Evolution of Anaerobe Susceptibility Testing in the United States

Transcription

1 SUPPLEMENT ARTICLE Evolution of Anaerobe Susceptibility Testing in the United States David W. Hecht Infectious Diseases Division, Loyola University Medical Center, and Hines VA Hospital, Maywood, Illinois Susceptibility testing of anaerobes has been described for 45 years, some 15 years fewer than descriptions for aerobic organisms. During that time period, 116 methods, 116 media, and a host of other variables have been described in the United States literature, culminating in the most recent standards published by the National Committee for Clinical Laboratory Standards (NCCLS) in These new guidelines include a single agar dilution reference method as well as an alternative minimal inhibitory concentration method validated by extensive multilaboratory collaborative trials. In addition, the Food and Drug Administration approved E-test has proved valuable as a user-friendly alternative to NCCLS methods. Highlights of the evolution of various methods, their variations and their failures, factors that affected the development of the current standards, and the rationale for susceptibility testing of anaerobes are discussed herein. ROLE OF SUSCEPTIBILITY TESTING OF ANAEROBES IN THE 21ST CENTURY Antibiotic resistance to multiple agents among anaerobes has been increasing steadily over the past few decades, such that some previously effective antimicrobials are now considered ineffective or second-line agents [1]. Until recently, antibiotic susceptibility testing of anaerobes has been limited in part because of budgetary reductions in hospital clinical laboratories, a concomitant loss of expertise at these institutions, a lack of automated testing for anaerobes, and a failure to consider resistance as important to clinicians [2, 3]. However, the need for increased testing is now evident and recommended, given reports of clinical failures associated with antibiotic-resistant strains [4]. The National Committee for Clinical Laboratory Standards (NCCLS) has now suggested that clinical laboratories strongly consider surveillance testing annually, with testing of Reprints or correspondence: Dr. David W. Hecht, Professor of Medicine, Microbiology and Immunology, Division Director, Infectious Diseases, Loyola University Medical Center, Hines VA Hospital, 2160 S. First Ave., Maywood, IL (Dhecht@lumc.edu). Clinical Infectious Diseases 2002; 35(Suppl 1):S by the Infectious Diseases Society of America. All rights reserved /2002/3505S1-0007$15.00 individual isolates performed in certain clinical settings. The suggested changes reflect recognition of the emerging resistance problem and the availability of validated reference and alternative methods published most recently [5]. The current recommendations are the result of extensive work performed by numerous investigators in the last half of the 20th century. EVOLUTION OF SUSCEPTIBILITY TESTING IN THE 20TH CENTURY : the beginnings. The earliest methods used in the susceptibility testing of anaerobes were naturally rooted in those used for aerobic bacteria. In 1951, Jackson and Finland [6] described the results of extensive comparisons among available methods for testing susceptibility of aerobic organisms and laid the foundation for methods used by others in the development of methods for anaerobic bacteria. Comparison of turbidimetric, tube dilution, plate dilution, and medicated disk methods revealed wide variation in MICs for all antibiotics and were method-dependent. Those authors also observed that inoculum, period of incubation, and choice of complete or partial inhibition as an end point were the factors that most profoundly influenced results. This led the authors to comment that undue re- S28 CID 2002:35 (Suppl 1) Hecht

2 liance was placed on the results of tests, without adequate appreciation of their limitations. These findings, identified early in the development of methods for aerobic susceptibility testing, remain recurring themes throughout the development of susceptibility testing methods for anaerobes, as seen below. Although there are a few scattered references to antibiotic sensitivity testing of Bacteroides prior to 1955, there are virtually no descriptions of methods, and they will not be discussed further [7 10]. Instead, one of the earliest method descriptions was offered in 1955 by Garrod [11], who provided some detail of a plate dilution (agar dilution) method that used peptone agar, lysed horse blood (to liberate V factor), and doubling antibiotic dilutions. An inoculum consisted of a 1-mm loop of broth culture spread over a 2-cm diameter on a 12-quadrant plate, incubated anaerobically in H 2 and 5% CO 2 for 48 h, and MICs read where no growth appeared. In that early study, penicillin, streptomycin, chloramphenicol, oxytetracycline, erythromycin, polymyxin, and bacitracin were tested against several isolates of Bacteroides spp. The author found the results to be helpful in providing information for empirical therapy but considered them to be impractical for routine testing. The following year, Gillespie and Guy [12] described the use of disk-diffusion testing, although little detail about media and inoculum were given. In that study, up to 57 strains were tested with 10 antibiotics by disk diffusion, whereas 20 strains were tested by agar dilution to obtain MIC values. Both methods were considered comparable. A third susceptibility test was introduced in the Antibiotic Annual. Finegold and Hewitt [13] reported the largest study to date of susceptibility testing for 50 Bacteroides spp. and 10 antibiotics, using a serial 2-fold tube dilution technique; 45 of 50 isolates were tested with brain-heart infusion (BHI) broth (considered more satisfactory than trypticase soy broth ), and 5 of 50 strains, which were more fastidious, were tested with 2-fold plate dilution. A wide range of inocula (from a 1:2 to a 1:10 6 dilution of a 48- h culture), incubation times ( h), and 10% CO 2 in Brewer anaerobic jars were used in repeated testing. When a 10 2 dilution of a 48-h broth culture and 48-h incubation period were used, both broth and plate dilution methods were considered comparable, validating this alternative MIC testing method. Of note, the most active agents in that study were tetracycline, chloramphenicol, and erythromycin. Over the next 14 years, sporadic reports of susceptibility testing that used one of these methods appear in the literature without significant modifications. Most significant were reports of the activity of lincomycin against anaerobes and its effects on fecal flora [14, 15]. In 1965, Finegold et al. [14] reported testing 138 anaerobic species from 15 patients against lincomycin using the plate dilution method and a blood agar base (not specified) plus 5% sheep blood. Incubation conditions included 90% N 2 and 10% CO 2. Readings were taken at both 2 and 6 days, although only the 6-day results were reported. Overall, a very high degree of activity of lincomycin was seen among all anaerobes. Lincomycin was, of course, followed by 7-chlorolincomycin or clindamycin, a mainstay of therapy for patients with anaerobic infections. Also of note during this time period was the first proposal that antibiotic susceptibility patterns would aid in the classification of gram-negative anaerobes [16]. This work also included the prophetic statement that the use of standardized procedures for antibiotic susceptibility testing is essential if comparable results are to be obtained in different laboratories. However, this theme would not return until the 1990s (see below). The 1970s: additional methods and the development of a standard. Several important observations were made during the early 1970s that affected the performance of susceptibility testing of anaerobes. In 1970, Ingham et al. [17] reported the effect of 10% CO 2 added to pure H 2 and consequent lower ph. In contrast to their earlier study that used only H 2 [15], the addition of CO 2 resulted in 4 32-fold higher MIC values when erythromycin and lincomycin were tested against B. fragilis. These studies included a plate dilution method with nutrient agar, 5% defibrinated horse blood, and a phage dispenser to deliver an inoculum from broth that contained 10 5 cfu/ml. This led the authors to conclude that CO 2 should in fact be included in the incubating environment for anaerobes to mimic conditions of those in vivo. Subsequently, nearly all studies have used a CO 2 - containing anaerobic atmosphere for incubation. These studies were confirmed by Rosenblatt and Schoenknect [18]. Also in 1970, Thornton and Cramer [19] compared plate dilution (trypticase soy blood agar base plus 5% defibrinated sheep blood) directly with disk diffusion for 29 Bacteroides strains and demonstrated a poor correlation between the 2 methods for 5 of 7 antibiotics. The authors concluded that disk-susceptibility tests should not be used for Bacteroides susceptibility until zone sizes could be correlated with results obtained by more reliable methods. The year 1972 produced a plethora of work covering a wide variety of susceptibility testing methods and findings. In January 1972, Kagnoff et al. [20] reported the susceptibility results for 48 isolates of Bacteroides spp. derived from blood culture sources against 13 antibiotics, using a modified 3-disk method. Growth around 3-disk concentrations yielded sensitive strains when inhibited by all 3 disks, partial sensitivity when growth was inhibited by 2 disks, and resistant strains if growth occurred around all 3 disks. Media and other details were not specified, but 18 of 19 patients treated with an antibiotic to which the organism was susceptible did improve, although 9 of 14 patients who received an antibiotic to which the strain was resistant also improved. Thus, this represented one of the earliest studies Anaerobe Susceptibility Testing CID 2002:35 (Suppl 1) S29

3 linking susceptibility of anaerobes with outcome but also confirmed that antibiotic resistance does not necessarily equate with failure. The largest anaerobe susceptibility study up to that date was published by Martin et al. in 1972 [21]; 601 isolates of various anaerobic species were tested against 10 antibiotics, using the agar dilution method and a Steers/Foltz replicator for inoculation. BHI agar plus 5% blood was used as the testing medium. MICs were interpreted at 48 h when the plates showed no growth, a barely visible haze, or 1 discreet colony. Minimum bactericidal concentrations for 25 strains were also reported. Overall, those authors found good correlation with studies elsewhere that had used the same method [16, 17, 19]. That study was also the first to use the specific criteria for interpretation of end points suggested by Ericsson and Sherris [22]. In the same month, Sutter et al. [23] published a standardized antimicrobial disk susceptibility testing method for tetracycline; 100 strains of B. fragilis were tested by a modified Bauer-Kirby- Sherris-Turck disk susceptibility method and compared with both agar and broth dilution methods. Brucella supplemented with 5% sheep blood and 0.5 mg/ml menadione (and agar as indicated) was used for all 3 methods. Of note, the introduction of Brucella agar was based on preliminary studies (discussed but not shown) that compared it with Columbia, BHI, Mueller Hinton (MH), Schaedler, and trypticase soy base agars (TSA). The authors specifically noted that Schaedler contained too high a glucose concentration, there was poor growth overall on supplemented BHI, and there was no growth of some isolates on supplemented MH and TSA. Although supplemented Brucella and Columbia agars were considered comparable, the choice of Brucella was noted as arbitrary but remained a mainstay for future testing and the most recent recommended standard. (Note: Brucella base was initially tested because of its ready availability in the Finegold laboratory; S. Finegold, personal communication.) Incubation conditions included 10% H 2 and 90% N 2, to which 10% CO 2 was added. Correlation between methods was considered good, with no false resistance or susceptibility identified when break points recommended by the authors were used. Subsequently, the same group used similar methods for 6 antibiotics and the testing of other non-bacteroides organisms [24, 25]. In March 1972, Kislak [26] reported testing 40 clinical isolates of Bacteroides against 24 antibiotics, using BHI agar supplemented with defibrinated horse blood and an inoculum of cfu/cm 2. Included in the results was a report of the excellent activity of clindamycin and the confirmation of earlier reports of significant resistance to tetracycline. In June 1972, Wilkins et al. [27] introduced a new method, single-disk diffusion, and compared it with broth dilution. In that study, actively growing cultures were added to melted BHI agar supplemented with heme, menadione, and 0.5% yeast extract (BHIS), to which commercially available disks were dispensed on the surface of solidified medium. Zone diameters were observed under high-intensity light. Inhibition of 80% or more growth around a disk was considered to be the zone of inhibition. Five different anaerobic genera were tested against 7 antibiotics, with good correlation for rapidly growing anaerobes, although the method was not recommended for slow growers because of a lack of distinct zones. In 1973, Wilkins and Thiel [28] published the broth disk method. In their initial description, 1 4 disks of various antibiotics were added to BHIS broth medium to achieve concentrations near the susceptible break point, and the broths were inoculated with 1 drop of an h culture. Susceptibility was interpreted as!50% turbidity compared with control. These results were correlated with both broth dilution and diskdiffusion methods. Compared with an MIC method (broth dilution), values were within 2 double dilutions. An overall correlation of 97% among 1015 comparisons was achieved. They noted that disk diffusion did not correlate as well, especially when testing Clostridium spp., Peptostreptococcus spp., and a variety of other organisms that grew poorly. The principle advantage of the broth disk method was the user-friendly approach that could help determine whether an organism was susceptible. However, by design, this method did not provide an MIC result. In the same year, support for the disk-diffusion method was tempered by limited correlation with agar dilution for erythromycin and lincomycin because a great deal of overlapping of zone diameters among strains classed as susceptible, intermediate, and resistant [25]. However, it retained good correlation for chloramphenicol, clindamycin, and penicillin G. Seven publications in 1975 were important in establishing parameters for standardized susceptibility testing of anaerobes. Tally et al. [29, 30] and Sutter et al. [31] each published susceptibility results on the basis of the agar dilution method that differed in both the media and inoculum used for testing. Tally et al. used BHIS medium and an inoculum that was 10-fold lower than that reported in most other publications (10 4 cfu/ spot) to test penicillin, cefoxitin, and other cephalosporins. Sutter et al. [32] published the second edition of the Wadsworth manual, describing the Wadsworth method and results of susceptibility to carbenicillin, cefoxitin, and related drugs, using Brucella agar supplemented with sheep blood and vitamin K 1 and an inoculum of 10 5 cfu/spot. Both groups used similar incubation conditions and interpretative criteria and obtained similar results, despite the differences in media and inoculum. From 1975 to 1997, both groups continued to perform susceptibility testing using their respective methods, until a unifying standard was published by NCCLS in 1997 (see below). The Wadsworth manual also included specific recommendations for testing with both the broth dilution and the agar diffusion methods [31]. S30 CID 2002:35 (Suppl 1) Hecht

4 Important and more user-friendly modifications of 2 testing methods were also introduced in The limited agar dilution method was described as an economical approach to determine whether isolates were susceptible to various antibiotics [33]. This method used a 4-quadrant plate that contained 3 different concentrations of the test antibiotic and a control. Concentrations at or around a potential break point were tested, and conclusions were drawn about the susceptibility of the organism without an actual MIC result. Similarly, a 3-tube broth dilution method that used antibiotic dilutions at or around the break point was also described by Stalons and Thornsberry [34]. Although offering an economical and timesaving advantage to the most widely used methods, these modifications were limited ultimately by the wide range of MIC values needed for validating quality control strains incorporated in the standards (see below). Instead, the broth microdilution method introduced by Rotilie et al. [35] provided an important modification of broth dilution that was economical, allowed testing of multiple antibiotics simultaneously, and provided MIC end points. In that method, 96-well microtiter trays containing 2-fold diluted antibiotics in Schaedler broth were inoculated with cfu/ml and incubated in 80% N, 10% H 2, and 10% CO 2 for up to 48 h. MICs were read as the lowest concentrations of antibiotics to inhibit growth, which was interpreted as diffuse turbidity, a single large dot, or a filamentous network of growth. Both a B. fragilis and a C. perfringens strain (not specified) were used as controls. The authors found their results to be comparable to others and suggested this method as convenient to test multiple antibiotics with commercially available materials. Thus, at the conclusion of 1975, significant development and adoption of techniques for susceptibility testing of anaerobes had occurred, although they were still remarkable for the number of variables incorporated into the different testing methods. In 1976, an alternative medium for agar dilution testing was proposed by Wilkins and Chalgren [36] that was ultimately adopted as the initial reference standard. Wilkins-Chalgren medium was produced and demonstrated growth of most anaerobes, with the advantage that blood supplementation was not needed. The authors acknowledged that they used this medium for only 1 year in the laboratory but found it advantageous because it could be made from reproducible components. No susceptibility results for this medium were offered. However, Sutter et al. [37] reported the results of a multilaboratory collaborative evaluation of a proposed reference standard that used the agar dilution method and Wilkins-Chalgren medium in That study was the first organized under the auspices of the NCCLS for anaerobe susceptibility testing and was performed in 10 laboratories. Results of 6 antibiotics and 10 selected strains were compared among all laboratories, using a 10 5 cfu/spot inoculum and incubation in anaerobic jars for 48 h. Each site also tested each combination on 3 separate days. Following strict protocols, results from this landmark study demonstrated a very high degree of reproducibility, with 193% agreement among all laboratories. However, all laboratories had difficulty in growing some strains, particularly gram-positive cocci. Therefore, the authors recognized at that early date that the method might have been limited for more fastidious organisms that may not grow as well, including those that were not tested in the study. Despite problems with growth of some anaerobic bacteria, the authors recommended Wilkins-Chalgren medium as a reference standard. In the same year, the first proposed standard for susceptibility testing of anaerobes (PSM-11) was published by the NCCLS [38]. In that first standard, the method described by Sutter et al. [37] for agar dilution was formally adopted. In addition, quality control strains and reference ranges were also proposed, but no break points were incorporated at that time. Simultaneously, work was also proceeding with modification of the broth microdilution method. Rosenblatt et al. [39] reported the direct comparison of 6 broth media, in which 3 anaerobic MIC broth (Wilkins-Chalgren formula), Schaedler, and thioglycolate broth performed the best. However, those authors also recognized the limitation of the method for slow-growing and fastidious anaerobes. During the same decade, there were numerous publications that either used published methods or reported the use of different media or further attempts at refining the methods. These included the use of a 24-component medium for agar dilution [40], disk testing of slow-growing anaerobic bacteria [41], a simplified tube test [42], and examples of agar dilution testing with either Brucella or BHIS media [43 45]. The 1980s: refinement of the standard and recognition of alternative methods. Early on, standardized methods were published in various reputable manuals as guides for susceptibility testing. The third edition of the Wadsworth manual, published in 1980, incorporated the proposed NCCLS standard but as an alternative method, along with macrobroth dilution [46]. Agar diffusion testing also remained, but new methods for slow-growing strains were performed as a separate methodology, to recognize the variations noted previously for this method. In the same year, the Manual of Clinical Microbiology included a chapter by Sutter and Washington [47] covering susceptibility testing of anaerobes. Methods included agar dilution, broth dilution, broth microdilution, and the broth disk tests, with modifications provided for aerobic incubation of broth disk tubes [48]. The first reported evaluation of a commercial broth microdilution method was made in 1982 by Jones et al. [49]. That work represented the first collaborative evaluation of commercially prepared microtiter trays and compared results directly with the reference standard. In summary, 98% interlabo- Anaerobe Susceptibility Testing CID 2002:35 (Suppl 1) S31

5 ratory agreement was achieved for the broth microdilution method, with 91% 95% agreement when interpretative end points were used, compared with that of agar dilution. These results were later confirmed by Baron and Bruckner [50]. Also in 1982, Murray and Niles [51] reported comparable results, using the broth microdilution method incubated in 5 different anaerobic environments: an anaerobic chamber, 2 gas-pack jars, an evacuated anaerobic jar, and a bio-bag. Although discordant results for metronidazole were noted for the bio-bag, results were otherwise highly correlative. As a result of their findings, clinical laboratories were no longer limited to use of a glove box or similar incubation device. The first tentative standard, M11-T, was published in the same year by the NCCLS [52] and described the same methods published in the proposed standard. From 1983 to 1985, 3 surveys of antibiotic susceptibility among anaerobes were reported by the nationwide study group that documented an increase in antibiotic resistance among B. fragilis [53 55]. These surveys used the agar dilution method published by Tally et al. [29], discussed above. The use of control strains in each of the studies supported the validity of the changing susceptibility patterns detected by this method. Furthermore, Aldridge et al. [56] were reporting similar findings, using the broth microdilution method and anaerobe MIC broth, although intermethod variability was noted [57]. In contrast, the consistency of the broth disk method was challenged in 1984 by Barry and Packer [58], who reported that the broth microdilution method was more reproducible than disk elution, especially in testing cephalosporins, which was confirmed by Aldridge et al. in 1990 [59]. The first approved standard for antimicrobial testing of anaerobes was published by the NCCLS in 1985 (M11-A). That document continued to recommend Wilkins-Chalgren medium as the standard but for the first time also included acceptable ranges for quality control strains and 14 antibiotics [60]. The NCCLS simultaneously published a separate document that described alternative methods for susceptibility testing of anaerobes (M17-P) [61]. This latter document detailed descriptions of limited agar dilution, broth microdilution, and broth disk elution procedures, which were published for the benefit of clinical laboratories unable or unwilling to perform the more time-consuming and labor-intensive agar dilution method. Neither document included a mention of the disk-diffusion method. The fourth edition of the Wadsworth manual was also published during the same year [62]. That guide included both broth macrodilution and microdilution methods, as well as broth disk elution, Wadsworth and reference agar dilution methods, and a b-lactamase test for the screening of isolates for penicillin resistance. In that edition, agar disk diffusion testing was no longer recommended; the explanation noted the complexities and variation introduced by slow growth rates of anaerobic bacteria. After 1985, several reports of anaerobe susceptibility testing appeared that used predominantly the agar dilution and broth microdilution methods [59, 63]. Meanwhile, the most significant changes in anaerobe susceptibility testing methods emerged predominantly through the consensus process of the NCCLS. The second edition of M11 (M11-T2) was published in 1989 [64]. That document essentially merged both M11-A and M17-P but with some additions. The reference standard agar dilution method, using Wilkins-Chalgren media, and the Wadsworth method were offered as essentially equivalent, although with a notation that MICs might be higher when Brucella medium was used. In addition, the limited agar dilution, broth dilution, broth microdilution, and broth disk elution methods were included and described in extensive detail. Of note, 5 different media were recommended for use in broth microdilution plates, and a comment was included that when this method was used, MIC values might be 2-fold lower than those obtained by the agar dilution method. Aside from methodology, M11-T2 also included, for the first time, resistant and susceptible MIC break points for 20 antibiotics as well as quality control ranges for the same agents. The 1990s: consolidation and validation of methods. M11-A2, the second approved standard, was published in 1990 [65]. Although highly similar to M11-T2, this version did not include broth disk elution because of recognized difficulties in performance when newer cephalosporins were tested, clustering of MIC values around the break point, and a lack of established control procedures [59]. However, that document did include recommendations regarding the use of b-lactamase testing as a screening tool similar to that of the Wadsworth manual [62] but also recognizing its limitations. In the same year, fastidious anaerobe agar was introduced as a potentially superior medium for testing of Fusobacterium spp. because of superior growth characteristics [66]. However, the medium was not incorporated into the NCCLS standards. In 1991, the E-test was introduced as the first new method for anaerobe susceptibility testing since commercial broth microdilution. The E-test was reported as a alternative userfriendly, agar gradient method with a high degree of correlation to agar dilution for testing anaerobic bacteria [67]. Subsequently, several studies have validated the correlation of this method to the reference standard agar dilution method, although strict adherence to proper incubation conditions is essential for metronidazole [68 71]. This FDA-approved method continues to be used as a relatively easy but somewhat expensive test to determine MIC values for individual anaerobic isolates. Despite its high degree of correlation, the E-test has not been specifically identified in NCCLS documents, including those S32 CID 2002:35 (Suppl 1) Hecht

6 testing anaerobes, because of the exclusion of proprietary products from their documents. The next significant change in anaerobe susceptibility testing appeared in 1993 with the publication of M11-A3. That document included important changes in the interpretation criteria without significant changes in methodology [72]. In particular, interpretation of end points in the agar dilution method was described in detail to include no growth, barely visible haze, multiple tiny colonies, or few large colonies. In addition, 3 interpretative end points were included to recognize the difficulty in the reading of end points. Thus, susceptible, intermediate, and resistant break points were listed, with a footnote indicating that strains with an intermediate break point would likely respond to therapy if maximum doses of antibiotics were used. The inclusion of the intermediate category also brought the anaerobic interpretative categories in line with those for aerobic bacteria. Early in 1995, the NCCLS anaerobe working group recognized the lack of consistent methods in the literature and the inconsistencies in interpretation of the multiple methods (as noted above for M11-T2, M11-A2, and M11-A3), which limited the comparability of data. Furthermore, rising antibiotic resistance among more fastidious anaerobes prompted the group to validate and recommend a single reference method that would allow the growth and testing of virtually all anaerobes, which would serve as the method to which others could be rigorously compared. To that end, several multicenter collaborative trials were designed and conducted over that time period. The first objective was to determine a supportive testing medium that would grow virtually all anaerobes. A 5-laboratory study was conducted that compared the growth characteristics of 5 different media, including Wilkins-Chalgren agar with and without whole or laked sheep blood (frozen and thawed) and Brucella agar supplemented with vitamin K 1, hemin, and either whole or laked sheep blood and 30 varied anaerobic isolates [73]. The results of these carefully designed studies supported the use of supplemented Brucella agar and laked sheep blood as equal to or better than most other media tested in the growth of all anaerobes and was used for later phases of testing. To answer whether choice of media affected susceptibility results, a second 5-laboratory study was conducted that compared the MIC results for 58 clinical isolates and 6 antibiotics among 3 testing media. Results for Wilkins-Chalgren agar with and without laked sheep blood were compared with supplemented Brucella blood agar [74]. In summary, a high degree of correlation was found when MIC values on Wilkins-Chalgren were compared with those of the other media for the organisms that would grow on both media. This observation was reassuring, given the observation elsewhere (M11-T2) that results might differ between the 2 methods, and essentially validated any previous data that used the guidelines and either reference method. The group concluded that supplemented Brucella agar would give results equivalent to the Wilkins-Chalgren reference standard while supporting the growth of fastidious organisms for susceptibility testing. As a result, the NCCLS published M11-A4, in 1997, which recommended only a single reference method that used supplemented Brucella agar, eliminating Wilkins-Chalgren medium. In addition, M11-A4 also contained, for the first time, color photographs of end point interpretations when the agar dilution method was used, to assist investigators and laboratory technicians in the reading of end points. Most recently, the anaerobe working group of the NCCLS has conducted additional studies to compare the results of broth microdilution with those of the new reference standard. The first multilaboratory study compared results of the growth of fastidious and nonfastidious anaerobes in 4 broths: anaerobe MIC with and without lysed horse blood and Brucella broth that contained vitamin K 1 and hemin with and without lysed horse blood. Those studies (data not shown) demonstrated the superior growth characteristics of Brucella blood broth over the other media. This medium, which is essentially equivalent to the reference agar dilution method, was used in the next set of studies to compare MIC results between the broth microdilution and reference agar dilution method. The first multicenter collaborative trial compared the susceptibility results between the 2 methods for 40 different anaerobic species but did not achieve statistical correlation. This was attributed in part to inadequate growth of the most fastidious organisms and the small number of isolates tested from each species. Subsequently, a second multilaboratory collaborative trial that compared these methods tested 120 B. fragilis group isolates. A very high degree of correlation was in fact achieved for 7 of 8 antibiotics: clindamycin, cefoxitin, ampicillin/sulbactam, piperacillin, metronidazole, trovafloxacin, and ertapenem (data not shown). Correlation was not established for imipenem, which was attributed to its inherent instability in solution. As a result of these large recent studies, the NCCLS published its most recent guideline for anaerobe susceptibility testing, M11-A5 [5], which recommends a single agar dilution reference method and a single broth dilution method for testing of anaerobic bacteria. However, because of the lack of correlation to the reference method for some anaerobes, the broth dilution method is currently recommended only for testing the members of the B. fragilis group and the 7 antibiotics listed above. An 8-laboratory study was also performed to establish quality control ranges for the new media and the broth dilution method. They are now listed in a separate table. Finally, additional color photographs of end point interpretations for both agar and broth microdilution were provided for consistency of readings. However, the document also eliminated 2 infrequently used methods, broth macrodilution and limited agar dilution. Studies to further compare results of the reference agar method and Anaerobe Susceptibility Testing CID 2002:35 (Suppl 1) S33

7 broth microdilution for anaerobes other than the B. fragilis group and the validation of additional antibiotics are ongoing; the results will be published in future editions of the M11 series, by NCCLS. EVOLUTION OF ANAEROBE SUSCEPTIBILITY TESTING: SURVIVAL OF THE FITTEST? From its initial descriptions, which used one method, to the rapid expansion of several methods and variables and, ultimately, the extinction of many, anaerobe susceptibility testing can truly be described as an evolutionary process. In total, 116 methods, 116 media, and a wide range of other variables have been described in the US literature alone over the past 45 years, not counting other reports worldwide. It is presumed that, through recent rigorous studies, the expansion and then contraction of methods will now allow investigators to compare the results of susceptibility testing with each other when identical methods and conditions are used. Ultimately, these methods may also become extinct with the development and deployment of modern technologies and with genetic means to identify and determine resistance. Acknowledgment I thank Dr. Gayatri Vedantam for valuable contributions to the reading and preparation of this manuscript. References 1. Snydman DR, Jacobus NV, McDermott LA, et al. Multicenter study of in vitro susceptibility of the Bacteroides fragilis group, 1995 to 1996, with comparison of resistance trends from 1990 to Antimicrob Agents Chemother 1999; 43: Goldstein EJC, Citron DM, Goldman RJ, Claros MC, Hunt-Gerrado S. United States national hospital survey of anaerobic culture and susceptibility methods, II. Anaerobe 1995; 1: National Committee for Clinical Laboratory Standards (NCCLS). Methods for antimicrobial susceptibility testing of anaerobic bacteria, 4th ed. Approved standard M11-A4. Villanova, PA: NCCLS, Nguyen MH, Yu VL, Morris AJ, et al. 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