Review of Methods for Antibiotic Susceptibility Testing of Anaerobic Bacteria

Review of Methods for Antibiotic Susceptibility Testing of Anaerobic Bacteria by Tracy D. Wilkins, Ph.D., and Maria D. Appleman, Ph.D. Several methods are available for testing the antibiotic susceptibility of anaerobes. The method used depends on the type of information needed and the amount of time, personnel, and effort that can be devoted to the tests. In this article we will discuss the main advantages and disadvantages of the principal methods used by clinical and research laboratories and the antibiotics used in these tests. Antibiotics The antibiotics tested should be those useful in the treatment of anaerobic infections (Tables I and II). Although Table II lists these antibiotics as effective, none are effective against all anaerobes and most can adversely affect some patients. In addition, antibiotic therapy alone is not always effective in the treatment of anaerobic infections in which large abscesses are present. Chemotherapy must be combined with surgical drainage in patients with such infections. Thus, in vitro test results Tracy D. Wilkins, Ph.D., and Maria D. Appleman, Ph.D., are with the Anaerobic Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va. Table I Antibiotics Inactive Against Most Anaerobes Aminoglycosides Kanamycin Neomycin Streptomycin Gentamicin Sulfonamides and Trimethoprim Polymyxin Colistin Table II Antibiotics Active On Most Anaerobes Clindamycin Chloramphenicol Tetracyclines Penicillins (high dosages) and in vivo response do not always correlate exactly. Clindamycin is the most effective drug now available for the treatment of anaerobic infections. Although resistant strains occur, particularly among the Clostridia, 8 more than 95% of routine clinical anaerobic isolates are susceptible to this drug. Unfortunately, clindamycin therapy can cause pseudomembranous colitis, a rare and unpredictable condition that can be fatal. Chloramphenicol is a bacteriostatic and antibiotic with a wide antibacterial spectrum in vitro. It also has some well-known side effects. Aerobic bacteria with a minimum inhibitory concentration (MIC) value of 2.5 /u-g/ml or less (blood level) are considered susceptible. Most anaerobes, however, have MIC values of 3-6 Mg/ml. A blood level of 2.5 /u,g/ml of a 6 pet criostatic does not insure growth inhibition for an organism with an MIC value of 6 pig/ml, since the actual concentration of the antibiotic in the anaerobic environment may be well below blood level concentrations. The tetracyclines are very effective in the treatment of anaerobic infections caused by tetracyclinesusceptible organisms. In the past, tetracycline was the drug most widely used in treating anaerobic infections. Unfortunately, the majority of Bacteroides fragilis strains now are resistant to tetracycline as are about 25% of the isolates of other species. Doxycycline offers a somewhat wider spectrum in vitro, but yet has to be proved clinically useful. There is a controversy about the use of penicillin and carbenicillin in the treatment of anaerobic infections. Most species of anaerobes (MIC < unit/ml) are very susceptible to penicillin, but B. fragilis strains have average MIC 2 LABORATORY MEDICINE VOL. 7, NO. 4 APRIL 976

values of 32-64 units/ml. Some investigators have claimed successful treatment of B. fragilis infections using large doses of carbenicillin, but not enough evidence is available now to establish a definite conclusion. Penicillin resistance also occurs occasionally in other species such as Bacteroides melaninogenicus, Bacteroides oralis, Clostridium ramosum, Bacteroides (Clostridium) clostridiiformis, and Peptostreptococcus anaerobius, Penicillinase and cephalosporinase are known to be produced by B. fragilis 2 ' 4 and B. melaninogenicus 8 and may prove to be of importance in the chemotherapy of mixed infections. 6,8 A new antibiotic, Cefoxitin (Merck & Co., Inc., Rahway, NJ), which is resistant to these cephalosporinase enzymes, has been shown to be more effective than cephalothin in the treatment of laboratory-produced B. fragilis mixed infection. This drug is now being administered on a trial basis to humans with anaerobic infections. Disk Diffusion Methods The Kirby-Bauer disk diffusion method cannot be used for anaerobes even if the plates are quickly placed into an anaerobic environment. The method was not developed or standardized for anaerobes, and anaerobes do not grow well on Mueller-Hinton agar. Table III Protocol for Sutter's Disk Diffusion Test. Pick two to three colonies and transfer into supplemented thioglycollate broth. 2. Dilute in freshly boiled Brucella broth to the Kirby-Bauer standard. 3. Swab fresh Brucella blood agar plates. 4. Incubate for 24 hours and compare zone diameters to Sutter's breakpoints. Antibiotic penicillin G ampicillin cephalothin tetracycline clindamycin chloramphenicol erythromycin Table IV Test Concentrations of Antibiotics Labeled Disk Content 0 units 0 nq 30 Mg 30 Mg?M 30 Mg 5/xg The most successful modification of the Kirby-Bauer method has been developed by Sutter and colleagues. 0,67 The steps in this procedure are outlined in Table III. Main Advantages of This Method Because of its similarity to the Kirby-Bauer method, technologists trust the results. A relatively large number of antibiotics can be tested at one time, and anaerobe jars are the only special equipment necessary. Main Disadvantages The Brucella blood agar plates must be made immediately prior to use and cannot be stored, and the Brucella and thioglycollate broth must be freshly boiled. Because there are different standards for interpreting zone diameters for different species, the identification must be done prior to reporting the data and must be correct. Despite its similarity to the Kirby-Bauer method, Sutter's method entails important differences in technique and interpretation. An error frequently made is the use of the Kirby-Bauer zone diameter breakpoints in interpreting the results. Broth-Disk Methods The broth-disk procedure is the simplest technique available for susceptibility testing of anaerobes. The basic technique was developed many years ago by Schneierson. 3 More recently it has been modified No of Disks per tube 2 8 2 Cal :ulated Test Concentration per ml 2 units 4 Kg 6/xg 6/xg 3.2/^g 2/Ltg 3^9 by Abramson and Smibert for testing treponemes and by Wilkins and Thiel 9 for routine clinical testing of anaerobes. The principle of the technique also is used in the Autobac I automated susceptibility testing device (Pfizer, Inc., New York, NY). The modified broth-disk procedure of Wilkins and Thiel 9 consists of adding regular antibiotic susceptibility disks to tubes of anaerobic broth in order to achieve an antibiotic concentration in the broth that approximates the concentration achieved in the blood (Table IV). From a pasteur pipette, one drop of a turbid culture in chopped meat medium is added to anaerobic, rubber-stoppered tubes containing 5 ml of prereduced brain-heart infusion broth. Oxygen is kept from entering the tubes during addition of both the antibiotic disks and the inoculum by inserting a cannula that carries a stream of oxygenfree carbon dioxide into the neck of each tube when the stopper is removed. Thus the culture never comes in contact with air, and the re-stoppered tube is its own anaerobic culture chamber. A simple set-up which costs less than an anaerobe jar consists of a tank of carbon dioxide, a regulator, two pieces of hose and two cannulas. Details of this set-up are given in the Virginia Polytechnic Institute (VPI) Anaerobe Laboratory Manual. 9 LABORATORY MEDICINE VOL 7, NO. 4 APRIL 976 3

Table V Protocol for Agar MIC Test. The organisms are diluted to one half No. McFarland standard so that the Steers Replicator 5 (Melrose Machine Shop, 76 Fairview Rd., Woodlyn, PA) applies an inoculum of 0 5 cells per spot. 2. The cells are spotted on a series of freshly prepared Brucella blood agar plates, each plate containing a different concentration of antibiotic. 3. The plates are incubated at 37 C for 48 hours in a Gaspak jar. Alternatively, the tubes can be used inside an anaerobic chamber. The tubes are incubated overnight (8 to 24 hours) at 37 C. After incubation, the turbidity of the antibiotic tubes is compared to that of an inoculated control tube not containing antibiotics. The organism is considered resistant to an antibiotic if that tube has a tu rbidity 50% or greater than the turbidity of the control tube. In 90% or more of the tests, the result can be read simply as "growth" or "no growth." The tubes can be examined for growth at any time: in many cases, resistance to some antibiotics can be reported within four hou rs after inoculation. However, susceptibility to an antibiotic cannot be reported until after 8 hours of incubation. More than 95% of clinical isolates will grow well enough within 8 to 24 hours for results to be reported. A few very slowly growing organisms may not reach turbidity until after 48 to 72 hours of incubation. Although resistance results recorded at this time are somewhat less reliable because the antibiotic could have decayed, susceptibility can be reported with confidence. Main Advantages of This Method Technologists can report reliable results after only a few minutes of instruction, because the method is simple. It has been shown in two studies to be 95% or more accurate. 3,9 Results can be reported more quickly than with any of the other methods, and correct identification is not necessary. It allows easy and quick identification of anaerobes by VPI techniques because the broth-disk tubes and the biochemical test media are inoculated at the same time. The medium is commercially available and has a shelf life of at least several months. Almost all anaerobes can be tested with this method. Main Disadvantages of This Method Because the method is simple, some people do not believe it will work. The method requires the use of prereduced media, which are more expensive than most media, and because of the need for a separate tube for each antibiotic, there is a limit to the number of antibiotics that can be tested conveniently. The procedure cannot be performed in a Gaspak jar; rather, a source of oxygen-free carbon dioxide or an anaerobic chamber is required. Agar Dilution MIC Methods The MIC methods set the standards with which all other procedures are compared and give the most information of any of the methods. Unfortunately, MIC determinations require too much time and expense to be used routinely in most clinical laboratories. However, it is important to have a standard MIC method available. A subcommittee of the National Committee of Clinical Laboratory Standards is now deciding on a standard protocol for the agar dilution MIC procedure, which it plans to publish in the fall of 976. The procedure should be similar to that given in Table V, except that Brucella blood agar base may be replaced by a completely new formulation that does not require the addition of blood. This procedure is designed to be done at the laboratory bench with no special apparatus other than anaerobe jars. Most clinical isolates should grow well enough under these conditions, but there will be some strains, particularly of B. melaninogenicus, that cannot tolerate this amount of oxygen exposure. If such cultures must be tested, the dilution of the inoculum and its application to the plates can be done completely inside an anaerobic glove box. Results are read after the plates have incubated for 48 hours. The MIC value is the lowest antibiotic concentration at which the inoculated spot contains less than three colonies or only a "barely visible haze." This haze is difficult to define. It occurs most often when fusobacteria are tested with penicillin. In such cases, good confluent growth occurs on the control plate and on the plates with the lowest dilutions of penicillin, but this growth changes within the space of two or three dilutions to a haze that continues for many more dilutions. The MIC value should occur close to the time that the major drop in growth is seen. To efficiently use the agar dilution MIC procedure, a minimum of 20 organisms should be tested at one time. Very few clinical laboratories have 20 strains of anaerobes to test each day, so the cultures have to be saved until enough are available to justify use of the MIC procedure. This causes a delay in reporting results; more time is added by the need for 48 hours of incubation. Susceptibility test results are seldom of value if they reach the physician two weeks after the specimen was taken. Broth Dilution MIC Methods Several broth dilution MIC methods have been used and the 4 LABORATORY MEDICINE VOL 7, NO. 4 APRIL 976

results seem comparable (for examples see Sutter 6 and Wilkins et al. 20 ). Broth dilution techniques also have been done with microtiter type of equipment inside an anaerobic chamber. 2 The most recent detailed study of the broth dilution method was by Stalons and Thornsberry, 4 who recommended the use of Schaedler broth prereduced in an anaerobic glove box for 24 hours. Using a spectrophotometer, the inoculum was adjusted to contain 0 6 cells per milliliter. The tubes were incubated in an anaerobic chamber or Gaspak jar at 35 C C for 8 to 24 hours or until turbidity appeared. The broth dilution method has some advantages over the agar dilution procedure for laboratories that need to determine MIC values for only a few organisms at a time. Other advantages are that the results are available in 24 hours instead of 48, that the end points are somewhat easier to read and that minimal bactericidal concentrations (MBC) can be determined. However, the method shares several problems with the agar dilution method: preparing accurate antibiotic solutions, performing dilutions, and making media. Categorization Method Several investigators have recommended that clinical laboratories use fewer antibiotic dilutions with both agar and broth 5,4 MIC methods. Usually, three concentrations are tested in order to categorize the organism as very susceptible, susceptible only to high doses, or extremely resistant. Advantages of These Methods They were designed to require less effort than that required for the entire MIC procedure. More information is obtained through these methods than by testing the growth response to a single concentration of antibiotic. Main Disadvantages of These Methods Antibiotic solutions must still be prepared accurately. If antibiotic disks of appropriate concentrations were made available, this problem would be solved. The broth-disk procedure could then be done as a categorization method. The amount of effort and expense required to test three concentrations of each antibiotic is still considerable. Conclusions Some laboratories do not perform susceptibility tests on anaerobes because no "standard methods" such as the Kirby-Bauer tests for aerobes are available. We do not believe that this is justified. Several of the methods described in this review probably will become standard methods within the next few years; in the meantime, all of the procedures give accurate results when performed exactly according to the published protocols. Clinical laboratories must refrain, however, from modifying published methods unless they are capable of doing the large amount of research necessary to prove that their modification works. It is unfortunate that the clinical laboratory often is forced to compromise between the speed and the thoroughness of reports. Although we recognize that MIC methods unquestionably offer the most reliable data, we cannot recommend them as routine tests for clinical laboratories because they are too slow and complicated. Such methods should be available, perhaps in a regional reference laboratory, for more detailed data in cases of chronic infections. We do recommend the broth-disk procedure for routine use because of its inherent speed and simplicity. References. Abramson I J, Smibert R M: Method of testing antibiotic sensitivity of spirochaetes using antibiotic discs. Br J Vener Dis 48:269-273, 2. Anderson J D, Sykes R B: Characterisation of a /3-lactamase obtained from a strain of Bacteroidesfragilis resistant to/3-lactam antibiotics. J Med Microbiol 6:20-206, 973 3. Blazevic D J: Evaluation of the modified brothdisk method for determining antibiotic susceptibilitiesof anaerobic bacteria. Antimicrob Agents Chemother 7:72-723, 975 4. Del Bene V, Farrar W E: Cephalosporinase activity in Bacteroides fragilis. Antimicrob Agents Chemother 3:369-372, 973 5. Fass R J, Prior R B, Rotilie C A: Simplified method forantimicrobial susceptibility testing of anaerobic bacteria. Antimicrob Agents Chemother 8:444-452, 975 6. Hackman A S, Wilkins T D: In vivo protection from penicillin by Bacteroides fragilis. Antimicrob Agents Chemother 7:698-703, 975 7. Hackman A S, Wilkins T D: Comparison of cefoxitin and cephalothin therapy of a mixed Bacteroides fragilis and Fusobacterium infection in mice. Antimicrob Agents Chemother 8:224-225, 975 8. Hackman A S, Wilkins T D: Influence of penicillinase production by strains of Bacteroides melaninogenicus and Bacteroides oralis on the success of penicillin therapy of an experimental mixed anaerobic infection. Arch Oral Biol, In press 9. Holdeman L V, Moore W E C (eds): Anaerobe Laboratory Manual, ed 3. Blacksburg, Va: Virginia Polytechnic Institute & State University, Anaerobe Laboratory, 975 0. Kwok Y Y, Disk susceptibility testing of slowgrowing anaerobic bacteria. Antimicrob Agents Chemother 7:-7, 975. Meny R, Webb C D, Fiedelman W: Carbenicillin therapy of anaerobic infections. Curr Ther Res 7:478-487, 975 2. Rotilie C A, et al: Microdilution technique for antimicrobial susceptibility testing of anaerobic bacteria. Antimicrob Agents Chemother 7:3-35, 975 3. Schneiersen S S: A simple rapid disc-tube method for determination of bacterial sensitivity to antibiotics. Antibiot Chemother 4: 25-32, 954 4. Stalons D R, Thornsberry C: Broth-dilution method for determining the antibiotic susceptibility of anaerobic bacteria. Antimicrob Agents Chemother 7:5-2, 975 5. Steers E, Foltz E L, Graves B S: An inocula replicating apparatus for routine testing of bacterial susceptibility of antibiotics. Antibiot Chemother 9:307-3, 959 6. Sutter V L: Antibiotic susceptibility testing of anaerobes, in Balows A (ed): Current Techniques for Antibiotic Susceptibility Testing, Springfield, Illinois: Charles C Thomas, 974, pp09-8 7. Sutter V L, Vargo V L, Finegold S M: Wadsworth Anaerobic Bacteriology Manual, ed 2. Los Angeles, 975 8. Wilkins T D, Thiel T: Resistance of some species of Clostridium to clindamycin. Antimicrob Agents Chemother 3:36-37, 973 9. Wilkins T D, Thiel T: Modified broth-disk method fortesting the antibiotic susceptibility of anaerobic bacteria. Antimicrob Agents Chemother 3:350-356, 973 20. Wilkins T D, et al: Standardized single-disc method for antibiotic susceptibility testing of anaerobic bacteria. Antimicrob Agents Chemother :45-459, 972 ftt] LABORATORY MEDICINE VOL 7, NO 4 APRIL 976 5

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