waves are nonionizing radiation. Of these, only UV light has sufficient energy to be a practical antimicrobial agent. Visible light and microwaves (radio waves of extremely short wavelength) have little value in microbial control, though microwaves Non-irradiated heat food, inhibiting microbial growth and reproduction if the food gets hot enough. UV light with a wavelength of 260 nm is specifically absorbed by adjacent pyrimidine nucleotide bases in DNA, causing them to form covalent bonds with each other rather than forming hydrogen bonds with bases in the complementary DNA strand. Such pyrimidine dimers distort the shape of DNA, making it impossible for the cell to accurately transcribe or replicate its genetic material. If dimers remain uncorrected, an affected cell may die. The effectiveness of UV irradiation is tempered by the fact that UV light does not penetrate well. UV light is therefore suitable primarily for disinfecting air, transparent fluids, and the surfaces of objects such as barber s shears and operating tables. Some cities use UV irradiation in sewage treatment. By passing wastewater past banks of UV lights, they reduce the number of bacteria without using chlorine, which might damage the environment. Table 4 summarizes the physical methods of microbial control discussed in the previous pages. CRITICAL THINKING Irradiated Figure 11 A demonstration of the increased shelf life of food achieved by ionizing radiation. The circular radura symbol is used in the United States to label irradiated foods. During the fall 2001 bioterrorist attack in which anthrax endospores were sent through the mail, one news commentator suggested that people should iron all their incoming mail with a regular household iron as a means of destroying endospores. Would you agree that this is a good way to disinfect mail? Explain your answer. Which disinfectant methods would be both more effective and more practical? Richard Megna/Fundamental Photographs 275

6 4 Physical Methods of Microbial Control TABLE Method Conditions Action Representative Use(s) Moist heat Boiling Autoclaving (pressure cooking) 10 min at 100 C Denatures proteins and 15 min at 121 C Denatures proteins and Pasteurization 15 sec at 72 C Denatures proteins and Ultrahigh-temperature sterilization 1 3 sec at 140 C Denatures proteins and Disinfection of baby bottles and sanitization of restaurant cookware and tableware Autoclave: sterilization of medical and laboratory supplies that can tolerate heat and moisture; pressure cooker: sterilization of canned food Destruction of all pathogens and most spoilage microbes in dairy products, fruit juices, beer, and wine Sterilization of dairy products Dry heat Hot air Incineration 2 h at 160 C or 1 h at 171 C 1 sec at more than 1000 C Denatures proteins, destroys membranes, oxidizes metabolic compounds Oxidizes everything completely Sterilization of water-sensitive materials such as powders, oils, and metals Sterilization of inoculating loops, flammable contaminated medical waste, and diseased carcasses Refrigeration 0 7 C Inhibits metabolism Preservation of food Freezing Inhibits metabolism Long-term preservation of foods, drugs, and cultures Desiccation (drying) Lyophilization (freeze drying) Varies with amount of water to be removed -196 C for a few minutes while drying Inhibits metabolism Inhibits metabolism Filtration Filter retains microbes Physically separates microbes from air and liquids Osmotic pressure Ionizing radiation (electron beams, gamma rays, X rays) Nonionizing radiation (ultraviolet light) Exposure to hypertonic solutions Seconds to hours of exposure (depending on wavelength of radiation) Irradiation with 260-nmwavelength radiation Inhibits metabolism Destroys DNA Formation of thymine dimers inhibits DNA transcription and replication Preservation of food Long-term storage of bacterial cultures Sterilization of air and heat-sensitive ophthalmic and enzymatic solutions, vaccines, and antibiotics Preservation of food Sterilization of medical and laboratory equipment and preservation of food Disinfection and sterilization of surfaces and of transparent fluids and gases Biosafety Levels Describe four levels of biosafety and give examples of microbes handled at each level. The Centers for Disease Control and Prevention (CDC) has established guidelines for four levels of safety in microbiological laboratories dealing with pathogens. Each level raises personnel and environmental safety by specifying increasingly strict laboratory techniques, use of safety equipment, and design of facilities. Biosafety Level 1 (BSL-1) is suitable for handling microbes, such as E. coli, not known to cause disease in healthy humans. Precautions in BSL-1 are minimal and include handwashing with antibacterial soap and washing surfaces with disinfectants. BSL-2 facilities are similar to those of BSL-1 but are designed for handling moderately hazardous agents, such as hepatitis and influenza viruses and methicillin-resistant Staphylococcus aureus (MRSA). Access to BSL-2 labs is limited when work is being conducted, extreme precautions are taken with contaminated sharp objects, and procedures that might produce aerosols are conducted within safety cabinets (see Figure 10). BSL-3 is stricter, requiring that all manipulations be done within HEPA safety cabinets and specifying special design features for the laboratory. These include entry through double sets of doors and ventilation such that air moves into the room only through an open door. Air leaving the room is HEPAfiltered before being discharged outside the room. BSL-3 is designed for experimentation on microbes such as tuberculosis and anthrax bacteria and viruses of yellow fever and Rocky Mountain spotted fever. The most secure laboratories are BSL-4 facilities, designated for working with dangerous or exotic microbes that cause severe or fatal diseases in humans, such as Ebola, smallpox, and Lassa fever viruses. BSL-4 labs are either separate buildings or completely isolated from all other areas of their buildings. Entry and 276

7 exit are strictly controlled through electronically sealed airlocks with multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designed to destroy all traces of the biohazard. All air and water entering and leaving the facility are filtered to prevent accidental release. Personnel wear space suits supplied with air hoses (Figure 12). Suits and the laboratory itself are pressurized such that microbes are swept away from workers. Chemical Methods of Microbial Control Compare and contrast nine major types of antimicrobial chemicals, and discuss the positive and negative aspects of each. Although physical agents are sometimes used for disinfection, antisepsis, and preservation, more often chemical agents are used for these purposes. As we have seen, chemical agents act to adversely affect microbes cell walls, cytoplasmic membranes, proteins, or DNA. As with physical agents, the effect of a chemical agent varies with temperature, length of exposure, and the amount of contaminating organic matter in the environment. The effect also varies with ph, concentration, and freshness of the chemical. Chemical agents tend to destroy or inhibit the growth of enveloped viruses and the vegetative cells of bacteria, fungi, and protozoa more than fungal spores, protozoan cysts, or bacterial endospores. The latter are particularly resistant to chemical agents, as demonstrated by numerous failed attempts to decontaminate a United States Senate office building of anthrax endospores sent there by bioterrorists in In the following sections we discuss nine major categories of antimicrobial chemicals used as antiseptics and disinfectants: phenols, alcohols, halogens, oxidizing agents, surfactants, heavy metals, aldehydes, gaseous agents, and enzymes. Some chemical agents combine one or more of these. Additionally, researchers and food processors sometimes use antimicrobials substances normally used to treat diseases as disinfectants. Figure 12 A BSL-4 worker carrying Ebola virus cultures. Phenol and Phenolics Distinguish between phenol and the types of phenolics, and discuss their action as antimicrobial agents. In 1867, Dr. Joseph Lister began using phenol (Figure 13a) to reduce infection during surgery. As stated previously, the efficacy of phenol remains one standard to which the actions of other antimicrobial agents can be compared. Phenolics are compounds derived from phenol molecules that have been chemically modified by the addition of halogens or organic functional groups (Figure 13b). For instance, chlorinated phenolics contain one or more atoms of chlorine and have enhanced antimicrobial action and a less annoying odor than phenol. Natural oils such as pine and clove oils are also phenolics and can be used as antiseptics. Ramon Flick CH 3 O CH 2 Orthocresol Orthophenylphenol Triclosan HO Hexachlorophene (a) Phenol (b) Phenolics Bisphenolics Precision Graphics Figure 13 Phenol and phenolics. (a) Phenol, a naturally occurring molecule that is also called carbolic acid. (b) Phenolics, which are compounds synthesized from phenol, have greater antimicrobial efficacy with fewer side effects. Bisphenols are paired, covalently linked phenolics. 277