Antianaerobic Antimicrobials: Spectrum and Susceptibility Testing · exhibiting multidrug...

Antianaerobic Antimicrobials: Spectrum and Susceptibility Testing

Itzhak Brook,a Hannah M. Wexler,b,c Ellie J. C. Goldsteinc,d

Department of Pediatrics, Georgetown University School of Medicine Washington, DC, USAa; Greater Los Angeles Veterans Administration Healthcare Systemb and DavidGeffen School of Medicine at UCLA,c Los Angeles, California, USA; R. M. Alden Research Laboratory, Culver City, California, USAd

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .526INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .526SUSCEPTIBILTY TESTING OF ANAEROBIC BACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .527

Standardization of Testing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .528Surveillance Tests for Particular Hospitals or Geographic Regions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .528Testing in a Clinical Setting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .528Testing in a Research or Reference Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .529

Agar dilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .529Broth microdilution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .529Etest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .529Spiral gradient endpoint system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .529

Commercially Available Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .530�-Lactamase Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .531Factors Contributing to Variability in MIC Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .531Detection of Resistance by Using Molecular Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .532Is a Rapid Test on the Horizon? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .532

ANTIMICROBIAL AGENTS EFFECTIVE AGAINST ANAEROBIC BACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .533�-Lactam Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .533

Resistance to �-lactam antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .536Chloramphenicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .537The Macrolides: Erythromycin, Azithromycin, and Clarithromycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .538Clindamycin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .538

Clindamycin resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .538Metronidazole and Tinidazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .538

Metronidazole resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .539Tetracyclines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .539

Tetracycline resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .539Fluoroquinolones. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .539

Fluoroquinolone resistance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .540Other Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .540

GENERAL CONSIDERATION OF ANTIMICROBIAL SELECTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .540CURRENT PRACTICE OF SELECTION OF ANTIBIOTIC FOR ANAEROBIC BACTERIA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .541CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .542REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .542AUTHOR BIOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .546

SUMMARY

Susceptibility testing of anaerobic bacteria recovered from se-lected cases can influence the choice of antimicrobial therapy. TheClinical and Laboratory Standards Institute (CLSI) has standard-ized many laboratory procedures, including anaerobic suscepti-bility testing (AST), and has published documents for AST. Thestandardization of testing methods by the CLSI allows compari-sons of resistance trends among various laboratories. Susceptibil-ity testing should be performed on organisms recovered from ster-ile body sites, those that are isolated in pure culture, or those thatare clinically important and have variable or unique susceptibilitypatterns. Organisms that should be considered for individual iso-late testing include highly virulent pathogens for which suscepti-bility cannot be predicted, such as Bacteroides, Prevotella, Fusobac-terium, and Clostridium spp.; Bilophila wadsworthia; and Sutterellawadsworthensis. This review describes the current methods forAST in research and reference laboratories. These methods in-clude the use of agar dilution, broth microdilution, Etest, and the

spiral gradient endpoint system. The antimicrobials potentiallyeffective against anaerobic bacteria include beta-lactams, combi-nations of beta-lactams and beta-lactamase inhibitors, metroni-dazole, chloramphenicol, clindamycin, macrolides, tetracyclines,and fluoroquinolones. The spectrum of efficacy, antimicrobial re-sistance mechanisms, and resistance patterns against these agentsare described.

INTRODUCTION

Infections caused by anaerobic bacteria are common and may beserious and life-threatening. Anaerobes are the predominant

components of the bacterial flora of normal human skin and mu-cous membranes (1), and they are a common cause of bacterial

Address correspondence to Itzhak Brook, [email protected].

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infections of endogenous origin. Because of their fastidious na-ture, they are difficult to isolate from infectious sites and are oftenoverlooked. Their isolation requires appropriate methods of col-lection, transportation, and cultivation of specimens (2–5). Treat-ment of anaerobic bacterial infections is complicated by the rela-tively slow growth of these organisms (which makes diagnosis inthe laboratory possible only after several days), by the frequentpolymicrobial nature of the infection, and by the growing resis-tance of anaerobic bacteria to antimicrobial agents.

Failure to direct therapy against anaerobic organisms oftenleads to clinical failures. The inadequate isolation, identification,and subsequent performance of susceptibility testing of anaerobesfrom an infected site can prevent detection of antimicrobial resis-tance. Therefore, correlation of the results of in vitro susceptibilityand clinical and bacteriological responses can be difficult or im-possible (1, 3, 6). This discrepancy occurs because of a variety ofreasons. Individuals may improve without antimicrobial or surgi-cal therapy, and others can get better because of adequate drain-age. In some instances of polymicrobial infection, eradication ofthe aerobic component may be adequate, although it is well estab-lished that it is important to eliminate the anaerobic pathogens (2,7–14).

Reasons that may lead to failure of therapy include variation inthe duration, severity, and extent of infection; lack of surgicaldrainage or poor source control; the patient’s age, nutritional sta-tus, and comorbidities; impaired host defenses; poor penetrationand low levels of the antimicrobial at the site of infection; enzy-matic inactivation of antimicrobials; low pH at the infection site;and inaccuracies in the susceptibility testing procedure.

Despite all of these factors, a correlation between the antimi-crobial resistance of the anaerobic pathogens and poor clinicaloutcome has been reported in several retrospective studies (7–9).There are a number of studies showing that inappropriate therapywill directly affect clinical outcome (10–15).

A prospective study of Bacteroides bacteremia reported adverseclinical outcomes for 128 individuals who received an antibiotic towhich the organism was not susceptible (14). Clinical outcomewas correlated with results of in vitro susceptibility testing of Bac-teroides isolates recovered from blood and/or other sites and wasdetermined by three endpoints: mortality at 30 days, clinical re-sponse (cure versus failure), and microbiological response (erad-ication versus persistence). The mortality rate among those whoreceived inactive therapy (45%) was higher than that among pa-tients who received active therapy (16%; P � 0.04). Clinical failure(82%) and microbiological persistence (42%) were higher forthose who received inactive therapy than for patients who receivedactive therapy (22% and 12%, respectively; P � 0.0002 and 0.06,respectively). In vitro activity of agents directed at Bacteroides spp.reliably predicts outcome (specificity of 97% and positive predic-tive value of 82%). The authors of this study concluded that anti-microbial susceptibility testing may be indicated for patientswhose blood specimens yield Bacteroides spp. (14).

These findings emphasize that it is important to perform sus-ceptibility testing of organisms recovered from certain selectedcases to guide therapeutic choices. Susceptibility testing should beperformed for organisms recovered from sterile body sites, thosethat are isolated in pure culture, and those that are clinically im-portant and have variable or unique susceptibility. Anaerobic in-fections for which susceptibility testing is indicated include (i)serious or life-threatening infections (e.g., brain abscess, bactere-

mia, or endocarditis), (ii) infections that failed to respond to em-pirical therapy, (iii) infections that relapsed after initially respond-ing to empirical therapy, (iv) infections where an antimicrobialwill have a special role in the patient’s outcome, (v) when an em-pirical decision is difficult because of an absence of precedent, (vi)when there are few susceptibility data available on a bacterial spe-cies, (vii) when the isolate(s) is often resistant to antimicrobials, or(viii) when the patient requires prolonged therapy (e.g., septicarthritis, osteomyelitis, undrained abscess, or infection of a graftor a prosthesis). The standardization of testing methods by theClinical and Laboratory Standards Institute (CLSI) (Wayne, PA)allows for comparison of resistance trends among various labora-tories (15–17). Organisms that should be considered for individ-ual isolate testing include highly virulent pathogens for which sus-ceptibility cannot be predicted, such as Bacteroides, Prevotella,Fusobacterium, and Clostridium spp.; Bilophila wadsworthia; andSutterella wadsworthensis.

The routine susceptibility testing of all anaerobic isolates isextremely time-consuming and is not cost-effective. However,susceptibility testing should be performed for epidemiologicaland survey purposes for a limited and selected number of anaer-obic isolates. Antibiotics tested should include penicillin, a beta-lactam– beta-lactamase inhibitor combination (BL-BLIC), clin-damycin, metronidazole, and a carbapenem (i.e., imipenem,meropenem, or ertapenem). If needed, ancillary susceptibilitytesting can be performed for cefoxitin, tigecycline, and moxifloxa-cin, which have approved antianaerobe indications.

Antimicrobial resistance among anaerobes has consistently in-creased in the past 3 decades, and the susceptibility of anaerobicbacteria to antimicrobial agents has become less predictable. Themost commonly isolated antibiotic-resistant anaerobes are spe-cies within the Bacteroides fragilis group (18). Resistance to severalantimicrobial agents by B. fragilis group species and other anaer-obic Gram-negative bacilli (AGNB) has increased over the pastdecade (15–17, 19–22). Resistance has also increased among otheranaerobes, such as Clostridium spp., that were previously very sus-ceptible. This increase makes the choice of appropriate empiricaltherapy even more difficult. Resistance patterns have been moni-tored through national and local surveys, but susceptibility testingof anaerobic bacteria at individual hospitals is rarely done (20).

This review describes the antimicrobial agents that are effectiveagainst anaerobic bacteria and the methods used to perform anti-microbial susceptibility testing of these organisms.

SUSCEPTIBILTY TESTING OF ANAEROBIC BACTERIA

The antibiograms of anaerobic bacteria have become increas-ingly unpredictable, and multiresistant clinical isolates are ap-pearing, confounding the concept of foolproof anaerobic ther-apy (21, 23, 147). Resistance to even the most active drugs, suchas imipenem, piperacillin-tazobactam, ampicillin-sulbactam,and metronidazole, has been reported (23, 25, 26). Further-more, there are clear differences in the geographic patterns ofresistance and even differences in resistance patterns in differ-ent hospitals in a single city, perhaps due in part to the vari-ability in prescribing patterns (27). Numbers of reports ofbroadly multidrug-resistant B. fragilis strains as well as num-bers of reports of resistance arising during treatment have in-creased (6, 11, 14, 22, 28). There is evidence that suboptimaltherapy can actually select for antibiotic resistance and eveninduce transfer of resistance determinants. Also, more strains

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exhibiting multidrug resistance (MDR) have been found(21, 23).

Taken together, these factors emphasize the need for antimi-crobial susceptibility testing of anaerobes as well as periodic sur-veillance antimicrobial susceptibility testing to detect geographicor temporal trends. The most appropriate susceptibility testmethod will differ depending on whether the test is being done fora specific isolate in a hospital laboratory (or a commercial labora-tory used by the hospital) or whether surveillance testing is beingperformed at a hospital or reference laboratory. In the last fewdecades, testing methodologies used have been standardized.

Standardization of Testing

The CLSI is a U.S. organization that evolved from a voluntaryconsensus organization in 1967 to become a World Health Orga-nization Collaborating Center for Clinical Laboratory Standardsand Accreditation. The CLSI has standardized many clinical pro-cedures, including anaerobic susceptibility testing, and has pub-lished documents for anaerobic susceptibility testing (commonlycalled M11) (16). CLSI policy does not permit it to advocate anycommercial technique; rather, it describes two reference methods(agar dilution and broth microdilution) and emphasizes thatother techniques, such as gradient techniques (generally referringto Etest) or commercial broth microdilution plates, may be usedas long as equivalence to the reference methods is established.Current recommendations of the CLSI limit the broth microdilu-tion method to testing of the B. fragilis group. Surveillance studiesperformed in reference laboratories in the United States andworldwide most commonly use the CLSI method (see below). Thenewest document, M11-A8, was published in 2012 (16). The CLSIreference standard is not intended for testing of single isolates;rather, it provides a standard against which other methods may bemeasured.

The European Committee on Antimicrobial Susceptibility Testing(EUCAST) publishes its own breakpoints; these are not always equiv-alent to those of the CLSI (29). However, EUCAST does not actuallyspecify a testing method for anaerobes. Most susceptibility studiesemanating from European countries use CLSI methodology, al-though breakpoint interpretation is often based on EUCAST rec-ommendations, and differences in reported resistance rates maybe due to differences in breakpoint determination.

Both Argentina (31) and Japan have published testing methods,but these are closely based on CLSI methodology. In 2007, TheCommittee on Antimicrobial Susceptibility Testing of the Japa-nese Society of Chemotherapy recommended the use of the CLSImethod (32). Recent large surveillance studies in Europe have alsoused CLSI methodology and often include both CLSI andEUCAST breakpoints (29, 147). Some studies refer to othermethod documents; a recent German multicenter study referredto a specific German document for testing methodology (24). Thedifferences between different technical methods may seem trivial;however, in cases where MICs cluster around breakpoint values,small changes in MICs (due to differences in media, inocula, orendpoint reading methods) may lead to perceived significant dif-ferences in resistance rates. Therefore, when trying to evaluate orcompare published studies, the method used should be taken intoaccount. At this point, the most commonly used method by far isM11-A8 of the CLSI (16).

Surveillance Tests for Particular Hospitals or GeographicRegions

Surveillance tests have been conducted for years by groups world-wide and reflect sweeping general trends (18, 24, 27, 33, 34, 147)However, these results will not necessarily reflect the patterns ofspecific patients or hospitals. Because of this, the CLSI stronglyrecommends that hospitals conduct at least annual surveillanceantimicrobial susceptibility testing to elucidate their local pat-terns. The numbers and choice of species of strains tested shouldreflect the frequency with which they are isolated. At least 50 to100 strains should be tested in order to obtain an accurate pictureof the pattern of local isolates, and if isolates from different bodysites are available, they should be included. At least 20 isolates ofBacteroides spp. and 10 isolates of other frequently isolated generashould be tested. If the expertise is not available in the hospitalclinical laboratory, the strains should be sent to a reference labo-ratory for testing.

Large reference laboratories may use CLSI-approved methods(described briefly below), which are more laborious and requiremore in-house preparation than commercially available methods.In these cases, the antimicrobials tested can be tailored to reflectthe hospital’s particular formulary. Laboratories should ideallyinclude at least one agent from each antimicrobial class, even if itis not included on the formulary. Many reference laboratories willuse commercially prepared panels; in these cases, the agents testedwill depend on whichever antibiotics are included in the commer-cial panel that the laboratories are using for testing. The CLSImicrodilution method is approved only for B. fragilis group or-ganisms, because many other anaerobes will not consistently growwell in broth media. The CLSI recognized that there are commer-cially available broth microdilution panels that are FDA approvedfor testing of all anaerobes and may work satisfactorily for certainnon-B. fragilis group species. At the time of writing of this publi-cation, the only ready-made commercially available broth mi-crodilution panel identified was produced by Sensititre (Trek Di-agnostic Systems). Other laboratories performing large-scalesurveillance testing may prepare their own broth microdilutionpanels (e.g., the R. M. Alden Laboratory [Santa Monica, CA] usesthe CLSI method of broth microdilution and prepares frozen pan-els according to need).

The results of the surveillance study should be maintained andrecorded so that local trends in emerging resistance may be recog-nized and documented. If formal surveillance testing cannot bedone, hospitals should collect and summarize their antimicrobialsusceptibility test results and create a hospital-specific antibi-ogram that can be consulted if needed. A 2008 survey of clinicalhospital laboratories (20) revealed that fewer than half of the lab-oratories did any kind of anaerobic testing at all, either in-house orany kind of batch testing sent to an outside laboratory. At the veryleast, periodic batch testing should be strongly encouraged.

Testing in a Clinical Setting

Susceptibility testing may not be necessary for many routinepatient isolates. The CLSI suggests testing of isolates fromblood, brain abscess, endocarditis, osteomyelitis, joint infec-tion, infection of prosthetic devices, or vascular grafts (seeabove). Also, any bacteria isolated from normally sterile bodysites should be tested (as long as they are not likely to be con-taminants). Isolates from patients likely to undergo long-term

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therapy should be tested so that any development of resistancecan be recognized. Obviously, any isolate from a therapy failureor in a case in which the therapeutic decisions will be influ-enced by the results should be tested.

Organisms to test should include those most likely to be themost resistant (such as B. fragilis group species) or highly virulent(certain Bacteroides, Prevotella, Fusobacterium, Clostridium, Bilo-phila, and Sutterella species), especially if their susceptibility pat-terns are unpredictable. Agents to test should include those on thehospital formulary, and the agent that is being considered or usedfor therapy should be included if at all possible.

The most recently published surveys of anaerobic susceptibilitytesting performed in a clinical laboratory indicated that, as of the timeof the survey, only 21% (21/98) of hospital laboratories performedanaerobic susceptibility testing in-house (20). This indicated a steepdecline from earlier rates; in 1990, 70% of hospital laboratories per-formed susceptibility testing (36), which declined to 33% in 1993(37). When testing was performed, blood isolates were always tested.Testing was performed by 85% (17/20) of laboratories for sterile bodysite isolates and by 70% (14/20) of laboratories for selected surgicalwound isolates. An additional 40% (8/20) of laboratories surveyedwould also perform susceptibility testing by special request. In thissurvey, most hospital laboratories used the Etest (62%; 13/21) forsusceptibility testing, while only 17% of reference laboratories used it.All the reference laboratories used broth microdilution for suscepti-bility testing, as did 40% (8/20) of hospital laboratories (all commer-cially prepared). Since nearly two-thirds of laboratories do not per-form testing (and even those that test do it on only a limited basis), theclinicians often choose therapy based on manufacturers’ informa-tion, FDA indications, published studies, or their clinical judgment(20).

At this time, most commercial laboratories use Etest method-ology for performing anaerobic susceptibility testing on isolatessent to them for testing. The Etest is particularly suitable for test-ing of one or a few isolates against multiple agents (as long as theparticular agent is available on an Etest strip). Currently, we arenot aware of any commercially available ready-made broth mi-crodilution panels that are “FDA approved” for clinical diagnosticuse. The only commercially available ready-made panel is pro-duced by Sensititre (Trek Diagnostic Systems); it is designated forresearch purposes only and is not FDA approved for diagnostictesting. Thus, a clinical laboratory would have the option of eitherusing Etest (which is FDA approved), using noncommercial pan-

els with CLSI-approved methodology (thus, FDA approval wouldnot be relevant), or sending the isolates to a commercial or refer-ence laboratory for testing.

Testing in a Research or Reference Laboratory

Agar dilution. Agar dilution involves the incorporation of differ-ent concentrations of the antimicrobial agent into a nutrient agarmedium followed by the application of a standardized number ofbacterial cells to the surface of the agar plate (Fig. 1). Plates areread after �48 h of growth by visually comparing the growths ofdifferent strains in the series, and the MIC is designated the low-est antimicrobial concentration that inhibits growth. The CLSImethod specifies the use of control strains including B. fragilisATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, andClostridium difficile ATCC 700057.

Broth microdilution. In the broth microdilution assay, apolystyrene tray (usually containing 96 wells) is filled withsmall volumes of serial 2-fold dilutions of different antibiotics(Fig. 2). If made in-house, trays can be tailored to the particularneeds of the laboratory, using the drugs and concentrationranges needed. The panels can be prepared in advance, frozen,and used as needed. Details for this procedure are described inthe CLSI manual (16).

Etest. For the Etest (AB Biodisk, bioMérieux) procedure, anindividual isolate is suspended in broth or saline and swabbedonto a Brucella blood agar plate. The Etest is a plastic strip with apredetermined antimicrobial concentration gradient on one sideand an interpretative MIC scale on the other. The MIC is read asthe concentration where the elliptical zone of inhibition intersectsthe strip. In general, the Etest correlates well with the referenceprocedure, although for certain drugs, there are some discrepan-cies (39, 41). This has become the most popular test for testing ofindividual isolates (Fig. 3).

Spiral gradient endpoint system. The Autoplate 4000 (Ad-vanced Instruments, Inc., Boston, MA) spiral gradient endpoint(SGE) system deposits a specific amount of antimicrobial stocksolution in a spiral pattern on a 150-mm agar plate, producing aconcentration gradient that decreases radially from the center ofthe plate (Fig. 4, left). After the antimicrobial agents are allowed todiffuse, the isolates are deposited onto the plate with an auto-mated inoculator or manually streaked from the center to the edgeof the plate. After incubation, endpoints of growth are marked,and the distance is measured in millimeters from the center of the

FIG 1 Agar dilution technique. (Left) A Steers replicator is used to apply inocula onto a agar plate. (Right) Series of agar dilution plates. Each spot represents adifferent strain. The arrow indicates a spot of dye, which is generally added to orient the plate. The plate for which the growth is no longer present should beconsidered the MIC.




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plate to the point where growth stops (Fig. 4, middle). The data arethen entered into a computer software program provided by themanufacturer, which determines the concentration of drug fromthe radius of growth and the molecular weight (i.e., diffusioncharacteristics) of the antimicrobial agent. Details of the proce-dure are described in the manufacturer’s guidelines. Comparisonsof this procedure with standard agar dilution have been favorable(46–48). Also, any tendency for spontaneously resistant mutants

to develop (i.e., colonies that grow beyond the “endpoint”) can beeasily determined (Fig. 4, right).

Commercially Available Testing

ThermoFisher Scientific (Cleveland, OH) now owns Remel, Oxoid,and Sensititre (Trek Diagnostic Systems). These manufacturers cur-rently offer two ready-made panels. There is a dried anaerobic panel(AN02B; Sensititre [Trek Diagnostic Systems]) that includes 15 anti-

FIG 2 Broth microdilution. (Left) Sensititre pipette for filling microdilution plate. (Middle) Plate being inoculated with strains. (Right) Plate after growth ofstrains. The MIC is read as the lowest dilution of antimicrobial resulting in no growth. (Left and right panels courtesy of Trek Diagnostics Systems, Inc.,reproduced with permission.)

FIG 3 Etest (AB Biodisk). (Courtesy of bioMérieux, reproduced with permission.)

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microbials in a variety of dilution ranges, depending on the antibiotic(http://www.trekds.com/products/sensititre/c_pltformats.asp). Thedried panels are stable at room temperature for 1 to 2 years. Thepanels are specifically designated for research purposes and not fordiagnostic testing. Sensititre will also prepare custom panels whichcan be either frozen or air dried. There is also a frozen panel that waspreviously sold by Remel (ANA MIC panel, catalog numberR8320100), now marketed through Oxoid; this can be ordered in theUnited States through ThermoFisher (it is shipped from the UnitedKingdom and requires a lead time of 4 to 6 weeks).

The question of whether these tests are FDA approved for diag-nostic purposes is a bit confusing. FDA approval for a panel re-quires that all agents on the panel are FDA approved for use inanaerobic infections. If any agent does not meet this requirement,the panel is not FDA approved for diagnostic purposes, eventhough the testing method is, in fact, approved. In practice, mosthospitals that use microbroth panels order the panels that reflectthe needs of their physicians based on hospital formulary and drugused and not on FDA approval.

Specialty Laboratories (operated by Quest Diagnostics) pro-vides testing services for 6 antimicrobials (cefoxitin, penicillin,clindamycin, piperacillin-tazobactam, metronidazole, and imi-penem) using Etest methodology (anaerobic susceptibility panel5711). Focus Diagnostics (also a subsidiary of Quest Diagnostics,Inc.) has discontinued the microdilution test for anaerobic testingand now offers routine testing using Etest (anaerobic susceptibil-ity panel 51477). Six to nine drugs are offered routinely, depend-ing on the organism being tested. For B. fragilis group organisms,ampicillin-sulbactam, clindamycin, imipenem, meropenem, met-ronidazole, and piperacillin-tazobactam are included in the rou-tine panel. The addition of penicillin, cefoxitin, and cefotetan maybe ordered for testing of Clostridium. Other drug tests can be cus-tom ordered (depending on the availability of the Etest strip).Mayo Medical Laboratories (Rochester, MN) also uses Etestmethodology.

�-Lactamase Test

Anaerobic organisms may be tested for the presence of the �-lac-tamase (BLA) enzyme by using a chromogenic cephalosporin test,

such as nitrocefin disks. These are colorimetric tests that are veryeasy to perform, and results can be read quickly (5 to 30 min) andwould be useful if penicillin or ampicillin therapy is being consid-ered. The great majority of B. fragilis group isolates are �-lacta-mase producers; therefore, testing is generally not recommendedfor this group. Other isolates have less predictable patterns, andcertain anaerobes (some Clostridium, Fusobacterium, and Pre-votella species, for example) may be penicillin-ampicillin resistantdue to �-lactamase. Isolates with a positive �-lactamase testshould be considered resistant to penicillin and ampicillin. A neg-ative test does not necessarily predict susceptibility to these drugs,as some anaerobes are resistant to �-lactam antimicrobial agentsby other mechanisms.

Increased activity of efflux pumps or changes in penicillin-binding proteins (PBPs) have been shown to affect MICs of �-lac-tams for many Bacteroides isolates; systematic surveys of thesemechanisms have not been conducted, so the percentage of strainsthat have or utilize these mechanisms is not known (35, 49).

Factors Contributing to Variability in MIC Results

A 1991 review by Wexler (35) reported the major reasons forvariability in reporting of MIC results. At that time, technical vari-ability among laboratories was a major factor. Laboratories useddifferent media and different inoculum sizes and may have readresults after different incubation times. Since that time, the CLSI(formerly NCCLS) procedures have been extensively revised andhave been adopted by virtually all testing laboratories in theUnited States and even worldwide. Therefore, the technical vari-ability among laboratories has been greatly minimized. Differingbreakpoints will also not influence the individual MIC results fora particular strain but will change the percentage of strains re-ported as susceptible or resistant in surveillance studies. Moststudies conform to CLSI breakpoints, but certain EUCAST break-points are different. Many studies will acknowledge these differ-ences and report results with both breakpoints.

Other factors that impact results reported in survey antibi-ograms include the particular makeup of the groups of strainsincluded. The different species belonging to a particular genusmay have markedly different susceptibility patterns. For example,

FIG 4 Spiral gradient endpoint technique. (Left) Dye representing the gradient application of antimicrobial stock solution, decreasing from the center of theplate. (Middle) Growth of bacterial strains inoculated in a radial manner onto a plate. The radius from the center of the plate to end of growth is measured andtranslated into an MIC by a software program. (Right) Detail of the endpoint and observation of resistant colonies past the endpoint.




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within the B. fragilis group, MICs for B. thetaiotaomicron and B.ovatus are often higher than those for B. fragilis. Studies usingdifferent proportions of the various B. fragilis group species mayreflect different antibiograms for the B. fragilis group as a whole,when in reality, the only difference is the proportion of the variousspecies used. The source of isolates (i.e., stool, abscess, or appen-diceal) may also influence the resistance profile of the species andshould be considered when evaluating the survey results.

By far, the most prevalent cause of variability in MIC reports isthe variation in interpreting what the MIC is in cases where end-points are not very clear. CLSI reference protocol M11-A8 hasincluded extensive discussion on breakpoint interpretation andincluded several sets of photographs to aid in reading of break-points.

There is a certain margin of error (usually �1 2-fold dilution)for any of these techniques. With certain antimicrobial agents, theMICs for a large percentage of B. fragilis group strains clusterwithin one 2-fold dilution range of the breakpoint. Clusteringaround the breakpoint is a characteristic of the organism-druginteraction and is seen, to some degree, in all of the testing meth-ods. When an MIC is near the breakpoint (e.g., in the case of the B.fragilis group and chloramphenicol as well for as many �-lactamagents), an organism called susceptible on one occasion may beretested and called resistant, all within the accepted variability ofthe technique. Therefore, in the case of single isolates, it is usefulfor the clinician to know the MIC of a drug for the strain as well asthe established breakpoint rather than just the categorical deter-mination. In cases where other factors that can cause variabilityare also involved (e.g., differences in techniques or media or dif-ferent people reading results), it becomes clear that minor changes(e.g., less than 15%) in percent susceptibility may not be signifi-cant in reports of large groups of strains; still, survey studies pro-vide useful information on trends and patterns in antimicrobialsusceptibility of anaerobes.

On the other hand, changes in susceptibility that are known tobe due to specific mechanisms (such as the presence of the nitro-imidazole reductase gene [nim], which causes metronidazole re-sistance, or cfiA genes, which can result in carbapenem resistance)may begin to appear as relatively modest changes in resistancerates and then quickly increase as the resistance determinant be-comes disseminated. Research laboratories that conduct large sur-veys, as well as pinpointing the mechanisms of resistance that maybe relevant in the antibiograms, can help monitor, understand,and perhaps even control these shifts by making recommenda-tions based on the molecular traits of the pathogen.

Detection of Resistance by Using Molecular Methods

Molecular methods are currently limited to research laboratoriesstudying resistance mechanisms of anaerobic bacteria. The mostcommonly used molecular techniques are PCR amplifications toidentify nim genes responsible for metronidazole resistance orcfiA-type genes that confer resistance to carbapenems.

Metronidazole resistance is generally attributed to the nim gene.This gene codes for an enzyme that converts 4- or 5-nitroimidaz-ole (4- or 5-Ni, respectively) to 4- or 5-aminoimidazole (thusavoiding the formation of toxic nitroso radicals that are essentialfor antimicrobial activity). nim homologs were found in bothGram-positive and -negative genera of aerobic and anaerobic bac-teria and Archaea, suggesting that the nim gene family is ancientand widespread. More significantly, nim genes are often found on

mobilizable plasmids and therefore pose a significant threat to thecontinuing utility of 5-Ni drugs, including metronidazole, whichis the most frequently prescribed drug for anaerobic infectionsworldwide (50).

PCR is used to detect the presence of the nim gene. Detection ofnim genes was described in 1996, using the universal primersNIM-3 and NIM-5 (51), followed by restriction analysis to iden-tify the specific nim type (52). Since then, nine nim genes weredescribed for B. fragilis (nimA to nimI [nimA-I]), and an addi-tional nimI gene was described for Prevotella (53, 54). However,increasing numbers of clinical metronidazole-resistant isolatesthat do not possess any of the nimA-H genes are being found. Also,metronidazole resistance could be induced in nim-negative strainsby exposure to sub-MICs of metronidazole; the mechanisms be-hind the increased MICs are not clear (54, 55). However, it is clearthat there is also a non-nim-based mechanism of resistance tometronidazole.

Clindamycin resistance is conveyed by a macrolide-lincos-amide-streptogramin (MLS)-type 23S methylase, typically en-coded by one of several erm genes that are regulated and expressedat high levels (56).

Carbapenem resistance in B. fragilis is associated with cfiA- orccrA-encoded class B metallo-�-lactamase. Although not all cfiA-positive B. fragilis strains are resistant to carbapenems, they allhave the possibility of becoming resistant to this group of antibi-otics by acquisition of an appropriate insertion sequence (IS) ele-ment for full expression of the cfiA gene, leading to possible treat-ment failure. The presence of the cfiA gene, as well as associated ISelements, can be determined by a PCR technique (57–59). Tworecent studies used matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to identify B.fragilis strains that carry the cfiA gene; these strains are restricted todivision II type strains (60, 61). Although this type of test is notcurrently appropriate for clinical settings, there is increasing in-terest in using this technology for clinical laboratory identification(58), and it is conceivable that it could be potentially developed fortesting.

Five genes conferring ribosomal protection have been found inanaerobes; these are tet(Q) (the most common, found in 12 gen-era), tet(M) (found in nine genera), tet(W) (found in seven gen-era), tet(32), and tetB(P) (the ribosomal protection gene found inthe P operon in Clostridium). Four tetracycline efflux genes havebeen found in anaerobes; these are tet(B) (Treponema), tet(K)(Eubacterium and Peptostreptococcus), tet(L) (Actinomyces, Pepto-streptococcus, Veilonella, and Clostridium), and tetA(P) (the effluxgene in the P operon in Clostridium) (63). Three genes encodingenzymes that inactivate tetracycline, i.e., tet(X1), tet(X2), andtet(32), have been identified in Bacteroides (62, 63). Five genesconferring MLS resistance have been identified in anaerobes, in-cluding erm(B), erm(C), erm(F), erm(G), and erm(Q). In contrast,genes coding for MLS-resistant efflux proteins or inactivating en-zymes are not generally described for anaerobic species (63), al-though homologs of the mefA efflux pump have been found onconjugative transposons in Bacteroides (154).

Is a Rapid Test on the Horizon?

It would be very appealing to wish that there could be a simplemolecular test, or even a complex test, such as multiplex PCR, thatwould determine the actual or potential resistance of an organismto multiple antibiotics. One can envision a test that could, in fact,

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measure many genetic determinants that confer drug resistance,including enzymes that confer resistance to carbapenems (e.g.,cfiA), metronidazole (nim), chloramphenicol (cat), erythromycin(erm), tetracycline (tet), or quinolones (changes in gyr or parCgenes). A multiplex PCR test that could detect multiple resistancedeterminants in B. fragilis isolates was recently described by Pum-bwe et al. (64) and could be helpful to predict likely resistancepatterns (Fig. 5). However, the presence of systems of multidrugefflux pumps may prove to be the confounding problem that willnot permit a definitive determination of a resistance profile bymolecular techniques. At least in aerobes, much of the multidrugresistance seen in the last several years is due to the action ofmultidrug efflux pumps, and we have indications that a similarphenomenon may be operative in anaerobes as well. In B. fragilis,16 homologs of tripartite efflux pumps of the resistance nodula-tion division (RND) family have been described (Bacteroides mul-tidrug efflux [Bme] pumps 1 to 16) and are apparently important

in conferring multidrug resistance (23, 65, 68); pump activity re-lated to resistance has also been described for Clostridium (69–72).Several multidrug-resistant isolates appear to have significantlyincreased efflux pump activity. Genes for efflux pumps are presentin all strains of bacteria, so a PCR test to detect the gene wouldalways be positive. It is likely that the levels of efflux pump genestranscribed and expressed are important. At this time, the onlyway to measure these genes in clinical isolates is to quantitativelyidentify and sequence RNA transcripts, which is not a practicalsolution.

ANTIMICROBIAL AGENTS EFFECTIVE AGAINST ANAEROBICBACTERIA

Table 1 illustrates the antimicrobials effective against anaerobicbacteria and their efficacy against both aerobic and anaerobic bac-teria. Many of the older antimicrobials do not have an FDA-ap-proved indication for anaerobic infections, and many of the neweragents have only a limited number of indications for anaerobicinfections (Table 2). Because of this, many of these agents are usedfor the treatment of anaerobic infections without an FDA indica-tion. Table 3 illustrates the resistance of the B. fragilis group andother anaerobes to antimicrobial agents.

�-Lactam Antibiotics

Penicillin G is the classical drug of choice when the infectingstrains are susceptible to this drug in vitro. Most Clostridiumstrains (with the exception of some strains of Clostridium ramo-sum, Clostridium clostridioforme, and Clostridium innocuum) andPeptostreptococcus spp. remain susceptible to penicillin. Most clin-ical isolates of the B. fragilis group are resistant to penicillin G, andit should not be used for the treatment of infections caused bythese organisms. Other strains that may show resistance to peni-cillins are growing numbers of AGNB, such as the pigmented Pre-votella and Porphyromonas spp., Prevotella oralis, Prevotella bivia,Bacteroides disiens, strains of clostridia, Fusobacterium spp. (Fuso-bacterium varium and Fusobacterium mortiferum), and mi-croaerophilic streptococci. Some of these strains show MICs of 8to 32 units/ml of penicillin G. In these instances, administration of

FIG 5 Multiplex PCR assay to detect common resistance determinants in B.fragilis. Amplification was done with a set of primers designed for detecting fiveresistance genes, including carbapenems (cfiA), cephalosporins (cepA), clinda-mycin (ermF), metronidazole (nimA-F), and tetracycline (tetQ), plus a set ofprimers for the B. fragilis 16S rRNA gene (positive control). Lane 0, negativecontrol; lane M, DNA standards. Lanes 1 to 11 were either single or multiple B.fragilis clinical isolates with previously determined resistance determinants.The multiplex PCR assay was able to determine all resistance determinantspresent in either single- or multiple-strain samples.

TABLE 1 Antimicrobial agents effective against mixed infectiona

Antimicrobial agent

Degree of activity

Anaerobic bacteria Aerobic bacteria

Beta-lactamase-producing AGNB

Otheranaerobes

Gram-positivecocci Enterobacteriaceae

Penicillinb 0 �� 0Chloramphenicolb �� Cephalothin 0 � �� /�Cefoxitin �� Carbapenems �� Clindamycinb �� 0Ticarcillin � �� Amoxicillin � clavulanateb �� Piperacillin � tazobactam �� Metronidazoleb �� 0 0Moxifloxacin �� Tigecycline �� a Degrees of activity from 0 to ��.b Also available in an oral form.




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very high doses of penicillin G (for non-beta-lactamase produc-ers) may eradicate the infection.

Clinical experience with penicillin G in the management of sus-ceptible anaerobic bacterial infections has been good. Ampicillin,amoxicillin, and penicillin generally are equally active, but thesemisynthetic penicillins are less active than the parent com-pound. Methicillin, nafcillin, and the isoxazolyl penicillins (oxa-cillin, cloxacillin, and dicloxacillin) are ineffective against the B.fragilis group, have unpredictable activity, and frequently are in-ferior to penicillin G against anaerobes (73).

Penicillin, ampicillin, and amoxicillin are of limited utility dueto the production of beta-lactamases by many oral and most intra-abdominal anaerobes. Clavulanate, sulbactam, and tazobactamare beta-lactamase inhibitors that resemble the nucleus of penicil-lin but differ in several ways. They irreversibly inhibit beta-lacta-mase enzymes produced by some Enterobacteriaceae, staphylo-

cocci, and beta-lactamase-producing Fusobacterium spp. andAGNB (73–75). When used in combination with a beta-lactamantibiotic (such as ampicillin-sulbactam, amoxicillin-clavulanate,and piperacillin-tazobactam), they are effective in treating anaer-obic infections caused by beta-lactamase-producing bacteria(BLPB).

Beta-lactam–beta-lactamase inhibitor combinations (BL-BLICs)are popular and appropriate choices for mixed aerobic-anaerobicinfections. They have maintained good activity against the vastmajority of anaerobes. While 89% of B. fragilis strains are suscep-tible to ampicillin-sulbactam, 98% are susceptible to piperacillin-tazobactam (17), compared to 86% and 92%, respectively, of B.thetaiotaomicron isolates. Recently, the Infectious Diseases Societyof America (IDSA) has removed ampicillin-sulbactam from therecommended list of treatments for intra-abdominal infectionsdue to increased Escherichia coli resistance worldwide, although it

TABLE 2 FDA-approved indications for antimicrobials for the treatment of anaerobic infections

Antimicrobial Indication

Ertapenem Complicated intra-abdominal infections caused by Clostridium clostridioforme, Eubacterium lentum, Peptostreptococcus spp., B.fragilis, B. distasonis, B. ovatus, B. thetaiotaomicron, or B. uniformis

Complicated skin/skin structure infections, including diabetic foot infections without osteomyelitis due to B. fragilis,Peptostreptococcus spp., Porphyromonas asaccharolytica, or Prevotella bivia; acute pelvic infections, including postpartumendomyometritis, septic abortion, and postsurgical gynecologic infections caused by B. fragilis, P. asaccharolytica,Peptostreptococcus spp., or P. bivia

Ertapenem is indicated for adults for prophylaxis of surgical site infection

Imipenem Intra-abdominal infections, including acute gangrenous or perforated appendicitis and appendicitis with peritonitis caused byBacteroides spp. including B. fragilis, B. distasonis, B. intermedius, and B. thetaiotaomicron, Fusobacterium spp., andPeptostreptococcus spp.; skin and skin structure infections, including abscesses, cellulitis, infected skin ulcers, and wound infectionscaused by and Bacteroides spp. including B. fragilis

Gynecological infections, including postpartum endomyometritis, caused by group D Streptococcus, Bacteroides intermedius, orPeptostreptococcus spp.

Meropenem Complicated appendicitis and peritonitis caused by susceptible isolates of B. fragilis, B. thetaiotaomicron, or Peptostreptococcus spp.Complicated skin and skin structure infections caused by susceptible isolates of B. fragilis and Peptostreptococcus spp.

Doripenem Complicated intra-abdominal infections caused by Bacteroides caccae, B. fragilis, B. thetaiotaomicron, B. uniformis, B. vulgatus,Streptococcus intermedius, Streptococcus constellatus, or Peptostreptococcus micros

Tigecycline Complicated skin and soft tissue infection caused by B. fragilis; complicated intra-abdominal infections caused by B. fragilis, B.thetaiotaomicron, B. uniformis, B. vulgatus, C. perfringens, or Peptoniphilus micra (Peptostreptococcus micros)

Moxifloxacin Complicated intra-abdominal infections caused by B. fragilis, B. thetaiotaomicron, C. perfringens, or Peptostreptococcus spp.

Cefoxitin Intra-abdominal infections caused by Bacteroides spp. including B. fragilis or Clostridium spp.Gynecological infections caused by Bacteroides spp. including B. fragilis, Clostridium spp., Peptococcus niger, or Peptostreptococcus spp.Septicemia caused by Bacteroides spp. including B. fragilisSkin and skin structure infections caused by Bacteroides spp. including B. fragilis, Clostridium spp., Peptococcus niger, or

Peptostreptococcus spp.Metronidazole Intra-abdominal infections, including peritonitis, intra-abdominal abscess, and liver abscess, caused by Bacteroides spp. including the

B. fragilis group, Clostridium spp., Eubacterium spp., Peptococcus niger, or Peptostreptococcus spp.Skin and skin structure infections caused by Bacteroides spp. including the B. fragilis group, Clostridium spp., Peptococcus niger,

Peptostreptococcus spp., or Fusobacterium spp.Gynecological infections, including endometritis, endomyometritis, tubo-ovarian abscess, and postsurgical vaginal cuff infection,

caused by Bacteroides spp. including the B. fragilis group, Clostridium spp., Peptococcus niger, or Peptostreptococcus spp.Bacteremia and septicemia caused by Bacteroides spp. including the B. fragilis group and Clostridium spp.Bone and joint infections caused by Bacteroides spp. including the B. fragilis groupCentral nervous system infections, including meningitis and brain abscess, caused by Bacteroides spp. including the B. fragilis groupLower respiratory tract infections, including pneumonia, empyema, and lung abscess, caused by Bacteroides spp. including the B.

fragilis groupEndocarditis caused by Bacteroides spp. including the B. fragilis group

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has maintained good activity against B. fragilis and other anaer-obes (76). Amoxicillin-clavulanate remains the agent of choice forhuman and animal bite wound infections (77), especially whenanaerobes may be involved. Piperacillin-tazobactam is also a fre-quently and appropriately prescribed agent for serious intra-ab-dominal infections. It has also maintained good activity againstthe vast majority of anaerobes (17).

The semisynthetic penicillins, the carboxypenicillins (carbeni-cillin and ticarcillin), and ureidopencillins (piperacillin, azlocillin,and mezlocillin) generally are administered in large quantities toachieve high serum concentrations. These drugs are effectiveagainst Enterobacteriaceae and have good activity against most an-aerobes in these concentrations. However, up to 30% of strains ofthe B. fragilis group are resistant to these agents (78).

Many anaerobes possess cephalosporinases, and therefore, as aclass, cephalosporins have very limited utility (41). The activity ofcephalosporins against the beta-lactamase-producing AGNB var-ies. The antimicrobial spectrum of the narrow-spectrum cephalo-sporins against anaerobes is similar to that of penicillin G, al-though on a weight basis, they are less active. Most strains of the B.fragilis group and many Prevotella, Porphyromonas, and Fusobac-terium spp. are resistant to these agents by virtue of cephalospori-nase production (79). The enzyme has little or no hydrolytic ac-tivity for the second-generation antimicrobial cefoxitin (acephamycin). Cefoxitin is therefore the most effective cephalo-sporin against the B. fragilis group. However, susceptibility mayvary by geographic location and is generally related directly to itsclinical use. Cefoxitin is relatively inactive against most species ofClostridium, including C. difficile, with the exception of Clostrid-ium perfringens (6, 7, 79).

Studies done in the 1980s found cefoxitin to be effective ineradication of anaerobic infections (80–82). It has often been usedfor surgical prophylaxis at most body sites that are in proximity tomucus membranes. With the exception of moxalactam, the third-generation cephalosporins are not as active against B. fragilis ascefoxitin. However, these agents have improved activity againstEnterobacteriaceae.

At present, approximately 85% of B. fragilis isolates are suscep-tible to cefoxitin, but the other B. fragilis group species are moreresistant (17). Cefotetan is less effective than cefoxitin against B.fragilis and other members of the B. fragilis group.

The B. fragilis group is composed of more than 20 Bacteroidesspp. that were promoted to the genus level (Table 4) (83).

Among the B. fragilis group, B. fragilis accounts for 40% to 54%of the Bacteroides isolates recovered from intra-abdominal as wellas other infections (4, 84–86). Another important pathogen thatbelongs to the B. fragilis group is B. thetaiotaomicron, which ac-counts for 13% to 23% of the isolates. Other members of the B.fragilis group account for 33% to 39%. The antimicrobial suscep-tibilities of some members of the B. fragilis group vary, especiallyto the second- and third-generation cephalosporins. B. fragilis isgenerally the most susceptible, and B. thetaiotaomicron and Para-bacteroides distasonis generally are more resistant (87, 88).

The cephamycins cefoxitin and cefotetan are often not usedappropriately. This is because clinicians are not aware of theiractivity against the B. fragilis group locally and are unlikely to haveknowledge of the specific antibiotic susceptibility of the isolaterecovered from their patients. Sometimes, these agents are usedfor surgical prophylaxis for abdominal surgery and for the treat-ment of aspiration pneumonia. Recently, the IDSA has removed

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cefotetan from the recommended list of therapies for intra-ab-dominal infections due to poor B. fragilis group activity and resul-tant clinical failures (89–91).

The carbapenems (imipenem, meropenem, doripenem, and er-tapenem) have excellent activity against anaerobes (92). Imipenem, athienamycin, is a beta-lactam antibiotic that is effective against a widevariety of aerobic and anaerobic Gram-positive and Gram-negativeorganisms, including normally multiresistant species such as Pseu-domonas aeruginosa, Serratia spp., Enterobacter spp., Acinetobacterspp., and enterococci (93, 94). It also possesses excellent activityagainst beta-lactamase-producing Bacteroides. It has low MICs for theB. fragilis group. Despite the emergence of carbapenemase-resistantEnterobacteriaceae, it is also effective against most Enterobacteriaceae,with about 5% to 15% of Pseudomonas species strains being resistant(95). The pharmacokinetics of imipenem are characterized by poorabsorption from the gastrointestinal tract, high plasma concentra-tions after intravenous administration, a small degree of systemic me-tabolism, and renal excretion. In the kidney, imipenem is metabo-lized by breakage of the beta-lactamase bond in the proximal tubularcells. The result is low-level urinary excretion of active imipenem,which may impair its ability to inhibit certain urinary pathogens. Toovercome the problem of renal metabolism of imipenem, it is com-bined at a 1:1 ratio with an inhibitor of the renal dipeptidase cilastatin.This increases the urinary excretion of the active drug and its half-lifein serum. This agent is an effective single agent for the therapy ofmixed aerobic-anaerobic infections.

Meropenem is a carbapenem antibiotic that has a very broadspectrum of activity against aerobic and anaerobic bacteria, simi-lar to that of imipenem. Imipenem has more activity than mero-penem against staphylococci and enterococci, but meropenem

provides better coverage of aerobic and facultative Gram-negativebacteria such as Pseudomonas, Enterobacter, Klebsiella, Providen-cia, Morganella, Aeromonas, Alcaligenes, Moraxella, Kingella, Acti-nobacillus, Pasteurella, and Haemophilus spp. (96, 97). Mero-penem has been effective in abdominal infections, meningitis inchildren and adults, community-acquired and nosocomial pneu-monia, and neutropenic fever (98).

Ertapenem is a newer 1-beta-methyl carbapenem, stable to de-hydropeptidase. It has a broad antibacterial spectrum for penicil-lin-susceptible Streptococcus pneumoniae, Streptococcus pyogenes,methicillin-sensitive Staphylococcus aureus, Haemophilus influen-zae, Moraxella catarrhalis, Escherichia coli, Citrobacter spp., Kleb-siella spp., Serratia spp., Proteus spp., C. perfringens, Fusobacte-rium spp., Peptostreptococcus spp., and AGNB (99). It is indicatedfor complicated intra-abdominal and skin structure infections,including diabetic foot infections without osteomyelitis, and acutepelvic infections, including postpartum endomyometritis, septicabortion, and postsurgical gynecological infections. In compari-son to other available carbapenems, ertapenem has a long half-lifeof 4.5 h and is given as a single daily dose. It is not active against P.aeruginosa, Enterococcus spp., and Acinetobacter spp. Doripenem,a synthetic 1-beta-methyl carbapenem, is the newest carbapenemto be commercially released. Its antimicrobial spectrum moreclosely resembles those of meropenem and imipenem than that ofertapenem (94). Thus, it has significant in vitro activity againststreptococci, methicillin-susceptible staphylococci, Enterobacteri-aceae (including extended-spectrum-beta-lactamase-producingstrains), P. aeruginosa, Acinetobacter spp., and the B. fragilis group.Doripenem does not have activity against methicillin-resistantStaphylococcus aureus (MRSA), vancomycin-resistant entero-cocci, and the majority of Gram-negative bacilli that are resistantto meropenem or imipenem (94). In vitro, resistant P. aeruginosamutants appear to be more difficult to select with doripenem thanwith other carbapenems. Doripenem has been approved in theUnited States for use in treatment of complicated intra-abdominalinfection and complicated urinary tract infection.

In general, clinicians recognize the generally good activity ofcarbapenems against anaerobes and prescribe them appropriately.Consequently, they are employed in more serious anaerobic in-fections, such as intra-abdominal and skin and soft tissue infec-tions (89–91). Two recent reports have noted the development ofsome carbapenem resistance among anaerobes (22), ranging from1.1 to 2.5% in a multicenter U.S. survey but with a higher rate fora small number of isolates from Taiwan (33).

Resistance to �-lactam antibiotics. Anaerobes manifest threemajor mechanisms of resistance to �-lactam antibiotics: inactivat-ing enzymes, mainly beta-lactamases (BLAs), which include peni-cillinases and cephalosporinases; low-affinity penicillin-bindingproteins (PBPs); and decreased permeability through alterationsin the porin channel (35). The production of BLAs is the mostcommon mechanism of resistance to �-lactam antibiotics in an-aerobes, especially among the B. fragilis group and Prevotella spp.(100) The cephalosporinases are most often of the 2e class typeand can be inhibited by three beta-lactamase inhibitors, clavulanicacid, sulbactam, and tazobactam. Each individual cephalosporinmay have either a class or specific inhibitor enzyme that is able toinactivate it.

BLA hydrolyzes the cyclic amide bond of the penicillin or ceph-alosporin nucleus, causing its inactivation. There are a variety ofBLAs which are produced by different organisms. These enzymes

TABLE 4 Members of the Bacteroides fragilis group

Genus Species

Bacteroides B. acidifaciensB. caccaeB. coprocolaB. coprosuisB. eggerthiiB. finegoldiiB. fragilisB. helcogenesB. intestinalisB. massiliensisB. nordiiB. ovatusB. thetaiotaomicronB. vulgatusB. plebeiusB. uniformisB. salyersaiB. pyogenesB. doreiB. johnsonii

Parabacteroides P. distasonisP. merdaeP. goldsteiniiP. chartaeP. gordoniiP. johnsonii

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can be exoenzymes, inducible or constitutive, and genetically, theycan be of either chromosomal or plasmid origin (101). There aredifferent classifications of the enzymes. A classification based onamino acid sequence was created by Ambler (102), and a classifi-cation based upon substrate-of-inhibition profiles, molecularweight, and isoelectric points was proposed by Richmond andSykes (103).

Most B. fragilis group strains produce constitutive BLs that areprimarily cephalosporinases (104). Over 97% of Bacteroides iso-lates in the United States and 76% in Great Britain produce BLAs(105). Of the non-B. fragilis strains, 65% produce BLs (106, 107).Pigmented Prevotella and Porphyromonas spp., Prevotella bivia,Prevotella disiens, and Fusobacterium nucleatum produce primar-ily penicillinases (107).

Carbapenemases are active against the carbapenems as wellas all �-lactam antibiotics. Although these enzymes are gener-ally chromosomally mediated, a plasmid-mediated metallo-BLhas been reported in Japan (108). Carbapenem resistance oc-curs in �1% of U.S. isolates, and up to 3% of Bacteroides strainsharbor one of the genes that is expressed at a very low level. BLAinhibitors cannot inactivate the carbapenemases, which arezinc metalloenzymes encoded by either ccrA or cfiA genes of theB. fragilis group (109).

An evaluation of the molecular characterization of 15strains of imipenem-resistant, cfiA-positive B. fragilis strains(109) noted that the cfiA genes of 10 of the strains were upregu-lated by insertion sequence (IS) elements, while 5 others didnot harbor an IS but produced carbapenemase. These findingssuggest that some isolates possessed novel inactivation mech-anisms, suggesting that more than one mechanism of inactiva-tion exists. A recent report from Taiwan noted increased car-bapenem resistance in B. fragilis and other B. fragilis groupspecies as well as some Prevotella species strains (33).

With some exceptions among some Clostridium spp., strains ofClostridium, Porphyromonas, and Fusobacterium have also beenfound to express resistance by one or more of the BLAs. BLA-producing Fusobacterium and Clostridium spp. express enzymesthat are generally inhibited by clavulanic acid (110). Resistance to�-lactam antibiotics through changes in the outer membrane pro-tein (OMP)/porin channels, decreased PBP affinity, and effluxpumps (111) is less well studied.

B. fragilis group species are generally resistant to penicillins(average, 90%), piperacillin (25%), cefoxitin (25%), cefotetan (30to 85%), and third-generation cephalosporins (27, 88).

The combinations of BL-BLICs and carbapenems have main-tained their excellent antibacterial activity. The combinationagents of ampicillin-sulbactam, amoxicillin-clavulanate, ticarcil-lin-clavulanate, and piperacillin-tazobactam are generally veryactive against members of the B. fragilis group (27). However,species-to-species variations in susceptibility occur, and manynon-BLA-producing P. distasonis strains have elevated MICs at orapproaching the susceptible breakpoint (112). B. fragilis groupresistance rates for piperacillin-tazobactam are generally �1%(27). However, the rate of resistance of P. distasonis to ampicillin-sulbactam has risen to 20% in 2002 to 2004, but resistance ratescontinued to be low for the other B. fragilis group species.

The carbapenems (imipenem, meropenem, doripenem, andertapenem) are very effective against all members of the B. fragilisgroup, and resistance is rare, at �0.1% (27, 112, 113). Geometricmean MICs for imipenem and meropenem for P. distasonis, B.

thetaiotaomicron, and Bacteroides ovatus have been reported to be1-fold dilution lower than those for ertapenem (27) in 2004. Gold-stein et al. (80) reported that all Bacteroides isolates recoveredfrom pediatric intra-abdominal infections were beta-lactamaseproducers and susceptible to carbapenems and BL-BLICs. How-ever, cefoxitin has poor activity against B. thetaiotaomicron iso-lates.

�-Lactams are generally effective against non-B. fragilis groupspecies, and resistance to them is generally low, except that morethan half of Prevotella species isolates may also produce BLAs. Amulticenter survey (93) found penicillin resistance for Fusobacte-rium spp., Porphyromonas spp., and Peptostreptococcus spp. atrates of 9%, 21%, and 6%, respectively. No resistance to cefoxitin,cefotetan, �-lactam–BLA inhibitor combinations, and carbapen-ems was found in that survey, with the exception of Peptostrepto-coccus spp. and Porphyromonas spp. (4% and 5% resistance toampicillin-sulbactam, respectively). Beta-lactamases were identi-fied in several Prevotella and Porphyromonas species strains recov-ered from pediatric intra-abdominal infections.

Chloramphenicol

Chloramphenicol, a bacteriostatic agent, is active against mostanaerobic bacteria but is rarely used in the United States (3, 79).Resistance to this drug is rare, although it has been reported forsome Bacteroides spp. (113). One must be aware that MICs ofchloramphenicol often cluster around the susceptibility break-point. Although several failures to eradicate anaerobic infections,including bacteremia, with chloramphenicol have been reported(114), this agent has been used for over 64 years for treatment ofanaerobic infections. Chloramphenicol was regarded in the past asthe drug of choice for treatment of serious anaerobic infectionswhen the nature and susceptibility of the infecting organisms areunknown and of infections of the central nervous system (CNS).However, the drug has potential significant toxicity. The risk offatal aplastic anemia with chloramphenicol is estimated to be ap-proximately 1 per 25,000 to 40,000 patients treated. This seriouscomplication is unrelated to the reversible, dosage-dependent leu-kopenia. Other side effects include the production of the poten-tially fatal “gray baby syndrome” when given to neonates, hemo-lytic anemia in patients with glucose-6-phosphate dehydrogenase(G6PD) deficiency, and optic neuritis in those who take the drugfor a prolonged time.

Serum level measurements are often advocated for infants,young children, and occasionally adults, owing to their wide vari-ations (115). The usual objective is therapeutic levels of 10 to 25�g/ml. Levels exceeding 25 �g/ml are commonly considered po-tentially toxic in terms of reversible bone marrow suppression,and levels of 40 to 200 �g/ml have been associated with gray syn-drome in neonates or encephalitis in adults (115).

Chloramphenicol is widely distributed in body fluids and tissue,with a mean volume of distribution of 1.4 liters/kg of body weight(115). The drug has a somewhat unique property of lipid solubilityto permit penetration across lipid barriers. A consistent observa-tion is the high concentrations achieved in the CNS, even in theabsence of inflammation. Levels in the cerebrospinal fluid, with orwithout meningitis, usually are one-third to three-fourths of theserum concentrations. Levels in brain tissue may be substantiallyhigher than serum levels (116).




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The Macrolides: Erythromycin, Azithromycin, andClarithromycin

The macrolides, which possess low human or animal toxicity,have moderate to good in vitro activity against anaerobic bacteriaother than B. fragilis group strains and fusobacteria (79). Macro-lides are active against pigmented Prevotella and Porphyromonasspp. and microaerophilic streptococci, Gram-positive non-spore-forming anaerobic bacilli, and certain clostridia. They are less ef-fective against Fusobacterium and Peptostreptococcus spp. (117).They show relatively good activity against C. perfringens and pooror inconsistent activity against AGNB.

Clarithromycin is the most active macrolide against Gram-pos-itive oral cavity anaerobes, including Actinomyces spp., Propi-onibacterium spp., Lactobacillus spp., and Bifidobacterium den-tium. Azithromycin is slightly less active than erythromycinagainst these species (117). Azithromycin is, in general, the mostactive macrolide against AGNB such as Fusobacterium spp., Bac-teroides spp., Wolinella spp., and Actinobacillus actinomycetem-comitans, including strains resistant to erythromycin. Clarithro-mycin showed similar activity to that of erythromycin againstmost AGNB (118).

Emergence of erythromycin-resistant organisms during ther-apy has been documented (119, 120). Erythromycin is effective inthe treatment of mild to moderately severe anaerobic soft tissueand pleuropulmonary infections when combined with adequatedebridement or drainage of infected tissue. Phlebitis is reported todevelop in one-third of patients receiving intravenous erythromy-cin, but the oral preparation is well tolerated.

Clindamycin

Clindamycin has a broad range of activity against anaerobic or-ganisms and has proven its efficacy in clinical trials. It is used fordental infections, especially in patients who are allergic to penicil-lin, and for aspiration pneumonia. Clindamycin hydrochloride israpidly and virtually completely absorbed from the gastrointesti-nal tract (121–123). It rapidly penetrates into body tissues andfluids, including saliva, sputum, respiratory tissue, pleural fluid,soft tissues, prostate, semen, bones, and joints (124), as well as intofetal blood and tissues. Clindamycin does not cross the blood-brain barrier or eye efficiently and should not be administered inCNS infections.

The side effect of most concern is C. difficile-associated colitis(125, 126). Colitis has also been associated with a number of otherantimicrobials, such as ampicillin, cephalosporins, and quino-lones, and occasionally also in the absence of previous antimicro-bial therapy.

Clindamycin resistance. Although the patterns differ by re-gion, B. fragilis resistance to clindamycin is increasing worldwide.This is why it is no longer recommended as empirical therapy forintra-abdominal infections (22, 27, 76, 113). An 8-year study(1997 to 2004) revealed that 19.3% of 2,721 B. fragilis group,29.6% of P. distasonis, 33.4% of B. ovatus, 33.3% of B. thetaiotao-micron, and 35.6% of B. vulgatus isolates were clindamycin resis-tant. This is a significant increase compared to only 3% clindamy-cin resistance in 1987 (88). A study of pediatric intra-abdominalisolates revealed clindamycin resistance in only 6% of B. fragilisisolates, compared to 80% for B. thetaiotaomicron and 45% forother B. fragilis group isolates (80).

Resistance has also increased for many non-Bacteroides anaer-

obes. Up to 10% resistance was noted for Prevotella spp., Fusobac-terium spp., Porphyromonas spp., and Peptostreptococcus spp., withhigher rates for some Clostridium spp. (especially C. difficile) (93).Propionibacterium acnes isolates have also become more resistantto clindamycin, and this has been associated with prior therapy foracne (127).

Clindamycin has lost some of its activity against anaerobicGram-positive cocci (Finegoldia magna [30% resistant] and Pep-toniphilus spp., etc.) and Prevotella spp. (P. bivia [70% resistant],P. oralis, and P. melaninogenica [both 40% resistant]), although itsactivity against Fusobacterium and Porphyromonas spp. remainsgood.

Among the other resistant anaerobes are various species ofclostridia, especially C. difficile. Approximately 20% of Clostrid-ium ramosum strains are resistant to clindamycin, as are a smallernumber of C. perfringens strains.

Metronidazole and Tinidazole

The nitroimidazoles metronidazole and tinidazole have similar invitro efficacy against anaerobic bacteria. Metronidazole has excel-lent in vitro activity against most obligate anaerobic bacteria, suchas the B. fragilis group, other species of Bacteroides, fusobacteria,and clostridia (99). Only six strains of the B. fragilis group wereever reported to be clinically resistant and associated with thera-peutic failure (2).

Resistance of anaerobic Gram-positive cocci is rare, and resis-tance of nonsporulating bacilli is common. Microaerophilic strep-tococci, P. acnes, and Actinomyces spp. are almost uniformly resis-tant (128). Aerobic and facultative anaerobes, such as coliforms,are usually highly resistant. Over 90% of obligate anaerobes aresusceptible to less than 2 �g/ml metronidazole (79).

Some clinicians may not appreciate metronidazole’s limited ac-tivity against anaerobic and microaerophilic Gram-positive cocci,especially the microaerophilic streptococci, which are oftenlumped together with anaerobes. Because of metronidazole’s lackof activity against aerobic bacteria, an antimicrobial effectiveagainst these organisms (e.g., a cephalosporin or a fluoroquin-olone) needs to be added when treating a polymicrobial infection.There are occasions when clinicians are unsure of a specific drug’sactivity against anaerobes and may use redundant coverage withmetronidazole, such as a carbapenem or a BL-BLIC. Antibioticstewardship personnel should review this practice.

Adverse reactions to metronidazole therapy are rare and in-clude CNS toxicity, such as ataxia, vertigo, headaches, and con-vulsions, and peripheral neuropathy. Peripheral neuropathy is as-sociated with prolonged metronidazole use. Gastrointestinal sideeffects are common and include nausea, vomiting, metallic taste,anorexia, and diarrhea. Tinidazole may be better tolerated in pa-tients with gastrointestinal side effects due to metronidazole.Other adverse reactions include reversible neutropenia, phlebitisat intravenous infusion sites, and drug fever. The tolerance ofmetronidazole in patients is generally very good.

Some studies in mice (129) have shown possible mutagenicactivity associated with administration of large doses of this drug.It should be noted that in these animal toxicity studies, the drughas generally been administered for the lifetime of the animal, asituation that may not be relevant for humans. Other experiments(130) have shown that administration of metronidazole to ratsand hamsters does not induce any pathology. Furthermore, evi-dence of mutagenicity was never found in humans despite metro-

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nidazole use for over 2 decades for other diseases (131). Because ofsafety concerns, the FDA approved the use of metronidazole forthe treatment of serious anaerobic infections only in adults.

Clinical experiences of adults (132) illustrated metronidazole’sefficacy in the treatment of infections caused by anaerobes, in-cluding CNS infections (133). Available data on the safety of thedrug during pregnancy are contradictory, and valid data on thesafety of metronidazole in pregnancy are still needed. The nonter-atogenicity of metronidazole is difficult to prove, but the existingavailable data indicate no major risks and no indication for thetermination of pregnancies (134).

Metronidazole resistance. Although rare, resistance to metro-nidazole among B. fragilis group isolates has been observed world-wide (33, 135). Resistant B. fragilis group isolates carry one of nineknown nim genes (nimA-I) on either the chromosome or a mobi-lizable plasmid that seems to encode a nitroimidazole reductase,which converts 4- or 5-Ni to 4- or 5-aminoimidazole, preventingthe formation of toxic nitroso residues necessary for the agent’sactivity. These nim genes were found in 50/206 (24%) Bacteroidesspecies isolates and resulted in MICs of 1.5 to 256 �g/ml formetronidazole, including 16 isolates with MICs of �32 �g/ml(54). These findings suggested incomplete mobilization of nimgene-associated resistance. Gal and Brazier (54) speculated thatother mechanisms of resistance can occur and that prolonged ex-posure to metronidazole may select them. The mechanism of metro-nidazole resistance for non-Bacteroides anaerobes is unknown. Resis-tance among Gram-positive organisms that are not strict anaerobes isfrequent, especially for P. acnes and Actinomyces spp.

Tetracyclines

Tetracycline, once the drug of choice for anaerobic infections, ispresently of limited usefulness because of the development of re-sistance to it by virtually all types of anaerobes, including Bacte-roides and Prevotella spp. Resistance to P. acnes has been related toprevious use (127). Only about 45% of all B. fragilis strains arepresently susceptible to this drug (79). The newer tetracycline an-alogs doxycycline and minocycline are more active than the parentcompound. Because of the significant resistance to these drugs,they are useful only when susceptibility tests can be performed orin less severe infections in which a therapeutic trial is feasible. Theuse of tetracycline is not recommended for patients less than 8years of age because of the adverse effect on teeth.

Tigecycline is the first antibiotic approved in a new class calledglycylcyclines. Glycylcyclines are tetracycline antibiotics contain-ing a glycylamido moiety attached to the 9-position of a tetracy-cline ring; tigecycline is a direct analog of minocycline with a9-glycylamide moiety. It has activity against both aerobic Gram-negative and Gram-positive bacteria, anaerobes, and certain drug-resistant pathogens (136). These pathogens include MRSA, peni-cillin-resistant Streptococcus pneumoniae, vancomycin-resistantenterococci, Acinetobacter baumannii, beta-lactamase-producingstrains of H. influenzae and M. catarrhalis, and extended-spec-trum-beta-lactamase-producing strains of E. coli and Klebsiellapneumoniae. In contrast, MICs for Pseudomonas and Proteus spp.are markedly elevated. It is active against the Streptococcus angino-sus group (including S. anginosus, S. intermedius, and S. constella-tus), B. fragilis, B. thetaiotaomicron, Bacteroides uniformis, Bacte-roides vulgatus, C. perfringens, C. difficile, and Parvimonas micra(Peptostreptococcus micros) (137). Resistance of members of the B.fragilis group varied from 3.3% to 7.2% (27).

Tigecycline has been approved by the FDA for use in compli-cated skin and soft tissue infections, including those due to B.fragilis (153), and intra-abdominal infections, including those dueto B. fragilis, B. thetaiotaomicron, B. uniformis, B. vulgatus, C. per-fringens, and Ps. micros (138). In a study that investigated its effi-cacy in the treatment of abdominal infections, tigecycline wascompared to imipenem-cilastatin. Six tigecycline-treated patientscompared to two imipenem-treated patients developed sepsis/shock, again lending caution to its clinical use.

Tetracycline resistance. Tetracycline resistance is prevalentamong Bacteroides species, Prevotella species, and many other an-aerobic bacteria, limiting its clinical use (139). Several tetracyclineresistance genes have been described among several anaerobes,which encode protective proteins leading to protection of the ri-bosomes. Tetracycline resistance and the inducible transfer of re-sistance determinants can occur after exposure to low levels ofthese agents. The appearance of tetracycline resistance in P. acneshas been correlated with previous tetracycline therapy (127).Other antimicrobials, such as doxycycline and minocycline, aremore effective than tetracycline.

Tigecycline is effective against anaerobic bacteria (137, 140).Overall, tigecycline has a low rate (5.5%) of resistance against B.fragilis group species (22). Jacobus et al. (140) found that 90% of831 B. fragilis group isolates were susceptible to �8 �g/ml of tige-cycline and that P. distasonis isolates were the most resistant. Sny-dman et al. (27) observed that 5% of B. fragilis, 3.6% of B.thetaiotaomicron, 3.3% of B. ovatus, and 7.2% of the unusual B.fragilis group species isolates showed resistance to tigecycline.Goldstein et al. (137), who tested 396 unusual anaerobes, found allGram-positive anaerobes and 228/232 Gram-negative anaerobesto be susceptible to �1 �g/ml of tigecycline.

Fluoroquinolones

The earlier fluoroquinolones, such as ciprofloxacin and ofloxacin,are inactive against most anaerobic bacteria. However, somebroad-spectrum quinolones have significant antianaerobic activ-ity. Quinolones with low activity against anaerobes include cipro-floxacin, ofloxacin, levofloxacin, fleroxacin, pefloxacin, enoxacin,and lomefloxacin. Compounds with intermediate antianaerobicactivity include sparfloxacin and grepafloxacin (141). Trovafloxa-cin, gatifloxacin, and moxifloxacin yield low MICs against mostgroups of anaerobes (40). The use of trovafloxacin has been lim-ited because of hepatotoxicity. Quinolones with the greatest invitro activity against anaerobes include clinafloxacin and sitafloxa-cin (142).

Moxifloxacin has been studied and approved by the FDA asmonotherapy in intra-abdominal infections in adults (76, 90) andhas shown activity against intra-abdominal anaerobic isolates(143, 144). However, concern over increasing fluoroquinoloneresistance in both E. coli and B. fragilis group species has madeclinicians cautious in its use in intra-abdominal infections (76). Avariety of studies (19, 22, 144) have reported increases in B. fragilisresistance to moxifloxacin.

A recent study (143) reported a pooled analysis of 4 randomizedclinical trials (2000 to 2010) that assessed the comparative efficacyof moxifloxacin in complicated intra-abdominal infections of 745microbiologically evaluable cases and focused on its efficacyagainst B. fragilis. Of pretherapy anaerobes from moxifloxacin-treated patients, 561 (87.4%) were susceptible at �2 mg/liter, 34(5.3%) were intermediate at 4 mg/liter, and 47 (7.3%) were resis-




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tant at �8 mg/liter. Moxifloxacin achieved similar clinical successrates against all anaerobes, including those isolated from patientsinfected with B. fragilis (158 [82.7%] of 191 patients), B.thetaiotaomicron (74 [82.2%] of 90 patients), and Clostridium spp.(37 [80.4%] of 46 patients). The overall clinical success rate for allanaerobes was 82.3%. For all anaerobes combined, the clinicalsuccess rates were 83.1% (466 of 561 patients) for an MIC of �2mg/liter, 91.2% (31 of 34 patients) for an MIC of 4 mg/liter, 82.4%(14 of 17 patients) for an MIC of 8 mg/liter, 83.3% (5 of 6 patients)for an MIC of 16 mg/liter, and 66.7% (16 of 24 patients) for anMIC of �32 mg/liter. This suggests that moxifloxacin can be cau-tiously used for anaerobic intra-abdominal infections providedthat the patient has mild or moderate disease and has not beenexposed to a fluoroquinolone recently. It may be an alternative forhighly penicillin-allergic patients.

The use of the quinolones is restricted in growing children be-cause of their possible adverse effects on cartilage. The major con-cerns with expanding the use of fluoroquinolones to treat anaer-obic infections have been reports of increasing resistance in strainsof the B. fragilis group as well as anaerobic Gram-positive cocciand the impact of these antibiotics on the growing incidence of C.difficile-associated disease (142).

Fluoroquinolone resistance. Bacteroides species resistance tofluoroquinolones has been attributed to either an alteration inefflux of the antibiotic or a mutation in the quinolone resistance-determining region (QRDR) of the gyrase A gene (gyrA) fromsingle or multiple mutations (144). High-level resistance can becaused by both mechanisms.

A study (145) of 4,434 B. fragilis group isolates recovered from12 U.S. medical centers between 1994 and 2001 illustrated thatfluoroquinolone resistance was dependent on species and sourceof isolation, with B. vulgatus isolates from decubitus ulcers beingthe most resistant (71%). Moxifloxacin resistance rates variedfrom 17% for B. fragilis strains isolated from the female genitouri-nary tract to 52% for all blood culture isolates (moxifloxacin MICbreakpoint, 4 �g/ml). The most recent national survey (27) re-ported that 27% of B. fragilis, 26% of B. thetaiotaomicron, 38% ofB. ovatus, and 55% of B. vulgatus isolates were resistant to moxi-floxacin. A study of strains isolated from intra-abdominal infec-tions (2001 to 2004) found 87% of B. fragilis and 87% of B.thetaiotaomicron isolates to be susceptible to moxifloxacin (143).The overall results showed that 86% (303/363) of all B. fragilisgroup isolates and 417/450 isolates of all other anaerobic generaand species, including Fusobacterium, Prevotella, Porphyromonas,C. perfringens, Eubacterium, and Peptostreptococcus spp., were sus-ceptible to �2 �g/ml of moxifloxacin. Wexler et al. (146), whostudied 179 respiratory tract anaerobes, identified a single resis-tant strain of C. clostridioforme. A study by Edmiston et al. (40),who evaluated 550 anaerobes recovered from intra-abdominaland diabetic foot infections, reported that 97% were susceptible tomoxifloxacin. Liu et al. (33) observed that 90% of B. fragilis iso-lates recovered from nosocomial infections and bacteremias inTaiwan were susceptible to moxifloxacin. In contrast, a studyfrom Europe (147) found an overall fluoroquinolone resistancerate of 15%, with geographic variations from 7% in southern Eu-rope to 30% in northern Europe. Factors that could account forthese variations include differences in susceptibility that dependon the sources of isolation and antimicrobial utilization patterns.In support of this theory, Goldstein et al. (80) found that 41/42 B.fragilis group strains recovered from pediatric intra-abdominal

infections were susceptible to moxifloxacin, which is infrequentlyused in children.

Fusobacterium canifelinum, recovered from cat and dog bitewound infections, is intrinsically resistant to fluoroquinolones be-cause of Ser79 replacement with leucine and Gly83 replacementwith arginine on gyrA (148).

Moxifloxacin has been approved by the FDA for the treatmentof complicated skin and skin structure infections, including thosedue to B. fragilis, and for mixed intra-abdominal infections causedby B. fragilis, B. thetaiotaomicron, Peptostreptococcus spp., and C.perfringens.

Other Agents

Bacitracin was active in vitro against pigmented Prevotella andPorphyromonas spp. but is inactive against B. fragilis and Fusobac-terium nucleatum (79). Vancomycin and daptomycin are effectiveagainst all Gram-positive anaerobes but are inactive againstAGNB (149). Quinupristin-dalfopristin shows antibacterial activ-ity against anaerobic organisms including C. perfringens, Lactoba-cillus spp., and Peptostreptococcus spp. (150). Linezolid is activeagainst Fusobacterium nucleatum, other Fusobacterium spp., Por-phyromonas spp., Prevotella spp., and Peptostreptococcus spp.(117). However, little clinical experience been gained in the treat-ment of anaerobic bacteria using these agents.

GENERAL CONSIDERATION OF ANTIMICROBIAL SELECTION

Because anaerobic infection is often polymicrobial and is causedby aerobic and anaerobic organisms, antimicrobials that are effec-tive against both components of the infection should be adminis-tered. When such therapy is not given, the infection may persist,and serious complications may occur (2, 3, 151). A number offactors should be considered when choosing appropriate antimi-crobial agents: they should be effective against all target organ-isms, induce little or no resistance, achieve sufficient levels in theinfected site, have minimal toxicity, and have maximum stabilityand longevity.

When selecting antimicrobials for the therapy of mixed infec-tions, their aerobic and anaerobic antibacterial spectrum and theiravailability in oral or parenteral form should be considered (Table1). Some antimicrobials have a limited range of activity. For ex-ample, metronidazole is active against only anaerobic bacteria andtherefore cannot be administered as a single agent for the therapyof mixed infections. Other antimicrobials, such as carbapenems,tigecycline, and combinations of a beta-lactam and a beta-lacta-mase inhibitor, possess a broader spectrum of activity against aer-obic and anaerobic bacteria.

Selection of antimicrobial agents is simplified when a reliableculture result is available. However, this may be particularly diffi-cult in anaerobic infections because of the difficulties in obtainingappropriate specimens. For this reason, many patients are treatedempirically on the basis of suspected, rather than established,pathogens. Fortunately, the types of anaerobes involved in manyanaerobic infections and their antimicrobial susceptibility pat-terns tend to be predictable (2, 3). However, some anaerobic bac-teria have become resistant to antimicrobial agents, and many candevelop resistance while a patient is receiving therapy (111, 152).

Anaerobic bacteria have always been resistant to aminoglyco-sides and trimethoprim-sulfamethoxazole. Resistance amongsome anaerobes has increased significantly over the past 3 decades.The potential for growing resistance of anaerobes to antimicrobi-

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als is especially noted with penicillins, fluoroquinolones, clinda-mycin, and cephalosporins. Chloramphenicol is rarely used in theUnited States, and resistance is very rare; when present, it is due tochloramphenicol’s inactivation by acetyltransferase.

Aside from susceptibility patterns, other factors influencingthe choice of antimicrobial therapy include the pharmacologicalcharacteristics of the various drugs, their toxicity, their effect onthe normal flora, and their bactericidal activity (2, 3). Althoughidentification of the infecting organisms and their antimicrobialsusceptibility may be needed for selection of optimal therapy, theclinical setting and Gram stain preparation of the specimen mayindicate the types of anaerobes present in the infection as well asthe nature of the infectious process.

Antimicrobial therapy for anaerobic infections usually shouldbe given for prolonged periods because of their tendency to re-lapse. This period may range from 3 weeks to 3 months, depend-ing on the site and severity of the infection.

Because anaerobic bacteria generally are recovered mixed withaerobic organisms, selection of proper therapy becomes morecomplicated. In the treatment of mixed infection, the choice ofthe appropriate antimicrobial agents should provide for adequatecoverage of most of the pathogens, aerobic and anaerobic. Somebroad-spectrum antibacterial agents possess such qualities, whilefor some organisms, additional agents should be added to thetherapeutic regimen.

CURRENT PRACTICE OF SELECTION OF ANTIBIOTIC FORANAEROBIC BACTERIA

In our personal experience, clinicians are generally aware of theimportance of anaerobic bacteria in a wide variety of infections.These include mainly diabetic foot and intra-abdominal infec-tions and aspiration pneumonia. Clinicians are less aware of all thetaxonomic changes that have occurred and the names of new spe-cies. To most clinicians, B. fragilis group species are recognized asthe major anaerobic pathogen, but the individual B. fragilis groupsubspecies are less readily recognized. For many, there is only avague familiarity with Prevotella and Porphyromonas spp. andother Gram-negative organisms as anaerobic pathogens. Amongthe Gram-positive anaerobes, clinicians are also aware of Clostrid-ium species, especially C. perfringens and Clostridium botulinumand, more recently, the great importance of C. difficile. If asked,clinicians will know that anaerobic Gram-positive cocci exist butwould be unlikely to know either their current nomenclature ortheir involvement in specific infections. Consequently, whenasked, most clinicians will consider the presence and role of B.fragilis in an infectious process and will likely lump all other an-aerobes into the single category of “other anaerobes.” This nar-rowed view is also a result of current schemas of limited laboratoryidentification and reporting of anaerobes. Hence, the clinician islikely to consider only the in vitro activity of specific agents againstB. fragilis and assume that all the other anaerobes will likely besusceptible as well.

In addition, clinicians may be less aware of the variability be-tween laboratories in their capabilities and interest in anaerobicbacteriology and the specific level of performance and capabilityof their own clinical microbiology laboratories to both isolate an-aerobes and perform susceptibility testing (20, 36, 37). Cliniciansare likely to be unaware of recent changes in CLSI breakpoints forany specific bacterium-drug combination (17). Coupled with thisdilemma is the often unappreciated need for proper specimen

collection and transport media to facilitate the viability of anaer-obic pathogens (2–4).

Controversy even about the importance of obtaining anaero-bic blood cultures has reemerged (12). This occurred after thedecline in rates anaerobic bacteremia in the 1970s and 1980s,which led many medical centers to discontinue obtaining bloodcultures for anaerobic bacteria. However, with the reemergence ofanaerobic bacteremia in the 1990s, many centers resumed pro-cessing of blood cultures for anaerobes. Because many medicalcenters do not process anaerobic cultures or do so in an inappro-priate manner, results of anaerobic isolation and susceptibilitytesting for the individual patient remain virtually inaccessible toclinicians, who must then rely on the occasional and periodic pub-lished surveys from a small cadre of scattered research institutions(22, 27, 78). All of these factors affect the clinician’s cognitivechoices in the selection of an antimicrobial agent to treat anaero-bic infections.

A U.S. national survey on anaerobic susceptibility testing (20)found that drugs tested by hospital laboratories that performedtesting were as follows: metronidazole (89% of laboratories), pen-icillin and clindamycin (83%), cefotetan and ampicillin-sulbac-tam (67%), cefoxitin (50%), imipenem (44%), piperacillin-tazo-bactam (39%), and all other drugs (�34%). The agar dilutionmethod seemed reserved for “centers of excellence” or researchcenters.

All this suggests that clinicians rely on “FDA indications, infor-mation from the manufacturers supplied by drug reps, publishedstudy/survey data or just make an educated guess at the appropri-ate empirical or directed therapy” (20).

The limited medical education that students and residents re-ceive about anaerobes and the limitations of microbiology labo-ratory culture, with delays in identification and anaerobic suscep-tibility testing, suggest that at best, the choice of an agent for aserious anaerobic infection is an empirical and educated guess(38). The general principles are limited to questions of whetheranaerobes are involved in this type of infection and if the agentchosen has activity against B. fragilis. Even though the CLSI pro-posed that laboratories perform anaerobic susceptibility testingagainst certain major anaerobic pathogens (17), these recommen-dations are rarely followed and are performed at only a limitednumber of medical centers. Anaerobes (including the B. fragilisgroup) are conspicuous in their absence from the microbiologicalreports of most hospitals (17, 27, 78).

For the treatment of anaerobic infections, the clinician willlikely choose an antimicrobial therapy empirically and continuethat agent as definitive therapy because specific anaerobic micro-biological data will not be forthcoming. The choice will be basedon published literature and surveys and local or specific patientdata. The CLSI has published an appendix to its recent documentthat reports the cumulative susceptibilities for B. fragilis groupspecies collected from three U.S. medical centers from 2007 to2009 (17). Consequently, the most likely agents to cover the vastmajority of anaerobes encountered in mixed infections will beeither a carbapenem or a BL-BLIC such as piperacillin-tazobac-tam. Metronidazole can be used but in combination with anotheragent. Should rapid diagnostics (such as MALDI-TOF MS) be-come available for anaerobes in the future, it is likely that thisparadigm would change.




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CONCLUDING REMARKS

This review describes current methods for antimicrobial testing inresearch or reference laboratories. These methods include the useof agar dilution, broth microdilution, Etest, and the spiral gradi-ent endpoint (SGE) system.

Antimicrobial resistance among anaerobes has consistently in-creased in the past decades, and the susceptibility of anaerobicbacteria to antimicrobial agents has become less predictable. Inthe last few decades, the need for testing of anaerobic isolates hasbeen increasingly recognized, and the testing methodologies usedhave been standardized.

Performance of susceptibility testing for anaerobic bacterialisolates recovered from selected cases can provide important in-formation that can influence the choice of antimicrobial therapy.Susceptibility testing should be performed on isolates recoveredfrom sterile body sites, those that are isolated in pure culture, orthose that are clinically important and have a variable or uniquesusceptibility. The CLSI has standardized many laboratory proce-dures, including anaerobic susceptibility testing, and has pub-lished documents for anaerobic susceptibility testing (commonlycalled M11) (16). The standardization of testing methods by theCLSI allows for comparison of resistance trends among variouslaboratories (15–17). Organisms that should be considered forindividual isolate testing include highly virulent pathogens forwhich susceptibility cannot be predicted, such as Bacteroides, Pre-votella, Fusobacterium, and Clostridium spp.; Bilophila wadswor-thia; and Sutterella wadsworthensis.

The decline in the number of hospital laboratories perform-ing anaerobic susceptibility tests during exactly the same time pe-riod that more multidrug-resistant strains of anaerobes are beingfound in serious infections is problematic. The performance ofsusceptibility tests for individual isolates when indicated and ob-taining local surveillance testing to monitor regional trends havebeen recommended by the CLSI. Most hospitals that send strainsout for susceptibility testing are getting test results by using Etestmethodology, which is within the expertise available in most hos-pitals. If these tests are done in-house, they might yield fasterresults (results can typically be read after 48 h, while most testresults from a commercial laboratory take up to 7 days) and wouldbe clinically useful in therapeutic decisions. Testing that does notdemand quick turnarounds, such as surveillance or batch testingor to monitor trends, could be done by reference laboratories.

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Itzhak Brook, M.D., is an Adjunct Professor ofPediatrics at Georgetown University, Washing-ton, DC. He is the past chairman of the Anti-Infective Drug Advisory Committee of the Foodand Drug Administration. He has done exten-sive research on anaerobic and respiratory tractinfections, anthrax, and infections following ex-posure to ionizing radiation. He is the author of6 medical textbooks, 128 book chapters, andover 700 scientific publications. He is an editor,associate editor, and member of the editorialboard of several medical journals and the Head and Neck Cancer Alliance.Dr. Brook is the recipient of the 2012 J. Conley Medical Ethics Award by theAmerican Academy of Otolaryngology-Head and Neck Surgery.

Hannah M. Wexler, Ph.D., is a research micro-biologist at the Greater Los Angeles VeteransAdministration Healthcare System (GLA-VAHCS) and Adjunct Professor of Medicine atthe UCLA School of Medicine. Dr. Wexler iscurrently Councilor of Division A (Antimicro-bial Agents and Chemotherapy) of the Ameri-can Society for Microbiology after serving asChair of the Division. She began her career withanaerobic bacteria as Director of the Wad-sworth Anaerobe Laboratory at the GLA-VAHCS from 1981 to 2008. Dr. Wexler has been active in the WorkingGroup for Anaerobes for the Subcommittee on Antimicrobial SusceptibilityTesting of the Clinical and Laboratory Standards Institute (CLSI) and wasone of the primary authors of the M11 Standard for determination of sus-ceptibility for anaerobic bacteria. She is currently a Career Scientist at theGLAVAHCS, focusing on regulation of efflux pump activity as an importantfactor in the development of antibiotic resistance in Bacteroides. Her workcurrently includes comparative genomic and transcriptomic studies of mul-tidrug-resistant and virulent strains of Bacteroides fragilis.

Ellie J. C. Goldstein, M.D., F.I.D.S.A.,F.S.H.E.A., is Clinical Professor of Medicine atthe David Geffen School of Medicine, UCLA;Director of the R. M. Alden Research Labora-tory; and in private practice in Santa Monica,CA. He has received the IDSA Clinician of theYear Award and has over 380 publications. Hisinterests include the diagnosis, pathogenesis,and therapy of anaerobic infections, includingintra-abdominal infections, diabetic foot infec-tions, C. difficile, human and animal bites, andthe in vitro susceptibility of anaerobic bacteria to new antimicrobial agents.He is active in the Anaerobe Society of the Americas, the IDSA, ASM, and theSurgical Infection Society. He founded, and served as President of, the In-fectious Diseases Association of California and the Anaerobe Society of theAmericas. He is currently a Section Editor for Clinical Infectious Diseases andchair of the publications committee of Anaerobe. In the past, he has served asan Associate Editor for Clinical Infectious Diseases and the Journal of MedicalMicrobiology.

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