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California Association for Medical Laboratory Technology Distance Learning Program ANAEROBIC BACTERIOLOGY FOR THE CLINICAL LABORATORY by James I. Mangels, MA, CLS, MT(ASCP) Consultant Microbiology Consulting Services Santa Rosa, CA Course Number: DL-974 3.0 CE/Contact Hour Level of Difficulty: Intermediate © California Association for Medical Laboratory Technology. Permission to reprint any part of these materials, other than for credit from CAMLT, must be obtained in writing from the CAMLT Executive Office. CAMLT is approved by the California Department of Health Services as a CA CLS Accrediting Agency (#0021) and this course is is approved by ASCLS for the P.A.C.E. ® Program (#519) 1895 Mowry Ave, Suite 112 Fremont, CA 94538-1766 Phone: 510-792-4441 FAX: 510-792-3045 Notification of Distance Learning Deadline All continuing education units required to renew your license must be earned no later than the expiration date printed on your license. If some of your units are made up of Distance Learning courses, please allow yourself enough time to retake the test in the event you do not pass on the first attempt. CAMLT urges you to earn your CE units early!. CAMLT Distance Learning Course # DL-974 1 © California Association for Medical Laboratory Technology Outline A. Introduction B. What are anaerobic bacteria? Concepts of anaerobic bacteriology C. Why do we need to identify anaerobes? D. Normal indigenous anaerobic flora; the incidence of anaerobes at various body sites E. Anaerobic infections; most common anaerobic infections F. Specimen collection and transport; acceptance and rejection criteria G. Processing of clinical specimens 1. Microscopic examination 2. Media: primary, selective, differential 3. Incubation systems H. Isolation and identification 1. Provide identification to level needed by physician 2. Role of Gram stain and plate morphology 3. Presumptive grouping and identification using cost effective rapid tests I. Anaerobic bacteriology cost containment concepts Measurable Course Objectives Upon completion of this course, the participant will be able to: • Recognize the most important genera and species of clinically important anaerobes and the infections they may cause • Describe the normal anaerobic indigenous flora • List appropriate techniques for specimen selection, collection and transport • Describe initial processing techniques and the media employed • Identify laboratory methods used for initial grouping, presumptive identification, and definitive identification, and determine when each level is appropriate • Identify techniques used for cost-effective clinical anaerobic bacteriology CAMLT Distance Learning Course # DL-974 2 © California Association for Medical Laboratory Technology A. INTRODUCTION Anaerobic bacteria cause a variety of infections in humans, including appendicitis, cholecystitis, otitis media, dental and oral infections, endocarditis, endometritis, brain abscess, myonecrosis, osteomyelitis, peritonitis, empyema, salpingitis, septic arthritis, liver abscess, sinusitis, wound infections following bowel surgery and trauma, perirectal and tuboovarian abscesses, and bacteremia (1). Many reports associate an incidence of at least 50% to 60% of important infections due to anaerobic bacteria (Table 1). Anaerobic bacteria are often overlooked or missed unless the specimen is properly collected and transported to the laboratory. Next, the specimen must be subjected to appropriate procedures for isolation, including the use of specialized media supplemented with growth factors and the use of proper incubation methods. Anaerobes vary in their nutritional requirements, but most isolates require vitamin K and hemin for growth. Anaerobes also vary in their sensitivity to oxygen: a brief exposure (10 min.) to atmospheric oxygen is enough to kill some organisms. This course will discuss procedures for proper collection and transport of anaerobes; appropriate specimen types for culture, processing, incubation, and isolation; and methods of characterization of anaerobes from properly collected specimens. Practical schemes for isolating the majority of clinically important anaerobes will be described, including their salient features and cost-effective procedures for their work-up and identification. Many laboratorians believe that the isolation and identification of anaerobes is difficult, expensive, and time consuming. This course will present methods that will permit rapid, yet cost-effective procedures for the recovery and identification of clinically significant anaerobes for any clinical laboratory. B. WHAT ARE ANAEROBIC BACTERIA? Anaerobes are microorganisms that do not require oxygen for metabolism, reproduction or growth. Most anaerobes are actually inhibited by oxygen or oxygen by-products, however they vary as a group in their sensitivity to oxygen. An obligate or strict anaerobe (e.g., Porphyromonas spp., Fusobacterium spp., or Peptostreptococcus spp.) will grow only in an absolute anaerobic environment (zero % O2). They are killed by exposure to air after only a few minutes. A moderate anaerobe (e.g., Bacteroides fragilis grp.) can tolerate more exposure to air, but damage can occur after 15-20 minutes of exposure to air. A microaerotolerant anaerobe (e.g., Clostridium tertium) is an organism that is capable of growing in both an anaerobic and a microaerophilic atmosphere. A microaerotolerant anaerobe may marginally grow when exposed to air or in a CO2 incubator on a chocolate blood agar medium, but growth is best under anaerobic conditions. Molecular oxygen itself can be lethal to some anaerobes, however even more toxic substances are produced when oxygen becomes chemically reduced. Initially, molecular oxygen - is reduced to superoxide anion (O2 ), a highly reactive free radical capable of causing severe damage to components of media, bacterial enzyme systems, proteins, lipids, and cell walls. Further reduction of oxygen leads to the production of other toxic compounds of oxygen - (hydrogen peroxide {H2O2}, and hydroxyl radicals {OH }) that can damage microorganisms or the components of media on which they are to grow. Thus, oxygen, superoxide anions, hydroxyl radicals, and hydrogen peroxides inhibit the growth of anaerobes and should be avoided to permit their recovery in culture. CAMLT Distance Learning Course # DL-974 3 © California Association for Medical Laboratory Technology All living creatures that use oxygen for metabolism have one or more enzymes to provide protection from superoxide anions and their toxic derivates. These enzymes are known as superoxide dismutases (SODs). Anaerobes have various amounts of SOD, ranging from none to some, that presumably allow some anaerobes to tolerate oxygen. However, there is not a direct correlation between levels of SOD and the anaerobe’s ability to tolerate oxygen. There are other factors, such as the presence of catalase, which may play a role in the inability of anaerobic organisms to tolerate oxygen (2). C. WHY ISOLATE AND IDENTIFY ANAEROBES? The recovery of anaerobes is very important because they are commonly resistant to empiric antibiotic therapy (antibiotics that may be used prior to isolation of any organism), and many anaerobes (including Bacteroides fragilis grp., the most commonly recovered anaerobe) contain virulence factors that lead to abscess formation and chronic disease if not treated correctly. The recovery of anaerobes aids the physician in making a specific diagnosis and may provide the clinician with the potential source of the infection. Further, in this era of concern about antibiotic resistance, the isolation and identification of anaerobes allows the clinician to use appropriate antibiotic therapy instead of the “big gun”—the antibiotic with the broadest spectrum which will inhibit both aerobes and anaerobes, but may also contribute to antibiotic resistance. It has been shown that correctly employed specific therapy against anaerobes can reduce mortality and morbidity, and reduce hospitalization (1). There are some general concepts regarding anaerobic infections that are important to mention now, but will be discussed in greater detail in this course. • First, most anaerobic infections derive from our own indigenous microflora, so specimen selection and collection are essential for quality results and to reduce contamination. • Second, anaerobic infections are often mixed, containing both aerobic and anaerobic organisms. Employing an enriched primary medium as well as using differential and selective media is essential to rapidly recover anaerobes from specimens that contain a mixture of organisms. • Third, despite the diversity of our normal indigenous flora (1, 2, 3, 4), most infections are due to a relatively limited number of anaerobic isolates (Table 2): almost 35% are members of B. fragilis group; 28% are Peptostreptococcus spp. or other genera of anaerobic Gram-positive cocci; 6 % are pigmented Gram-negative rods; and 8% are Fusobacterium spp. The recovery of Clostridium spp. is only about 2%. These three concepts of anaerobic bacteriology have a profound effect on how we isolate and identify anaerobes and should be part of your thought process during this course. D. NORMAL INDIGENOUS ANAEROBIC FLORA Almost all surfaces of the human body are colonized by microorganisms referred to as normal or indigenous microflora. These organisms normally inhabit the skin, mouth, nose, throat, lower intestine, vagina, and outer portion of the urethra. Anaerobes colonizing these regions are present in high numbers. For example,

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