Ebook Textbook of diagnostic microbiology (5th edition): Part 1

(BQ) Part 1 book Textbook of diagnostic microbiology presents the following contents: Introduction to clinical microbiology (host parasite interaction, control of microorganisms, specimen collection and processing, immunodiagnosis of infectious diseases,...), laboratory identification of significant isolates (staphylococci, anaerobes of clinical importance, enterobacteriaceae, neisseria species and moraxella catarrhalis,...).

tahir99 VRG YOU’VE JUST PURCHASED MORE THAN A TEXTBOOK! Evolve Student Resources for Mahon, Lehman and Manuselis: Textbook of Diagnostic Microbiology, 5th Edition, include the following: • Laboratory Manual Activate the complete learning experience that comes with each textbook purchase by registering at http://evolve.elsevier.com/Mahon/microbiology/ REGISTER TODAY! You can now purchase Elsevier products on Evolve! Go to evolve.elsevier.com/html/shop-promo.html to search and browse for products Evolve Student Resources are provided free with each book purchase only tahir99 VRG Textbook of Diagnostic Microbiology tahir99 VRG This page intentionally left blank tahir99 VRG Textbook of Diagnostic Microbiology FIFTH EDITION Connie R Mahon, MS Director, Staff and Organization Development Health Resources and Services Administration HIV/AIDS Bureau Rockville, Maryland Adjunct Faculty Department of Clinical Research and Leadership School of Medicine and Health Sciences George Washington University Washington, DC Donald C Lehman, EdD, MT(ASCP), SM(NRM) Associate Professor Department of Medical Laboratory Sciences University of Delaware Newark, Delaware George Manuselis, MA, MT(ASCP) Emeritus Medical Technology Division Ohio State University Columbus, Ohio Adjunct Faculty Department of Natural Sciences and Forensic Science Central Ohio Technical College Newark, Ohio tahir99 VRG 3251 Riverport Lane Maryland Heights, Missouri 63043 TEXTBOOK OF DIAGNOSTIC MICROBIOLOGY, FIFTH EDITION ISBN: 978-0-323-08989-0 Copyright © 2015 Saunders, an imprint of Elsevier, Inc 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 written permission of the publisher Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein) Notices Knowledge and best practice in this field are constantly changing As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein The views and opinions of contributors to the Work who are employees of the National Institutes of Health, Department of Defense, or other Departments of the U.S Government not necessarily state or reflect those of the U.S Government, nor does the NIH, Department of Defense, or the U.S Government endorse, warrant, or guarantee the information contained therein Previous editions copyrighted 2011, 2007, 2000, 1995 Library of Congress Cataloging-in-Publication Data Textbook of diagnostic microbiology / [edited by] Connie R Mahon, Donald C Lehman, George Manuselis.—Fifth edition    p ; cm   Includes bibliographical references and index   ISBN 978-0-323-08989-0 (hardcover)   I.  Mahon, Connie R., editor of compilation.  II.  Lehman, Donald C., editor of compilation.  III.  Manuselis, George, editor of compilation   [DNLM:  1.  Microbiological Techniques.  2.  Bacterial Infections diagnosis.  3.  Communicable Diseases—diagnosis.  4.  Mycoses—diagnosis.  5.  Virus Diseases—diagnosis.  QW 25]   QR67   616.9′041—dc23 2013045846 Vice President and Publisher: Andrew Allen Managing Editor: Ellen Wurm-Cutter Content Development Specialist: Amy Whittier Publishing Services Manager: Julie Eddy Project Managers: Celeste Clingan/Nisha Selvaraj/Devendran Kannan Design Direction: Karen Pauls Printed in China Last digit is the print number:  9  8  7  6  5  4  3  2  1  tahir99 VRG To my husband, Dan, for his love and continued support and understanding, my son, Sean, who inspires me, my daughter, Kathleen, for showing me courage, and my granddaughters, Kelly Amelia and Natalie Page, who have given us so much pleasure CRM To my wife, Terri, who has given me constant support and encouragement, and whose love makes anything I possible, and my parents, Gerald and Sherrie, who have always been proud of me—even though they have passed away, I know they still look over me DCL To my daughters, Kristina and Shellie, my sisters, Libby and Helen, and my brother, Demetrious and, in memory of my parents, Katherine and George, for their love and encouragement GM tahir99 VRG This page intentionally left blank tahir99 VRG Reviewers Dawn Nelson, MA, MT(ASCP) Director of Medical Laboratory Technology Program Florence Darlington Technical College Florence, South Carolina Daniel deRegnier, MS, MT(ASCP) Associate Professor Program Director of Clinical Laboratory Science Ferris State University Big Rapids, Michigan Jennifer Sanderson, MS, MLS(ASCP) Curriculum Consultant Siemans Healthcare Diagnostics Wilmington, Delaware Delfina Dominguez, MT(ASCP), MS, PhD Professor University of Texas at El Paso El Paso, Texas Lynne Steele, MS, MLS(ASCP) Chair and Professor Medical Laboratory Technology and Phlebotomy Oakton Community College Des Plaines, Illinois Frances Pouch Downes, HCLD(ABB), PhD Professor Biomedical Laboratory Diagnostics Program Michigan State University East Lansing, Michigan Ron Walker, MBA, CNMT, PET Professor The University of Findlay Findlay, Ohio Shawn Froelich, MLS(ASCP) Adjunct Instructor of Medical Laboratory Sciences Allen College Waterloo, Iowa Karen Golemboski, Ph.D., MLS(ASCP) Associate Professor Medical Laboratory Science Bellarmine University Louisville, Kentucky Amy Kapanka, MS, MT(ASCP)SC Director, Medical Laboratory Technology Program Hawkeye Community College Waterloo, Iowa Michael Majors, BS, MLS(ASCP) Microbiology Technical Specialist Providence Sacred Heart Medical Center and Children’s Hospital Spokane, Washington Nicholas Moore, MS, MLS(ASCP) Laboratory Director Kindred Healthcare, Inc Kindred Hospital Chicago North Chicago, Illinois Mildred K Fuller, PhD, MT(ASCP) Former Department Chair, Allied Health Medical Technology Program Norfolk State University Norfolk, Virginia Lori A Woeste, EdD Assistant Dean and Associate Professor College of Applied Science and Technology Illinois State University Normal, Illinois tahir99 VRG vii This page intentionally left blank tahir99 VRG 514 PART II  Laboratory Identification of Significant Isolates Gram reaction Positive Negative Cocci Bacilli Clostridium-like SPS disk Branching or diphtheroid Cocci Bacilli Nitrate disk V + Nitrate disk + No disks Nitrate disk K + CL + Nitrate disk + Bile disk FIGURE 22-14  Disks to add to the pure culture-subculture plate Clostridium-like organisms are large, unbranched, gram-positive rods, with or without spores Not all clostridia are large, and not all clostridia will stain as gram-positive rods CL, colistin (10 µg); K, kanamycin (1000 µg); SPS, sodium polyanethol sulfonate; V, vancomycin (5 µg) FIGURE 22-15  Typical special potency antimicrobial disk results for Clostridium ramosum, susceptible to vancomycin (left) and kanamycin (right) and resistant to colistin (center disk) (Courtesy Anaerobe Systems, Morgan Hill, CA.) presumptively identified as Peptoniphilus (Peptostreptococcus) asaccharolyticus Nitrate Disk.  The nitrate reduction disk test is a miniaturized version of the conventional nitrate reduction test This determines an organism’s ability to reduce nitrate to nitrite or nitrogen gas Bile Disk.  This disk can be used to determine an organism’s ability to grow in the presence of relatively high concentrations (20%) of bile; however, it is not necessary if BBE agar is used as a primary plating medium The bile disk may be added to the anaerobic subculture plate whenever the Gram stain reveals the isolate to be a gram-negative bacillus Good growth on a BBE plate or growth in 20% bile indicates bile tolerance A biletolerant, gram-negative anaerobic bacillus indicates that the isolate is likely a member of the B fragilis group Lecithinase, Lipase, and Proteolytic Reactions.  An EYA plate can be used to determine the activities of lecithinase, lipase, and proteolytic enzymes These reactions are of value in identifying many species of clostridia Lecithinase cleaves lecithin found in egg yolk, releasing insoluble fat (diglyceride) that produces an opaque zone around the colony This opacity is actually in the medium and is not a surface phenomenon (see Figure 22-16) Lipases hydrolyze triglycerides and diglycerides to fatty acids and glycerol Lipase-positive organisms produce a colony covered with an iridescent, multicolored sheen, sometimes described as resembling the appearance of gasoline on water or mother of pearl This multicolored sheen also can appear on the surface of the agar in a narrow zone around the colony In contrast to the lecithinase reaction, the lipase reaction is essentially a surface phenomenon (Figure 22-17) tahir99 VRG CHAPTER 22  Anaerobes of Clinical Importance 515 Organisms that produce proteolytic enzymes (proteases) have a completely clear zone, often quite narrow, around their colonies Proteolysis is best observed by holding the plate up to a strong light source It is reminiscent of the complete clearing seen with β-hemolytic organisms on SBA plates FIGURE 22-16  Positive lecithinase reaction on egg yolk agar The reaction occurs within the agar Clostridium perfringens is shown here (Courtesy Anaerobe Systems, Morgan Hill, CA.) TABLE 22-12  Interpretation of Special Potency Antimicrobial Disk Results Kanamycin† Colistin‡ Interpretation S V R S R R R R R R R R R R S V S S Probably a pink-staining, gram-positive bacillus such as Clostridium ramosum or C clostridioforme; however, if the kanamycin result is resistant, it could be a Porphyromonas sp Porphyromonas sp Probably a member of the Bacteroides fragilis group, but could be a Prevotella sp Prevotella sp Probably a Prevotella sp Bacteroides ureolyticus, Bilophila wadsworthia, or a Fusobacterium sp.; Veillonella Vancomycin* From Mangels JI: Anaerobic bacteriology In Isenberg HD, editor: Essential procedures for clinical microbiology, Washington, DC, 1998, American Society for Microbiology R, Resistant; S, susceptible (a zone of inhibition ≥ 10 mm is considered susceptible.); V, variable *Vancomycin, 5 µg † Kanamycin, 1000 µg ‡ Colistin, 10 µg TABLE Presumptive Identification of Gram-Positive Anaerobes Many gram-positive anaerobes can also be presumptively identified with simple procedures (Table 22-13) None of these organisms will grow on a BBE or LKVB medium so identification is based on the colony appearance on nonselective anaerobic blood and EYAs • Large, irregular-shaped colonies on SBA demonstrating a double zone of β-hemolysis can be identified as C perfringens (Figure 22-18) These organisms will stain as large, boxcar-shaped bacilli • Large, flat colonies that produce a so-called barnyard or horse stable odor and fluoresce chartreuse under long-wave UV light can be identified as C difficile FIGURE 22-17  Positive lipase reaction on egg yolk agar The reaction occurs on the surface of colonies and surrounding medium A positive reaction by Fusobacterium necrophorum is shown here (Courtesy Anaerobe Systems, Morgan Hill, CA.) 22-13  Presumptive Identification of Gram-Positive Anaerobes* Identification Clostridium difficile C perfringens C septicum C sordellii C tetani Peptostreptococci Propionibacterium acnes Colony Morphology on Blood Agar Cellular Morphology Spot Indole Large, flat colonies; barnyard odor, chartreuse fluorescence Large, irregular-shaped, double zone of β-hemolysis Smoothly swarming Very large, lobate, irregular, flat Smoothly swarming but slow growing Small, peaked, circular Small, opaque, enamel white, circular (catalase-positive) Thin rods, rare spores Boxcar, large, square rods Thin rods, subterminal spores Thin rods, subterminal spores Swollen terminal spores Cocci, pairs and chains Coryneform rods Negative Not done Negative Positive Positive Not done Positive *Additional information about presumptive identification of aerobes and facultative anaerobes can be found in Clinical and Laboratory Standards Institute: Abbreviated identification of bacteria and yeast: approved guidelines, ed 2, CLSI document M35-A2, Wayne, PA, 2008, CLSI tahir99 VRG 516 PART II  Laboratory Identification of Significant Isolates • A rapidly growing colony exhibiting smooth swarming (as opposed to the waves observed with Proteus) and staining as thin rods with subterminal spores is likely to be C septicum Colonies often show β-hemolysis at 48 hours • Small, peaked, circular colonies appearing after 24 hours that stain as gram-positive cocci can be considered Peptostreptococcus spp The CLSI M-35-A2 document suggests continued use of the terms Peptostreptococcus spp or anaerobic grampositive cocci as a presumptive identification rather than identifying these organisms with the newer nomenclature • Peptococcus niger produces colonies that are initially black to olive green and become light gray when exposed to air, but it is only rarely isolated from clinical specimens • Small, opaque colonies that are catalase- and indole-positive and stain as coryneform rods can be identified as P acnes ✓ FIGURE 22-18  Double zone of hemolysis produced by Clos- tridium perfringens—inner zone of complete β-hemolysis and outer zone of partial β-hemolysis (Courtesy Anaerobe Systems, Morgan Hill, CA.) Case Check 22-3  In the Case in Point, a Gram stain of the patient’s leg wound revealed boxcar-shaped, gram-positive rods suggestive of clostridia The presence of large gram-positive bacilli with colonies exhibiting a double zone of β-hemolysis in the absence of organisms recovered on aerobic cultures of the specimen is sufficient to report a presumptive identification of C perfringens Presumptive Identification of Gram-Negative Anaerobes Using Gram stain results, growth characteristics on primary plating media (e.g., nonselective anaerobic blood, BBE, KVLB), and a few rapid tests, many gram-negative anaerobes can be presumptively identified, often within 24 hours of inoculation (Table 22-14) • Growth of large (>1 mm) gray-black colonies on BBE agar with growth on the KVLB agar after an overnight incubation is sufficient to identify an isolate as a member of the B fragilis group (Figure 22-19) TABLE FIGURE 22-19  Appearance of Bacteroides fragilis on a kanamycin–vancomycin–laked blood agar (left) and Bacteroides bile esculin (BBE) agar biplate (right) Darkening of the BBE medium is the result of esculin hydrolysis (Courtesy Anaerobe Systems, Morgan Hill, CA.) 22-14  Presumptive Identification of Gram-Negative Anaerobes* Identification Colony Morphology on Blood or KVLB Agar Colony Morphology on BBE Cellular Morphology Spot Indole B fragilis group B ureolyticus Bilophila wadsworthia Large (>1 mm) Translucent, pitting (some) Tiny, translucent Regular Tiny rods or coccobacilli Regular to filamentous Not done Negative Negative Fusobacterium nucleatum Ground-glass or breadcrumb Large, convex, black-gray No growth Translucent, with black center at 72 h No growth Positive Porphyromonas Small, translucent or opaque, brick red fluorescence on blood agar, no growth on KVLB Small, translucent or opaque, brick red fluorescence on blood and KVLB agar Small, translucent or opaque, brick red fluorescence on blood agar, no growth on KVLB No growth Fusiform, thin with pointed ends Tiny coccobacilli No growth Tiny coccobacilli No growth Tiny diplococci Prevotella intermedia Veillonella Positive Positive Negative Negative BBE, Bacteroides bile esculin; KVLB, Kanamycin and vancomycin with laked sheep blood *Additional information about presumptive identification of aerobes and facultative anaerobes can be found in Clinical and Laboratory Standards Institute: Abbreviated identification of bacteria and yeast: approved guidelines, ed 2, CLSI document M35-A2, Wayne, PA, 2008, CLSI tahir99 VRG CHAPTER 22  Anaerobes of Clinical Importance • Translucent pitting colonies observed on the anaerobic blood agar plate, with no growth observed on BBE or KVLB agars, are characteristic of Bacteroides ureolyticus • Translucent colonies with a black fish eye center observed on BBE agar (usually at 48 to 72 hours) can be used to presumptively identify Bilophila wadsworthia (Figure 22-20) • Ground-glass or breadcrumb-like colonies of long, slender, gram-negative rods with pointed ends are usually Fusobacterium nucleatum (Figure 22-21) • Organisms that grow on anaerobic blood agar and KVLB but not BBE and fluoresce brick red can be identified as Prevotella • Small, translucent or opaque colonies of tiny gram-negative cocci or diplococci can be identified as Veillonella Definitive Identification of Anaerobic Isolates For sterile site and some surgical isolates, it is important for the laboratory to provide full identification A variety of techniques can be used to make a definitive identification (see Table 22-9) Most clinical microbiology laboratories now use one of the commercially available biochemical-based or preexisting enzymebased minisystems for making definitive identifications, but it is important to remember that none of these will identify all the anaerobes that could potentially be isolated from clinical FIGURE 22-20  Appearance of Bilophila wadsworthia on Bacteroides bile esculin (BBE) agar Note the fish eye appearance of the colonies FIGURE 22-21  Gram-stained appearance of Fusobacterium nucleatum subsp nucleatum illustrating the fusiform morphology of this organism (×1000) (Courtesy Bartley SL, Howard JD, Simon R: Centers for Disease Control and Prevention, Atlanta, GA.) 517 specimens Most are designed to identify anaerobes that are most frequently encountered in clinical specimens It is far more important for a system to identify these organisms than to identify obscure anaerobes that are only rarely isolated from clinical specimens or involved in infectious processes Biochemical-Based Multitest Systems A commercially available alternative to conventional tubed media is the biochemical-based identification systems manufactured by Analytab Products (API; bioMérieux, Durham, NC) This system provides many of the same tests as the conventional systems but in the form of a plastic strip or tray The biochemicalbased multitest system is easier and faster to inoculate than a conventional system of plates and tubes Although they can be inoculated aerobically, they require anaerobic incubation The larger model BBL anaerobic jars and some of the commercially available bags and pouches, can be used to incubate biochemicalbased trays and strips if an anaerobic chamber is not available After 24 to 48 hours of incubation, test results are read, a code number is generated for each isolate, and identification is determined from a compendium codebook or electronic database It should be noted that the databases from which the codebooks are developed not contain all the anaerobes that could be isolated from clinical specimens Preformed Enzyme-Based Systems Many of the newer commercial identification systems are based on the presence of preformed (preexisting) bacterial enzymes Because these systems not depend on enzyme induction or bacterial growth, the results are available in about hours The small plastic panels or cards are easy to inoculate, can be inoculated at the bench, and not require anaerobic incubation Most of the systems generate code numbers, which are referenced in a manufacturer-supplied codebook Like the biochemical-based multitest systems, these systems are primarily of value for identifying only the most commonly isolated anaerobes One potential pitfall with these systems is that the codebook compendium is divided by Gram-staining characteristics of the organism Hence, it is vital to have an accurate Gram stain to arrive at an accurate identification Special potency disks to determine the true Gram stain of an organism can play a major role in the use of the preformed enzyme-based identification systems Preexisting enzyme-based systems include the Vitek ANI Card (bioMérieux), AN-IDENT (bioMérieux), MicroScan Rapid Anaerobe Identification Panel (Siemens Biomedical Diagnostics, Deerfield, IL), BBL Crystal Anaerobe ID System (BD Diagnostic Systems), and the RapID-ANA II (Remel, Lenexa, KS), shown in Figure 22-22 In general, these systems use the same or similar substrates They contain a number of nitrophenyl and naphthylamide compounds, colorless substances that produce yellow or red products, respectively, in the presence of appropriate enzymes The results of some of these systems can be read via instrumentation Conventional Tubed Biochemical Identification Systems In general, conventional or traditional systems for the identification of anaerobic isolates use large test tubes containing various tahir99 VRG 518 PART II  Laboratory Identification of Significant Isolates FIGURE 22-22  RapID ANA-II preformed enzyme system (Remel, Lenexa, KS) This is one of many systems available for rapid definitive identification of commonly isolated anaerobes PRAS or non-PRAS biochemical test media Biochemical reactions are interpreted by measuring the pH with a probe or by color changes based on a bromthymol blue indicator Identification of anaerobes by the use of conventional biochemicals is expensive and time-consuming and has been replaced largely in clinical laboratories by the systems described earlier Gas-Liquid Chromatography.  Identification of anaerobes by GLC analysis of metabolic end products was pioneered at the Virginia Polytechnic Institute (VPI); the VPI guidelines were modified by the CDC The anaerobe to be tested is grown in a tube of peptone–yeast extract–glucose (PYG) medium After incubation, the metabolic end products are extracted with chloroform and ether, injected into the chromatograph, volatilized, and carried as a gas through a packed column Aliquots of the extraction solutions are used to analyze volatile (ether-extracted) and nonvolatile (chloroform-extracted) acids The acids are separated in the column and recovered in order, based on their chemical and physical properties The acids are identified based on their position on the chromatogram by their relative retention time as compared with a standard Short-chain volatile acids produced by anaerobes include formic, acetic, propionic, isobutyric, isovaleric, valeric, isocaproic, butyric, caproic, and heptanoic acids The chloroform extract recovers nonvolatile, low–molecular-weight aliphatic and aromatic acids These include pyruvic, lactic, oxaloacetic, oxalic, malonic, fumaric, succinic, benzoic, phenylacetic, and hydrocinnamic acids These procedures were used extensively in the early days of anaerobic bacteriology, but because of cost, safety, and time commitment, they have been largely abandoned for more rapid identification procedures Cellular Fatty Acid Analysis by High-Resolution Gas-Liquid Chromatography Cellular fatty acid analysis is another method of identifying anaerobes The term cellular fatty acid refers to fatty acids and related compounds (e.g., aldehydes, hydrocarbons, dimethylacetals) present within organisms as cellular components Cellular fatty acids are coded for on bacterial chromosomes, as opposed to plasmids, and are not affected by simple mutations or plasmid loss Thus, the fatty acid composition of a particular organism is relatively stable when grown under specific growth conditions— medium, incubation temperature, and time Although fatty acid profiles can be identified manually, computerized, high-resolution GLC and specialized software programs are available to analyze cellular fatty acids of unknown bacteria and compare the results with patterns of known species The MIDI Sherlock Microbial Identification System (MIDI, Newark, DE) is a fully automated gas chromatography system that can be used to identify bacteria (including anaerobes) and yeasts The organism is grown in pure culture in PYG broth or another standardized medium The bacterial cells are removed by centrifugation and then saponified to release the fatty acids from the bacterial lipids After extraction, the methyl esters are analyzed by GLC A chromatogram depicting the unknown organism’s fatty acid composition and comparison to a database of fatty acids of known anaerobes are generated as a computer report The report includes statistical values, or similarity indices, which are based on deviations in the unknown organism’s fatty acid composition from the known profiles contained in the database Because no subjective interpretations are required, the identifications are objective and highly reproducible 16S Ribosomal RNA Gene Sequencing Many of the techniques mentioned have been replaced largely in research and reference laboratories by 16S ribosomal RNA gene sequencing The 16S ribosomal RNA gene (rDNA) has highly conserved and highly variable regions that can be used for microbial identification The DNA is first extracted from the organism; the target segment of about 500 base pairs is amplified through polymerase chain reaction (PCR) technology and then sequenced on an automated sequencer The resulting nucleotide sequence is compared to known sequences in public databases such as GenBank, a repository of sequences maintained by the National Institutes of Health The MicroSeq Microbial Identification System (Applied Biosystems, Foster City, CA) has kits and an electronic database that can be used for bacterial or fungal identification The results can be obtained in approximately hours Identification of Clostridium Species An identification algorithm for some Clostridium spp is shown in Figure 22-23 Identification of clinically encountered clostridia using cultural and biochemical characteristics is summarized in Table 22-15 The clostridial cell wall structure is similar to that of other gram-positive bacteria However, some species appear gram-variable and some, such as C ramosum and C clostridioforme, routinely stain gram-negative The vancomycin special potency antimicrobial disk set up at the same time as the aerotolerance test is used to determine the true Gram stain reaction of a pink-staining anaerobic bacillus Gram-positive clostridia are always susceptible to vancomycin Certain cultural characteristics can be used initially to identify clostridial isolates As noted, a boxcar-shaped, gram-positive anaerobic bacillus that produces a characteristic double zone of hemolysis on SBA can be presumptively identified as C perfringens A double zone of hemolysis appears as an inner zone of tahir99 VRG CHAPTER 22  Anaerobes of Clinical Importance 519 Double zone of hemolysis on blood agar Yes No Clostridium perfringens* Lecithinase reaction on egg-yolk agar Positive Negative Lipase reaction on egg-yolk agar Lipase reaction on egg-yolk agar Positive Negative Positive Negative Clostridium novyi A Spot indole Clostridium sporogenes Use definitive methods outlined in Table 22-9 Positive Negative Rapid urease Gelatin hydrolysis Positive Negative Positive Negative Clostridium sordellii Clostridium † bifermentans Clostridium limosum Clostridium barati * Boxcar-shaped bacilli; lecithinase-positive; reverse CAMP (Christie, Atkins, and Munch-Petersen) test–positive; subterminal spores, but spores rarely observed † Produces chalk-white colonies on egg-yolk agar FIGURE 22-23  Identification of Clostridium spp Use this chart for organisms fulfilling the following three criteria: (1) anaerobic, (2) gram-positive, and (3) spore former (Data from Mangels JI: Anaerobic bacteriology In Isenberg HD, editor: Essential procedures for clinical microbiology, Washington, DC, 1998, American Society for Microbiology.) complete β-hemolysis and an outer zone of partial β-hemolysis because of two different hemolysins (see Figure 22-18) A swarming, gram-positive, anaerobic bacillus with terminal spores is probably C tetani, whereas one with subterminal spores is most likely C septicum EYA is useful for detecting lecithinase, lipase, and proteolytic enzymes produced by some clostridia Lecithinase-positive clostridia include C bifermentans, C sordellii, C perfringens, and C novyi type A Some lipase-positive clostridia are C botulinum, C novyi type A, and C sporogenes C sordellii is the only member of the clostridia to exhibit urease activity C bifermentans and C sordellii are spot indole– positive Finally, it is important to remember that C tertium will grow minimally on the aerotolerance plate and can be mistaken for a facultative anaerobe However, growth on the anaerobic subculture plate will exhibit much heavier growth than on the aerotolerance test plate C difficile can be recovered from feces by inoculating a cycloserine–cefoxitin–fructose agar (CCFA) plate CCFA is a selective and differential medium for the recovery and presumptive identification of C difficile On CCFA, C difficile produces yellow ground-glass colonies; the originally pink agar turns yellow in the vicinity of the colonies because of the fermentation of fructose In reduced conditions, the indicator, neutral red, turns yellow in the presence of an acid pH Although other organisms may grow on CCFA, their colonies are smaller and not resemble the characteristic colonies of C difficile In addition, C difficile has a characteristic odor, resembling a horse stable, and colonies on blood agar fluoresce chartreuse under UV light tahir99 VRG Spore Position Motility Indole Lecithinase Lipase Proteolysis in Milk Gelatin Hydrolysis Acid from Lactose Urease bifermentans clostridioforme difficile novyi type A perfringens ramosum septicum sordellii sporogenes tertium tetani Gram-Stain Reaction C C C C C C C C C C C Chartreuse Fluorescence Clostridium species Double-Zone β-Hemolysis 22-15  Characteristics of Some Clinically Encountered Clostridium Species Swarming TABLE PART II  Laboratory Identification of Significant Isolates Aerotolerant Growth 520 − − − − − − − − − + − − − − − − − + − − − + − − − − + − − − − − − − − + − − − − − − − − + − + + + − + + + + + ST ST ST ST −ST (T) ST ST ST T T + −+ + +− − − + + + + + + − − − − − − + − − −+ + − − + + − − + −+ − − − − − + − − − − + − − + − − − − − − + + − − − − −+ + + − − − −+ − + − + − − + + + − − + − − − − − − − − + − − − Modified from Engelkirk PG et al: Principles and practice of clinical anaerobic bacteriology, Belmont, CA, 1992, Star ST, Subterminal; T, terminal; −ST, usually not observed but subterminal when seen; (T), variable but usually terminal; +−, most strains positive; −+, most strains negative Organisms that are isolated must be tested for toxin production because a small percentage of the human population can carry C difficile as normal biota, and some isolates may not be toxin producers Culture for C difficile has largely been replaced by assays designed to detect the toxins in feces produced by the organism The cell culture cytotoxicity assay that detects toxin B in fecal samples by tissue culture is generally considered the gold standard However, this assay is technically demanding and can take to days for results to be obtained A number of rapid detection tests are available that can detect toxin A, toxin B, an enzyme called glutamate dehydrogenase, or a combination of toxins A and B Many of the kits use enzyme immunoassay (EIA) methodologies Glutamate dehydrogenase is not a virulence factor, but it is an enzyme frequently associated with C difficile Negative glutamate dehydrogenase and EIA for toxins A and/or B can be used as rapid screening to rule out C difficile–associated disease (CDAD) Because not all strains of C difficile produce toxins A and B, assays that detect both toxins are generally more sensitive Nucleic acid amplification tests (NAATs), such as the Illumigene C difficile (Meridian Bioscience, Cincinnati, OH), that can determine the presence of toxins A and B genes in feces are rapidly becoming the new gold standard for the detection of toxin-producing C difficile Positive glutamate dehydrogenase and EIA should be confirmed by an NAAT for toxin genes; some laboratories opt to use an NAAT This is the only stand-alone test for C difficile disease, meaning test results not need to be confirmed A study of 81 patients with symptoms suggestive of CDAD compared three NAATs and two non-NAATS for the detection of CDAD The authors reported sensitivities for the NAATs of 88.5% to 96.2% and 42.3% and 61.5% for the non-NAATS The NAATS detected 34.7% more cases of CDAD than the non-NAAT methods The specificity for all five assays ranged from 96.4% to 100% Because patients with formed stools are rarely positive for CDAD, EIA and NAATs should not be performed on these types of specimens Identification of Anaerobic Non–SporeForming, Gram-Positive Bacilli Identifying characteristics of clinically encountered non–sporeforming, gram-positive bacilli are listed in Table 22-16 In general, non–spore-forming, gram-positive bacilli are difficult to identify because the preformed enzyme system databases often not contain many of the less commonly isolated species GLC of cellular fatty acids may be required to definitively place isolates within a genus Sequencing of the 16S rDNA gene has been used most recently to reclassify many of these organisms into new genera Actinomyces spp Actinomyces spp are straight to slightly curved bacilli of varying lengths, from short rods to long filaments Short rods may have clubbed ends and may be seen in diphtheroid arrangements, short chains, or small clusters Longer rods and filaments may be straight or wavy and branched Although the Actinomyces are gram-positive, irregular staining can produce a beaded or banded appearance, much like that seen with Nocardia spp The typical branching, filamentous, Gram-stained appearance of an Actinomyces sp., depicted in Figure 22-2, is referred to as Actinomyces-like Investigators at the CDC Anaerobic Bacteria Branch have found that members of the genus Actinomyces are seldom obligate anaerobes However, some are fastidious, requiring special vitamins, amino acids, and hemin for adequate growth Young Actinomyces colonies are frequently spider-like or wooly, tahir99 VRG TABLE CHAPTER 22  Anaerobes of Clinical Importance 521 22-16  Characteristics of Some Clinically Encountered Gram−Positive Non–Spore-Forming Anaerobic Bacilli 48-h Colony (1 mm) growing on a BBE plate at 24 hours can presumptively be called a member of the Bacteroides fragilis group _ 12 A pleomorphic gram-positive bacillus that is spot indole– and catalase-positive can be presumptively identified as Propionibacterium acnes BIBLIOGRAPHY Baron EJ: Approaches to identification of anaerobic bacteria In Versalovic J, et al, editors: Manual of clinical microbiology, ed 10, Washington, DC, 2010, ASM Press, p 799 Brook I: Antimicrobial treatment of anaerobic infections, Expert Opin Pharmacother 12:1691, 2011 Centers for Disease Control and Prevention: Summary of notifiable diseases—United States, 2011, MMWR 60(53):1, 2013 Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm6053a1.htm Accessed November 13, 2013 tahir99 VRG 528 PART II  Laboratory Identification of Significant Isolates Chapin KC, et al: Comparison of five assays for detection of Clostridium difficile toxin, J Mol Diagn 13:395, 2011 Clinical and Laboratory Standards Institute: Methods for antimicrobial susceptibility testing of anaerobic bacteria: approved standard, ed 8, CLSI document M11-A8, Wayne, PA, 2012, CLSI Clinical and Laboratory Standards Institute: Abbreviated identification of bacteria and yeast: approved guideline, ed 2, CLSI Document M35-A2, Wayne, PA, 2008, CLSI Fader RC, et al: CACMLE self-study course 117: anaerobic bacteriology for today’s clinical laboratory, Denver, 2007, Colorado Association for Continuing Medical Laboratory Education Kõnõnen E, et al: Bacteroides, Porphyromonas, Prevotella, Fusobacterium, and other anaerobic gram-negative rods In Versalovic J, et al, editors: Manual of clinical microbiology, ed 10, Washington, DC, 2010, ASM Press, p 858 O’Connor JR, et al: Clostridium difficile infection caused by the epidemic BI/NAP1/027 strain, Gastroenterology 136:1913, 2009 Simmon KE, et al: Genotypic diversity of anaerobic isolates from bloodstream infections, J Clin Microbiol 46:1596, 2008 Song Y, Finegold SM: Peptostreptococcus, Finegoldia, Anaerococcus, Peptoniphilus, Veillonella, and other anaerobic cocci In Versalovic J, et al, editors: Manual of clinical microbiology, ed 10, Washington, DC, 2010, ASM Press, p 803 Stevens DL, et al Clostridium In Versalovic J, et al, editors: Manual of clinical microbiology, ed 10, Washington, DC, 2010, ASM Press, p 834 Wade W, Kõnõnen E: Propionibacterium, Lactobacillus, Actinomyces, and other non–spore-forming anaerobic gram-positive rods In Versalovic J, et al, editors: Manual of clinical microbiology, ed 10, Washington, DC, 2010, ASM Press, p 817 tahir99 VRG ... Use of Colony Morphology for the Presumptive Identification of Microorganisms, 16 9   Biochemical Identification of Gram-Negative Bacteria, 18 1 10 Immunodiagnosis of Infectious Diseases, 19 8 11 ... employees of the National Institutes of Health, Department of Defense, or other Departments of the U.S Government not necessarily state or reflect those of the U.S Government, nor does the NIH, Department... fifth edition of the Textbook of Diagnostic Microbiology This edition embodies our commitment to convey information on the ever-evolving, complex, and challenging field of diagnostic microbiology