California Association for Medical Laboratory Technology. Distance Learning Program. ANAEROBIC BACTERIOLOGY FOR THE CLINICAL LABORATORY by

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1 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 CE/Contact Hour Level of Difficulty: Intermediate. 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 Phone: FAX: 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

2 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

3 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 % O 2 ). 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 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 CO 2 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 (O 2 - ), 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 {H 2 O 2 }, 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

4 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, in the intestine anaerobes outnumber aerobic bacteria 1,000 to 1. CAMLT Distance Learning Course # DL-974 4

5 Under usual circumstances these organisms do no harm by their presence, and there is considerable evidence that they are actually beneficial to their host. However, in cases where host defenses are impaired or breaks in the normal skin or mucous membranes occur, or when organisms are found in normally sterile sites after trauma or surgery, these organisms are capable of producing serious infection. Knowledge of the microflora composition at specific anatomic sites is useful for predicting the particular organisms most apt to be involved in infectious processes that arise at or adjacent to those sites. Because some anaerobes have fairly predictable susceptibility patterns, such knowledge may also be of value to physicians considering empiric antimicrobial therapy prior to isolation of organisms from clinical specimens and obtaining their susceptibility profile. In addition, the finding of site-specific organisms at a distant and/or unusual site can serve as a clue to the underlying origin of an infectious process. For example, the isolation of oral anaerobes from a brain abscess may suggest communication between an oral lesion and the bloodstream. Examples of the incidence of anaerobes at various body sites Skin: The anaerobic microflora of the skin consists primarily of bacteria within the genera of: Propionibacterium (usually P. acnes) and Peptostreptococcus and other anaerobic Grampositive cocci, and occasionally non-sporeforming Gram-positive bacilli in the genus Eubacterium. Should a venipuncture site be inadequately disinfected before collection of a specimen for blood culture, the specimen could become contaminated with skin flora, including anaerobes. Upper Respiratory Tract: In the upper respiratory tract, the number of anaerobes equals or exceeds that of aerobic organisms obtained in specimens from nasal washings, saliva, and gingival and tooth scrapings. Ninety percent of the bacteria present in saliva are anaerobes. Because of the large numbers of anaerobes that live in the oral cavity, virtually all oral lesions involve anaerobes, as do the majority of cases of aspiration pneumonia, and ear, nose and throat (ENT) infections. A wide variety of anaerobes lives in the oral cavity, although their concentrations and relative proportions vary from one microenvironment to another. Most often Fusobacterium spp., Porphyromonas spp., Prevotella spp., anaerobic Gram-positive cocci, Propionibacterium spp., Eubacterium spp., Lactobacillus spp. and Actinomyces spp. are recovered from the oral cavity. Therefore, these particular anaerobes should be suspected as participants in any infectious process from the respiratory tract. Vagina: About 50% of the bacteria in cervical and vaginal secretions are anaerobes, the most common being anaerobic Gram-positive cocci, Prevotella bivia, and Prevotella disiens, some anaerobic lactobacilli, and Actinomyces spp. Other anaerobic organisms such as Clostridium spp., Eubacterium spp., B. fragilis grp., Porphyromonas spp., and others may be found in the indigenous microflora of the vagina because of its proximity to the anus. P. bivia and P. disiens tend to dominate among the Gram-negative rods, but pigmented anaerobic Gramnegative bacilli, the B. fragilis group, and other Prevotella and Bacteroides species may be recovered as well. Whenever anaerobes are recovered from vaginal and cervical swabs, neither the microbiologist nor the physician can distinguish the indigenous microflora contaminants from organisms actually contributing to the patient s infectious process. For this reason, genitourinary tract swabs, including swabs of the vagina and cervix, are unacceptable for anaerobic bacteriology. CAMLT Distance Learning Course # DL-974 5

6 Intestine: Studies concerning the microflora of the intestine have found that anaerobes outnumber aerobes by a factor of 1,000 to 1. Anaerobes occurring in the highest numbers in intestinal flora are B. fragilis grp., Bifidobacterium, Clostridium, Eubacterium, Lactobacillus, Peptostreptococcus and other anaerobic Gram-positive cocci, Prevotella spp., Porphyromonas spp., and others. Intestine, intestinal contents, bowel, and other material such as rectal abscess, may be unacceptable specimens unless collected properly to avoid the normal anaerobic indigenous flora. The distal ileum may have counts of 10 4 to 10 5 colony forming units (CFU)/ml and both coliforms and various anaerobes may be encountered. In the distal colon, total bacterial counts average to CFU/g of feces, with anaerobes outnumbering the aerobes. Within the B. fragilis group, the species that is most prevalent in the indigenous flora of the intestine is Bacteroides thetaiotaomicron. Beneficial Aspects of Indigenous Anaerobes Many anaerobes of the indigenous microflora are beneficial and play an active role in maintaining the health of humans and other animals. Anaerobes, together with other microorganisms, provide a natural barrier to colonization of mucous membranes by pathogenic organisms. Within the gastrointestinal tract, anaerobes provide a source of fatty acids, vitamins, and cofactors that are used by the host and which degrade potentially toxic and/or oncogenic (cancer-causing) compounds. Anaerobes also play a role in maturation of the immune system during early development of neonates (1). E. ANAEROBIC INFECTIONS Anaerobes are key pathogens in brain abscess, oral/dental infections, aspiration pneumonia, lung abscess, pelvic and abdominal infections, and soft tissue infections, but they may cause any type of infection (Table 1). In a number of infections, anaerobic bacteria are the predominant pathogen; in other infections they are often mixed with aerobic organisms and with a variety of anaerobic organisms. Anaerobes produce and possess a variety of virulence factors, including enzymes, toxins, capsules, and adherence factors that are thought to play a role in pathogenicity. Certain clinical hints may suggest the presence of anaerobes in a clinical specimen (1): 1. Foul odor of specimen 2. Location of infection in proximity to a mucosal surface 3. Infections secondary to human or animal bite 4. Gas in specimen 5. Previous antibiotic therapy with aminoglycoside antibiotics that may have failed 6. Tissue necrosis; abscess formation 7. Unique morphology on Gram stain 8. Failure of culture to grow aerobically when organisms were observed on original Gram stain Bacteroides fragilis grp. (34%), followed by anaerobic Gram-positive cocci (28%), pigmented Gram-negative rods (Prevotella and Porphyromonas) (6.4%), and Fusobacterium spp. (7.9%), are the most commonly recovered anaerobes from infections. Since B. fragilis grp. can be forgiving in its tolerance toward oxygen, its physiological requirements of highly enriched media, and its need of good transport and anaerobic environmental conditions, laboratories may recover this group even if they use generally poor techniques. The anaerobic Gram-positive cocci, pigmented Gram-negative rods, and Fusobacterium spp., however, are CAMLT Distance Learning Course # DL-974 6

7 much more demanding and many laboratories do not frequently recover these organisms despite their reported high incidence (3). See Table 2. F. SPECIMEN COLLECTION AND TRANSPORT Proper collection of specimens and prompt transport to the laboratory for processing are imperatives. Specimens must be collected in a manner that will avoid contamination with indigenous flora. The laboratory must reject specimens that have not been collected or transported correctly or are likely to be contaminated. The saying garbage in, garbage out certainly applies to the collection and transport of anaerobic specimens. If the specimen has been improperly obtained or improperly transported, it may not provide information to the clinician, and the laboratory may expend useless time and resources on an unsatisfactory specimen. Indigenous anaerobes are often present in such large numbers on mucosal sites (gastrointestinal, genital tract, oral cavity), that even minimal contamination with indigenous flora will yield very misleading results and lead to much wasted effort by the laboratory. Communication and Supplies The laboratory director or supervisor must provide the clinical staff (nurses, physicians, etc.) with clear guidelines for the appropriate specimen types required for anaerobic culture (Table 3). The clinical staff must be told to immediately transport the properly collected specimen to the laboratory in an approved anaerobic transport system, and that some specimens may not be appropriate for anaerobic culture and may be rejected (5). Rejection of a clinical specimen can be a touchy subject to many clinicians. It works best to have meetings with physicians and nurses prior to the initiation of any policy to reject specimens to explain rationale and seek buy-in. Work with specific departments or physicians (surgery, OB, medicine, Pathologists, and Infectious Disease physician if your hospital has one) to explain information about the extent of normal anaerobic flora, contamination, and requirements to adequately isolate anaerobes. The clinical staff will understand that a quality specimen will reduce treatment delays and costs associated with working up improper specimens. Nurses in OR, ER, and ICU can be particularly helpful because they often see the patient one-on-one and frequently obtain specimens for culture. Patient care units, clinics, OR, and emergency rooms must be supplied with appropriate collection devices and complete instructions for their use. Good communication between the clinical microbiology laboratory and the clinical staff will ensure the collection and transport of the best possible specimen for anaerobic culture (5). Ideal specimens The ideal specimens for anaerobic culture are fluid obtained using a needle and syringe or a tissue sample (Table 3). Aspirated fluid collected by needle and syringe can be expelled in oxygen-free tubes or vials (Anaerobe Systems, BD, Hardy, Fisher Healthcare, and Remel) and then promptly transported to the laboratory. Aspirated material should never be transported in the syringe. Tissue samples or biopsy material are very satisfactory specimens and can be placed into oxygen-free tubes or vials for immediate transport to the laboratory (5). All specimens should be transported and held at room temperature. Do not place the transported specimen in the incubator or in the refrigerator; incubator temperatures will cause overgrowth of some bacteria and loss of isolates, and cold temperatures will allow increased oxygen diffusion. Anaerobic transport vials may contain modified Cary-Blair or other media that contain substances to scavenge excess oxygen (Anaerobe Systems, BD, Hardy, Fisher Healthcare, and Remel) and provide some moisture to the specimen. CAMLT Distance Learning Course # DL-974 7

8 In a good transport medium, anaerobes survive for some time usually up to 24 hours, depending upon the nature of the specimen. This fact permits batching of specimens in the laboratory at convenient times throughout the day without jeopardizing the recovery of anaerobes. Purulent specimens contain numerous reducing compounds that also help protect anaerobes from the effects of oxygen. Least Desirable Specimens The least desirable specimen for anaerobes is one collected by swab, and it should not be cultured, even though swabs are the predominant specimen type collected by medical/nursing personnel. Many laboratories commonly reject swab specimens for anaerobic culture. Generally, the specimen volume when collected by a swab is small, reducing the probability of isolating organisms. The specimen may be easily contaminated during collection. Many organisms adhere to the fibers of the swab and therefore are not recovered. Further, swab specimens commonly produce smears of poor Gram stain quality, and the inherent dryness of a swab specimen reduces the viability of many anaerobes. If collecting a specimen by swab is unavoidable and is absolutely necessary, then collect as much specimen as possible and use a commercially available anaerobe transport swab system (Anaerobe Systems, BD, Copan, Fisher Healthcare, and Remel). Take special care to sample the active site of infection to prevent contamination, and then place the swab deep into the agar butt. Break the stick off below where it was handled and replace the cap quickly. The commercial anaerobe transport system that contains two glass tubes (tube within a tube) for swab specimens has been shown not to be reliable. Remember that if you supply only a swab anaerobic collection device to the medical/nursing units, you will certainly receive a swab back. Get around this by consistently providing transport systems for collecting fluid or tissue. See Table 3 for appropriate specimens for anaerobic culture. G. PROCESSING CLINICAL SPECIMENS FOR ANAEROBIC CULTURE Ideally, a specimen is processed immediately upon arrival to the laboratory and is promptly incubated under anaerobic conditions to prevent further exposure to oxygen. However, the operations of a busy laboratory may prevent this from happening. When specimens cannot be inoculated onto media and placed immediately into an anaerobic atmosphere, it is best to hold specimens in their transport containers and batch process them later (e.g., once in the morning, and perhaps right before the day shift is ending, or at other convenient times throughout the day). Holding the clinical specimen in an appropriate transport device will not jeopardize the recovery of anaerobes or their viability. Batch processing of media inoculation at convenient times is preferred to processing specimens one at a time, which would require opening an anaerobic incubation jar each time, using expensive anaerobic incubation bags, or using up anaerobic gas. Batch processing specimens for anaerobes clearly reduces costs and improves the efficiency of the laboratory. The specimen for anaerobic culture may require special preparation. For example, grossly purulent specimens may require the use of a vortex mixer (avoid excessive aeration) on the anaerobic transport vial to ensure even distribution of microorganisms. You may need to grind bone or tissue with thioglycollate (THIO) or chopped meat broth to permit inoculation of specimen onto solid media. Swab specimens (should you accept one) may require the addition of THIO or chopped meat broth to make a liquid specimen. Large volume specimens may require centrifugation to produce the sediment needed to inoculate media and prepare a Gram stain. CAMLT Distance Learning Course # DL-974 8

9 Once the specimen has been prepared for culture, it should be inoculated onto the appropriate anaerobic media, placed in a liquid back-up broth, and onto a glass slide for a Gram stain. Once you begin processing the sample, you should complete it as quickly as possible, at least within 15 minutes. Microscopic Examination Always prepare a direct Gram stain from the clinical material. This is very important, for it often allows early presumptive evidence of the presence of anaerobes and provides information about the quality of the specimen. Direct smears for anaerobes are best fixed in absolute methanol for 1 min, and then stained by standard Gram stain procedure and reagents (5). Even gentle heat fixation can distort bacterial cell morphology, preventing clues in early identification. A Gram stain reveals the types and relative numbers of microorganisms and host cells present, and serves as a quality control measure for the adequacy of anaerobic technique. Failure to recover all the morphotypes seen on the direct Gram stain smear may indicate a problem in specimen collection, transportation or processing, or another problem that inhibited the growth of anaerobic microorganisms. The following are Gram stain clues for the presence of anaerobic organisms: 1. Large Gram-positive rods with boxcar-shaped cells and no spores usually indicate Clostridium perfringens. Within the same microscopic field, organisms may appear Gram-negative with the same cell morphology as the Gram-positive rods. 2. Gram-negative coccobacillary forms suggest Prevotella group or Porphyromonas group. 3. Thin Gram-negative bacilli with tapered ends suggest Fusobacterium nucleatum. 4. Pleomorphic pale-staining Gram-negative bacilli, sometimes with vacuoles, suggest Bacteroides fragilis spp. Media Efficient, cost-effective anaerobe recovery in the laboratory requires good media. Skimping on media costs and using inferior media wastes time and money, as cultures may fail to grow or yield inconclusive results and then have to be repeated. Use a highly enriched basal medium for primary isolation, such as Brucella medium containing vitamin K and hemin, which will support the growth of all anaerobes and aerobes. It has been shown that a PRAS (prereduced anaerobically sterilized) medium gives a faster growth rate and the ability to recover more anaerobes within a shorter period of time. Anaerobe Systems is the sole source of PRAS media, which have never been exposed to oxygen during any step of preparation. Therefore, PRAS media have not been exposed to superoxide anions, or hydroxyl radicals which may damage components of the media and prevent the growth of anaerobes. PRAS media also have a prolonged shelf life compared to other anaerobic media. Other manufacturers produce media for anaerobes that require pre-reduction (placing in anaerobic environment for 24 hrs. before use) or media that contain oxygen-scavenging substances (Oxyrase) or other reducing substances. It is best to perform side-by-side comparison testing in your own laboratory to determine which type of media recovers more organisms. In addition to using a highly enriched primary medium, it is also important to include a combination of selective and differential media for the recovery of anaerobes and for presumptive identification (2). The following media are suggested for the isolation of anaerobic bacteria from clinical specimens: Brucella agar supplemented with 5% sheep blood and vitamin K 1 (1µg/ml) and hemin (5µg/ml) as a nonselective medium which supports the growth of both anaerobic and aerobic organisms. CAMLT Distance Learning Course # DL-974 9

10 Phenylethyl alcohol-sheep blood agar (PEA) for the inhibition of enteric and certain other facultatively anaerobic Gram-negative bacilli that may overgrow anaerobes. PEA also reduces the spreading or swarming characteristic of certain Clostridium spp. Kanamycin-vancomycin-laked blood agar (KVLB or LKV) for the selection of pigmented Prevotella and other Bacteroides spp. Bacteroides bile esculin agar (BBE) for the selection and presumptive identification of Bacteroides fragilis grp. and Bilophila wadsworthia. Fusobacterium mortiferum/varium grp. is also resistant to bile and may occasionally grow on this medium. Thioglycollate medium without indicator, supplemented with vitamin K 1, hemin, and a marble chip, for enrichment and back-up culture. Chopped meat broth with vitamin K 1 and hemin may also be used. Use either of these broths as backup only. If primary plates are positive, you may discard the backup broth. Do not subculture the broth. Subculture the backup broth only if primary plates are negative and the broth is turbid. Anaerobic Incubation Systems The choice of incubation system used for anaerobic specimens depends on the number of anaerobic cultures performed, the cost of the system, and the space limitations of the laboratory. In general, there are three methods for the incubation of anaerobes from clinical specimens: anaerobic bags, anaerobic jars, and anaerobic chambers. A clinical laboratory that receives very few requests for anaerobic culture (1 per day and/or receives a rare anaerobic specimen after normal laboratory hours) may consider the use of anaerobic bags or pouches. A clinical laboratory that receives perhaps 2-4 specimens per day for anaerobic culture may consider the use of anaerobic jars. The use of anaerobic jars may be economically employed if the laboratory batches the processing of specimens at convenient times rather than using one jar for one specimen. If the laboratory receives a specimen at odd times after jars have been closed, perhaps the new specimen may be incubated in a pouch or bag and then after 48 hrs. included in an anaerobic jar. A laboratory that may receive 3 or 4 or more specimens per day should consider using an anaerobic chamber, the most economical way of producing an anaerobic atmosphere. The laboratory would need to consider the initial expense and the space required for the chamber. The ability to examine cultures at 24 hrs. and report the presence of anaerobes earlier (compared to jar and bag systems) may also be a patient-care benefit for the hospital. Whichever anaerobic system you use, the first step is to immediately place the inoculated plates into the anaerobic environment, and incubate them at 35 to 37ºC for hrs. Growing cultures must not be exposed to oxygen until after 48 hrs. of incubation in an anaerobic jar or pouch system, since anaerobes are most sensitive to oxygen during their log (early) phase of growth. An obvious advantage of an anaerobic chamber is that it permits the processing, inoculation of plates, and their examination at 24 hrs. or at any time under anaerobic conditions. Any anaerobic environment needs to be monitored with a methylene blue strip or resazurin chemical indicators. These indicators, initially blue and pink (respectively), change to colorless with low concentrations of oxygen. The following is a more detailed description of the most common choices of anaerobic incubation systems. Anaerobic bag or pouch Some anaerobic bag or pouch systems use a sachet that absorbs atmospheric oxygen without the generation of hydrogen, without the addition of water, and without requiring a catalyst. The resulting carbon dioxide level in these systems is generally higher than 10%. In other bag or pouch anaerobic atmospheric producing systems, a gas-generating envelope or ampoule provides CAMLT Distance Learning Course # DL

11 an atmosphere of 80 to 90% nitrogen (N 2 ), 5% hydrogen (H 2 ), and 5 to 10% carbon dioxide (CO 2 ). Some heat is produced from these systems, and the bags require a new catalyst each time they are opened. There are some gas-generating systems that have a catalyst incorporated into the envelope. The procedure is as follows: place the plates in the bag, activate the generating envelope, ampoule, or sachet, add an anaerobic indicator, and seal the bag or pouch by heat-sealing or by using special clamps. Check the anaerobic indicator through the clear plastic bag after a few hours to see that the bag has not leaked. Incubate the bag at 35 to 37ºC in a standard incubator for 48 hrs. Examining plates before 48 hrs. is not recommended since any small colonies are particularly susceptible to oxygen exposure at this stage and may not survive. At 48 hrs., remove the plates from the bag to examine them and work up the organisms as quickly as possible (this process will be described in greater detail). Add a new anaerobic generating envelope, ampoule, or sachet and reseal the bag or pouch. Bags and pouches are convenient, easy to use, and they do not take up a lot of space. However, the bags occasionally leak, and they are the most expensive way of producing an anaerobic environment (about $6.00 per bag). (BD Biosciences, Oxoid, Mitsubishi Gas Chemical America, and Difco). Anaerobic jars Anaerobic conditions are maintained in a self-contained jar by using a catalyst; a gasgenerating system (usually an envelope, ampoule, or sachet) providing an atmosphere of 80 to 90% nitrogen (N 2 ), 5% hydrogen (H 2 ), and 5 to 10% carbon dioxide (CO 2 ); and an anaerobic indicator. If a sachet is employed, hydrogen is not produced and a catalyst is not required. (See Anaerobic Bag or Pouch from above). For most jar systems, the procedure is the same. Place the inoculated plates into the jar, add an anaerobic indicator to the jar, add the anaerobic producing or catalyst system, close the jar, and incubate it at 35 to 37ºC in a standard incubator for 48 hrs. before opening the jar. This prevents exposure of smaller colonies to oxygen. The catalyst, composed of palladium-coated alumina pellets, should be fresh or rejuvenated each time the jar is opened prior to use, unless the catalyst is included in the gas pack envelope, or a water-less anaerobic generating system is used. At 48 hrs., remove the plates from the jar to examine them and work up the organisms. Add a new generating envelope, ampoule, or sachet system and reseal the jar. (BD Diagnostic Systems, Hardy Diagnostics, PML Microbiologicals, and Remel). The recovery of anaerobes in an anaerobic jar compares well to an anaerobic chamber if the plates are continuously incubated for 48 hrs. Jars do not recover anaerobes well if plates are incubated for only 24 hrs. prior to initial examination. Anaerobic chamber Anaerobic conditions are maintained in a gas-tight box or chamber by a gas mixture containing 80-90% nitrogen (N 2 ), 5 % hydrogen (H 2 ), and 5 to 10% carbon dioxide (CO 2 ), and using a palladium catalyst. The hydrogen concentration should not exceed 5% to prevent hazardous conditions. Usually anaerobic chambers have a positive pressure inside to prevent oxygen from coming into the chamber in case of a leak. The catalyst converts oxygen and hydrogen to water, thus removing atmospheric oxygen from the chamber. Carbon dioxide is included because many anaerobes require it for growth. Humidity is controlled by using silica gel crystals to absorb the water formed in the catalytic conversion process. In other chambers, humidity is controlled with a cold spot that condenses excess humidity and allows the water formed to be removed through a drain. Plates are incubated at 35 to 37ºC and can be examined at any time within the CAMLT Distance Learning Course # DL

12 chamber (generally at 24 to 48 hrs.) without removing them from the anaerobic environment (Coy Laboratory Products, Forma Scientific, and Sheldon Manufacturing). H. ISOLATION AND IDENTIFICATION OF ANAEROBES Isolation: After the plates primary Brucella, PEA, BBE and LKU have been incubated in an anaerobic pouch, jar or chamber, the next step is to isolate the anaerobes from other organisms. The primary medium (Brucella) likely will have grown not only anaerobes, but also facultative anaerobes (organisms that grow under either aerobic or anaerobic conditions) and microaerophilic organisms (organisms that grow in an atmosphere of reduced oxygen tension). Remember that facultative anaerobes and microaerophilic organisms will grow under anaerobic conditions, so you will need to exclude these from your workup. To determine which isolates from the primary Brucella medium are anaerobes, test the organisms for aerotolerance using two media: Brucella agar incubated anaerobically, and chocolate blood agar incubated under 5-10% CO 2 conditions. The facultative anaerobes and the microaerophilic organisms will grow on both the Brucella incubated anaerobically and the chocolate blood agar incubated under 5-10% CO 2, but the anaerobes will grow only on the Brucella incubated anaerobically and not on the chocolate blood agar. Chocolate blood agar must be used for aerotolerance testing. You may incorrectly assume that you have isolated an anaerobe if you use only blood agar media for aerotolerance testing. Use the chocolate blood agar media under 5-10% CO 2 to permit organisms such as Haemophilus spp., Actinobacillus spp., or other fastidious, slow-growing organisms to grow under aerobic conditions. When you set up the aerotolerance testing, also set up the special disks on the Brucella plate incubated anaerobically, and do a Gram stain as well. The disks will help you identify the organism once it shows growth (these disks are explained in detail in the next section, Identification ). Setting up the special potency disks at this time will permit faster identification and reporting of the anaerobe. Here is the procedure: 1. Select a single, well-isolated colony of each morphotype seen from the primary set-up medium (Brucella), and subculture it to a single Brucella agar plate and to a chocolate blood agar plate. Pick and subculture any colonies on the PEA, BBE and LKV plates that appear different from the colonies isolated on the anaerobic primary Brucella medium. 2. Divide the chocolate blood agar plate into quadrants so that 4 organisms at a time can be tested for aerotolerance. Streak the Brucella agar plate for isolation. Label the Brucella plate and the spot on the chocolate blood agar with the same identification number. 3. Add special potency antibiotic disks and a nitrate disk (as explained in the Identification section below) to the heavy quadrant of the Brucella subculture plate. 4. Make a smear for Gram stain on each colony type you observed from the primary Brucella medium. Facultative and anaerobic bacteria may have similar colony appearances, so you need to work up all colonial morphotypes you see on the primary media. 5. Incubate the Brucella plate anaerobically. Incubate the chocolate blood plate in an atmosphere of 5-10% CO Observe after 24 hrs. Anaerobic organisms will grow only on the Brucella medium incubated anaerobically, facultative anaerobes will grow on both the Brucella and chocolate blood agar. CAMLT Distance Learning Course # DL

13 7. Record a detailed description of each colony type from the anaerobic primary Brucella medium that does not grow on chocolate. Describe such characteristics as pitting, swarming, hemolysis, pigment, greening of the medium, etc. These colony characteristics can provide clues to identify the isolates when used in conjunction with Gram stain and rapid identification tests (explained in the next section). (See Table 4. Anaerobic Organism Identification Clues from Colony Morphology). Identification: Once you know that you have isolated an anaerobic organism(s) from the clinical specimen (growth on brucella medium, but no growth on chocolate), and you know the Gram reaction of the isolate, you are ready to begin identification of the isolate. The extent of identification required may vary according to the type of isolate, the source of the specimen, the needs of the physician, the clinical need, the patient s type of illness, and the operational and financial issues of the laboratory. In general, there are three different methods that can allow rapid and cost-efficient identification of anaerobic isolates: Method 1: presumptive and preliminary grouping using Gram stain information, colonial morphology (Table 4) and various rapid spot and disk tests; Method 2: the use of a variety of individual preformed-enzyme tests along with rapid spot and disk tests; and Method 3: the use of commercially available identification systems. The identification of anaerobes using either one of the first two methods is less expensive (about 50 per isolate) than using the third method (commercial systems cost about $6.00 per identification). The identification of anaerobic isolates to a group level using either Method 1 or Method 2 may be all that is necessary for many laboratories to provide clinically relevant information, and to allow initiation of appropriate antibiotic therapy. Method 1: Presumptive and Preliminary Grouping. You may already have some significant information about the identity of the anaerobic isolate based upon the Gram stain and colonial morphology (See Table 4). Begin the identification process by describing the colonial morphology in detail, including colony size, shape, edge, opacity, color and any other distinctive characteristic. Describe cellular morphology, including size, shape, and Gram reaction. Examine colonies for hemolysis on Brucella agar. Examine colonies for pigment on Brucella or LKV. Test colonies for fluorescence on Brucella agar. Next, determine susceptibility to special potency antibiotic disks (vancomycin 5 µg, kanamycin 1,000 µg, and colistin 10 µg) (Anaerobe Systems, Becton Dickinson, Hardy, PML, and Remel). The disks are used as an aid in determining the true Gram reaction and in separating different anaerobic species and genera (See Table 5). Generally, Gram-positive organisms are sensitive to vancomycin and resistant to colistin, whereas the Gram-negative organisms are resistant to the vancomycin disk and variable to colistin. The special potency antibiotic disks test is especially helpful with those clostridia that consistently stain Gramnegative, since their susceptibility to vancomycin disk confirms their true Gram reaction. Place special-potency antibiotic disks of vancomycin, kanamycin, and colistin on a Brucella agar plate. If you know the organism is Gram-negative, also add a nitrate disk to the heavily inoculated section. Special potency antibiotic disks are not needed when the organism stains Gram-positive because they will all be vancomycin susceptible, and the colistin and kanamycin do not provide additional information on Gram-positive organisms. CAMLT Distance Learning Course # DL

14 After 24 hrs. anaerobic incubation, use the results obtained with special-potency antibiotic disks for grouping or species identification (See Table 5). Examples of identification of Gram-negative isolates using special potency disks are as follows: 1. The B. fragilis grp. can be identified by the special potency antibiotic disk pattern showing resistance to all three disks (RRR) and resistance to 20% bile or growth on BBE agar. 2. The Bacteroides ureolyticus grp. is susceptible to kanamycin and colistin special potency disks, and resistant to vancomycin. These organisms reduce nitrate; they are nitrate reductase positive. 3. Fusobacterium sp. are susceptible to special potency disks kanamycin and colistin, and resistant to vancomycin. These organisms are nitrate reductase negative. 4. Porphyromonas sp. are resistant to special potency disks kanamycin and colistin, and are susceptible to vancomycin. They produce a black pigment. 5. Prevotella sp. are resistant to special potency disks kanamycin and vancomycin, and vary in their susceptibility toward colistin. Some Prevotella sp. may have a special antibiotic disk pattern typical of the B. fragilis grp. (RRR), but these organisms do not grow in 20% bile or on BBE. 6. Bilophila sp. are susceptible to special potency disks kanamycin and colistin and are resistant to vancomycin. Phenotypically this organism resembles the B. ureolyticus group and some Fusobacterium sp. These organisms can be distinguished by their strong positive catalase reaction and resistance to 20% bile. In 3 to 4 days Bilophila wadswortha forms small colonies on BBE that are clear with black centers, resembling fish-eyes. Use the pure-culture growth on the brucella agar to perform additional tests as needed. Once the true Gram stain reaction is known from the special potency disks, the laboratorian may use other rapid tests to assist in anaerobe identification. One such rapid test is determining the fluorescence of anaerobes using a Woods Lamp at 366 nm. The presence and color of fluorescing colonies can aid in the rapid detection and presumptive identification of certain anaerobic bacteria. Fluorescence disappears when black pigment has developed. See Table 6. Additional spot tests may include spot indole, catalase, SPS, bile test, lipase, lecithinase, pigment, and urease. See Table 7 for tests for the rapid identification of anaerobes. If the isolate is a Gram-negative rod, use Table 8; if isolate is a Gram-positive rod with spores (Clostridium spp.), use Table 9; and if the isolate is an anaerobic Gram-positive coccus, use Table 10. For guidance on further tests to permit rapid identification, the clinical laboratorian can use the tables in this course or others listed in the Wadsworth KTL Anaerobic Bacteriology Manual (2) or Clinical Microbiology Procedures Handbook (5). When typical morphology (cell and colony) is apparent and is combined with rapid tests, the resulting preliminary identification may be useful until more exhaustive tests are completed or are needed by the clinician. Anaerobic Gram-positive bacilli of human clinical relevance are divided into two distinct groups: one genus of spore-formers (Clostridium spp.). and five genera of non-sporeformers (Actinomyces, Bifidobacterium, Eubacterium, Lactobacillus, and Propionibacterium). The anaerobic Gram-positive bacilli are part of the normal microbiota of the oral cavity, gastrointestinal and genitourinary tracts, and skin. Currently there are 130 species of clostridia. Fortunately for the clinical microbiologist, the percentage of clostridial isolates commonly recovered in properly collected specimens is relatively small (Table 2). Clostridium perfringens is the most common clostridial isolate, followed by C. clostridioforme, C. innocuum, and C. ramosum (2, 4, 5). See Table 9 for identification of some commonly isolated Clostridium spp. Clostridium spp. can cause acute, CAMLT Distance Learning Course # DL

15 severe, or chronic infections. Some Clostridium spp. are highly pathogenic or toxigenic, while others are rarely pathogenic. Some species are resistant to antimicrobial agents. A great source of confusion is that many Clostridium spp., and occasionally the non-sporeforming genera as well, can stain Gram-negative. The use of the special antibiotic disks can help resolve this problem. There are a few aerotolerant strains of clostridia (C. tertium, C. carnis, C. histolyticum) that will grow marginally under aerobic conditions, and also a few aerotolerant strains of nonsporeforming bacilli (Actinomyces spp., Lactobacillus spp., and Propionibacterium spp.). The identification of the anaerobic non-sporeforming Gram-positive bacilli can be a challenge for the Clinical Laboratory Scientist. In many instances the use of PRAS biochemicals, gas-liquid chromatography (GLC) and fatty acid analysis is necessary. Many laboratories do not have access to these methods, and they will not be discussed in this course. The non-sporeforming Gram-positive bacilli comprise several genera that are differentiated from each other by their metabolic end products detected by GLC. The group is resistant to special potency disk of colistin, variable to kanamycin, and generally susceptible to vancomycin. However, there are rare strains of Lactobacillus and Clostridium spp. that may be vancomycin resistant (2). The clinical laboratory may encounter Propionibacterium acnes occasionally from a blood culture and from wound sources as contaminants. However, these organisms have been reported as causing chronic disease, so you need to rule this out before discarding the organism as a contaminant. P. acnes has a typical Gram stain appearance of clubbing, palisading, and Chinese character. P. acnes is nitrate, catalase, and spot indole positive. For identification of the Gram-positive cocci, the use of DNA composition, hybridization data, and cellular fatty acid profiles has permitted significant changes and reclassification among species that were at one time in the genus Peptostreptococcus. Peptostreptococcus hydrogenalis is now Anaerococcus hydrogenalis; Peptostreptococcus prevotii is now Anaerococcus prevotti; Peptostreptococcus magnus is now Finegoldia magna; Peptostreptococcus micros is now Parvimonas micra, Peptostreptoccus asaccharolyticus is now Peptoniphilus asaccharolyticus; Peptostreptococcus indolicus is now Peptoniphilus indolicus. The good news is that Peptostreptococcus anaerobius has not changed its name and is susceptible to the SPS (sodium polyanethanol sulfonate) disk which is useful for its rapid identification. Anaerobic cocci can be identified by Gram stain, colony morphology, spot tests such as SPS disk and spot indole, and various biochemical preformed enzymatic reactions and commercial systems (See Table 10). In some instances, PRAS biochemical, GLC, or fatty acid analysis may be necessary for identification. Method 2: Rapid biochemical tests for identification Many anaerobic isolates may be further identified using a variety of commercially available preformed-enzyme tests in conjunction with some of the rapid spot tests previously described in this course. Individual enzymatic biochemical tests may permit anaerobe identification without excessive expense or delay. One example is the identification of some species of anaerobic Gram-positive cocci using the alkaline phosphatase enzyme test. The combination rapid enzymatic tests are simple to perform and can be purchased allowing two or more enzymatic tests to be performed in a single tube to detect enzymatic activity visible by color change, or by detecting 4-methylumbelliferone fluorescent end products when exposed to a Wood s Lamp at 366 nm. (WeeTabs, Key Scientific Co., Stamford, TX). The tablet is inoculated heavily from fresh 24 hr. growth from Brucella medium; the heavier the inoculum, the better (>2.0 McFarland turbidity). Incubate for at least 2 hrs. at 37ºC. Identification tables of some anaerobes using the rapid preformed enzymatic tests are included in CAMLT Distance Learning Course # DL