Abstract
SUMMARY
Susceptibility testing of anaerobic bacteria recovered from selected cases can influence the choice of antimicrobial therapy. The Clinical and Laboratory Standards Institute (CLSI) has standardized many laboratory procedures, including anaerobic susceptibility testing (AST), and has published documents for AST. The standardization of testing methods by the CLSI allows comparisons of resistance trends among various laboratories. Susceptibility testing should be performed on organisms recovered from sterile body sites, those that are isolated in pure culture, or those that are clinically important and have variable or unique susceptibility patterns. Organisms that should be considered for individual isolate testing include highly virulent pathogens for which susceptibility cannot be predicted, such as Bacteroides, Prevotella, Fusobacterium, and Clostridium spp.; Bilophila wadsworthia; and Sutterella wadsworthensis. This review describes the current methods for AST in research and reference laboratories. These methods include the use of agar dilution, broth microdilution, Etest, and the spiral gradient endpoint system. The antimicrobials potentially effective against anaerobic bacteria include beta-lactams, combinations of beta-lactams and beta-lactamase inhibitors, metronidazole, chloramphenicol, clindamycin, macrolides, tetracyclines, and fluoroquinolones. The spectrum of efficacy, antimicrobial resistance mechanisms, and resistance patterns against these agents are described.
INTRODUCTION
Infections caused by anaerobic bacteria are common and may be serious and life-threatening. Anaerobes are the predominant components of the bacterial flora of normal human skin and mucous membranes (1), and they are a common cause of bacterial infections of endogenous origin. Because of their fastidious nature, they are difficult to isolate from infectious sites and are often overlooked. Their isolation requires appropriate methods of collection, transportation, and cultivation of specimens (2–5). Treatment of anaerobic bacterial infections is complicated by the relatively slow growth of these organisms (which makes diagnosis in the laboratory possible only after several days), by the frequent polymicrobial nature of the infection, and by the growing resistance of anaerobic bacteria to antimicrobial agents.
Failure to direct therapy against anaerobic organisms often leads to clinical failures. The inadequate isolation, identification, and subsequent performance of susceptibility testing of anaerobes from an infected site can prevent detection of antimicrobial resistance. Therefore, correlation of the results of in vitro susceptibility and clinical and bacteriological responses can be difficult or impossible (1, 3, 6). This discrepancy occurs because of a variety of reasons. Individuals may improve without antimicrobial or surgical therapy, and others can get better because of adequate drainage. In some instances of polymicrobial infection, eradication of the aerobic component may be adequate, although it is well established that it is important to eliminate the anaerobic pathogens (2, 7–14).
Reasons that may lead to failure of therapy include variation in the duration, severity, and extent of infection; lack of surgical drainage or poor source control; the patient's age, nutritional status, and comorbidities; impaired host defenses; poor penetration and low levels of the antimicrobial at the site of infection; enzymatic 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 antimicrobial resistance of the anaerobic pathogens and poor clinical outcome has been reported in several retrospective studies (7–9). There are a number of studies showing that inappropriate therapy will directly affect clinical outcome (10–15).
A prospective study of Bacteroides bacteremia reported adverse clinical outcomes for 128 individuals who received an antibiotic to which the organism was not susceptible (14). Clinical outcome was correlated with results of in vitro susceptibility testing of Bacteroides isolates recovered from blood and/or other sites and was determined by three endpoints: mortality at 30 days, clinical response (cure versus failure), and microbiological response (eradication versus persistence). The mortality rate among those who received inactive therapy (45%) was higher than that among patients who received active therapy (16%; P = 0.04). Clinical failure (82%) and microbiological persistence (42%) were higher for those who received inactive therapy than for patients who received active 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 predictive value of 82%). The authors of this study concluded that antimicrobial susceptibility testing may be indicated for patients whose blood specimens yield Bacteroides spp. (14).
These findings emphasize that it is important to perform susceptibility testing of organisms recovered from certain selected cases to guide therapeutic choices. Susceptibility testing should be performed for organisms recovered from sterile body sites, those that are isolated in pure culture, and those that are clinically important and have variable or unique susceptibility. Anaerobic infections for which susceptibility testing is indicated include (i) serious or life-threatening infections (e.g., brain abscess, bacteremia, or endocarditis), (ii) infections that failed to respond to empirical therapy, (iii) infections that relapsed after initially responding to empirical therapy, (iv) infections where an antimicrobial will have a special role in the patient's outcome, (v) when an empirical decision is difficult because of an absence of precedent, (vi) when there are few susceptibility data available on a bacterial species, (vii) when the isolate(s) is often resistant to antimicrobials, or (viii) when the patient requires prolonged therapy (e.g., septic arthritis, osteomyelitis, undrained abscess, or infection of a graft or a prosthesis). The standardization of testing methods by the Clinical and Laboratory Standards Institute (CLSI) (Wayne, PA) allows for comparison of resistance trends among various laboratories (15–17). Organisms that should be considered for individual isolate testing include highly virulent pathogens for which susceptibility cannot be predicted, such as Bacteroides, Prevotella, Fusobacterium, and Clostridium spp.; Bilophila wadsworthia; and Sutterella wadsworthensis.
The routine susceptibility testing of all anaerobic isolates is extremely time-consuming and is not cost-effective. However, susceptibility testing should be performed for epidemiological and survey purposes for a limited and selected number of anaerobic isolates. Antibiotics tested should include penicillin, a beta-lactam–beta-lactamase inhibitor combination (BL-BLIC), clindamycin, metronidazole, and a carbapenem (i.e., imipenem, meropenem, or ertapenem). If needed, ancillary susceptibility testing can be performed for cefoxitin, tigecycline, and moxifloxacin, which have approved antianaerobe indications.
Antimicrobial resistance among anaerobes has consistently increased in the past 3 decades, and the susceptibility of anaerobic bacteria to antimicrobial agents has become less predictable. The most commonly isolated antibiotic-resistant anaerobes are species within the Bacteroides fragilis group (18). Resistance to several antimicrobial agents by B. fragilis group species and other anaerobic Gram-negative bacilli (AGNB) has increased over the past decade (15–17, 19–22). Resistance has also increased among other anaerobes, such as Clostridium spp., that were previously very susceptible. This increase makes the choice of appropriate empirical therapy even more difficult. Resistance patterns have been monitored through national and local surveys, but susceptibility testing of anaerobic bacteria at individual hospitals is rarely done (20).
This review describes the antimicrobial agents that are effective against anaerobic bacteria and the methods used to perform antimicrobial susceptibility testing of these organisms.
SUSCEPTIBILTY TESTING OF ANAEROBIC BACTERIA
The antibiograms of anaerobic bacteria have become increasingly unpredictable, and multiresistant clinical isolates are appearing, confounding the concept of foolproof anaerobic therapy (21, 23, 147). Resistance to even the most active drugs, such as imipenem, piperacillin-tazobactam, ampicillin-sulbactam, and metronidazole, has been reported (23, 25, 26). Furthermore, there are clear differences in the geographic patterns of resistance and even differences in resistance patterns in different hospitals in a single city, perhaps due in part to the variability in prescribing patterns (27). Numbers of reports of broadly multidrug-resistant B. fragilis strains as well as numbers of reports of resistance arising during treatment have increased (6, 11, 14, 22, 28). There is evidence that suboptimal therapy can actually select for antibiotic resistance and even induce transfer of resistance determinants. Also, more strains exhibiting multidrug resistance (MDR) have been found (21, 23).
Taken together, these factors emphasize the need for antimicrobial susceptibility testing of anaerobes as well as periodic surveillance antimicrobial susceptibility testing to detect geographic or temporal trends. The most appropriate susceptibility test method will differ depending on whether the test is being done for a specific isolate in a hospital laboratory (or a commercial laboratory used by the hospital) or whether surveillance testing is being performed at a hospital or reference laboratory. In the last few decades, testing methodologies used have been standardized.
Standardization of Testing
The CLSI is a U.S. organization that evolved from a voluntary consensus organization in 1967 to become a World Health Organization Collaborating Center for Clinical Laboratory Standards and Accreditation. The CLSI has standardized many clinical procedures, including anaerobic susceptibility testing, and has published documents for anaerobic susceptibility testing (commonly called M11) (16). CLSI policy does not permit it to advocate any commercial technique; rather, it describes two reference methods (agar dilution and broth microdilution) and emphasizes that other techniques, such as gradient techniques (generally referring to Etest) or commercial broth microdilution plates, may be used as long as equivalence to the reference methods is established. Current recommendations of the CLSI limit the broth microdilution method to testing of the B. fragilis group. Surveillance studies performed in reference laboratories in the United States and worldwide most commonly use the CLSI method (see below). The newest document, M11-A8, was published in 2012 (16). The CLSI reference standard is not intended for testing of single isolates; rather, it provides a standard against which other methods may be measured.
The European Committee on Antimicrobial Susceptibility Testing (EUCAST) publishes its own breakpoints; these are not always equivalent to those of the CLSI (29). However, EUCAST does not actually specify a testing method for anaerobes. Most susceptibility studies emanating from European countries use CLSI methodology, although breakpoint interpretation is often based on EUCAST recommendations, and differences in reported resistance rates may be 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, The Committee on Antimicrobial Susceptibility Testing of the Japanese Society of Chemotherapy recommended the use of the CLSI method (32). Recent large surveillance studies in Europe have also used CLSI methodology and often include both CLSI and EUCAST breakpoints (29, 147). Some studies refer to other method documents; a recent German multicenter study referred to a specific German document for testing methodology (24). The differences 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, or endpoint reading methods) may lead to perceived significant differences in resistance rates. Therefore, when trying to evaluate or compare published studies, the method used should be taken into account. At this point, the most commonly used method by far is M11-A8 of the CLSI (16).
Surveillance Tests for Particular Hospitals or Geographic Regions
Surveillance tests have been conducted for years by groups worldwide and reflect sweeping general trends (18, 24, 27, 33, 34, 147) However, these results will not necessarily reflect the patterns of specific patients or hospitals. Because of this, the CLSI strongly recommends that hospitals conduct at least annual surveillance antimicrobial susceptibility testing to elucidate their local patterns. The numbers and choice of species of strains tested should reflect the frequency with which they are isolated. At least 50 to 100 strains should be tested in order to obtain an accurate picture of the pattern of local isolates, and if isolates from different body sites are available, they should be included. At least 20 isolates of Bacteroides spp. and 10 isolates of other frequently isolated genera should be tested. If the expertise is not available in the hospital clinical laboratory, the strains should be sent to a reference laboratory for testing.
Large reference laboratories may use CLSI-approved methods (described briefly below), which are more laborious and require more in-house preparation than commercially available methods. In these cases, the antimicrobials tested can be tailored to reflect the hospital's particular formulary. Laboratories should ideally include at least one agent from each antimicrobial class, even if it is not included on the formulary. Many reference laboratories will use commercially prepared panels; in these cases, the agents tested will depend on whichever antibiotics are included in the commercial panel that the laboratories are using for testing. The CLSI microdilution method is approved only for B. fragilis group organisms, because many other anaerobes will not consistently grow well in broth media. The CLSI recognized that there are commercially available broth microdilution panels that are FDA approved for testing of all anaerobes and may work satisfactorily for certain non-B. fragilis group species. At the time of writing of this publication, the only ready-made commercially available broth microdilution panel identified was produced by Sensititre (Trek Diagnostic Systems). Other laboratories performing large-scale surveillance testing may prepare their own broth microdilution panels (e.g., the R. M. Alden Laboratory [Santa Monica, CA] uses the CLSI method of broth microdilution and prepares frozen panels according to need).
The results of the surveillance study should be maintained and recorded so that local trends in emerging resistance may be recognized and documented. If formal surveillance testing cannot be done, hospitals should collect and summarize their antimicrobial susceptibility test results and create a hospital-specific antibiogram that can be consulted if needed. A 2008 survey of clinical hospital laboratories (20) revealed that fewer than half of the laboratories did any kind of anaerobic testing at all, either in-house or any kind of batch testing sent to an outside laboratory. At the very least, periodic batch testing should be strongly encouraged.
Testing in a Clinical Setting
Susceptibility testing may not be necessary for many routine patient isolates. The CLSI suggests testing of isolates from blood, brain abscess, endocarditis, osteomyelitis, joint infection, infection of prosthetic devices, or vascular grafts (see above). Also, any bacteria isolated from normally sterile body sites should be tested (as long as they are not likely to be contaminants). Isolates from patients likely to undergo long-term therapy should be tested so that any development of resistance can be recognized. Obviously, any isolate from a therapy failure or in a case in which the therapeutic decisions will be influenced by the results should be tested.
Organisms to test should include those most likely to be the most resistant (such as B. fragilis group species) or highly virulent (certain Bacteroides, Prevotella, Fusobacterium, Clostridium, Bilophila, and Sutterella species), especially if their susceptibility patterns are unpredictable. Agents to test should include those on the hospital formulary, and the agent that is being considered or used for therapy should be included if at all possible.
The most recently published surveys of anaerobic susceptibility testing performed in a clinical laboratory indicated that, as of the time of the survey, only 21% (21/98) of hospital laboratories performed anaerobic susceptibility testing in-house (20). This indicated a steep decline from earlier rates; in 1990, 70% of hospital laboratories performed 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 body site isolates and by 70% (14/20) of laboratories for selected surgical wound isolates. An additional 40% (8/20) of laboratories surveyed would also perform susceptibility testing by special request. In this survey, most hospital laboratories used the Etest (62%; 13/21) for susceptibility testing, while only 17% of reference laboratories used it. All the reference laboratories used broth microdilution for susceptibility testing, as did 40% (8/20) of hospital laboratories (all commercially prepared). Since nearly two-thirds of laboratories do not perform testing (and even those that test do it on only a limited basis), the clinicians often choose therapy based on manufacturers' information, FDA indications, published studies, or their clinical judgment (20).
At this time, most commercial laboratories use Etest methodology for performing anaerobic susceptibility testing on isolates sent to them for testing. The Etest is particularly suitable for testing of one or a few isolates against multiple agents (as long as the particular agent is available on an Etest strip). Currently, we are not aware of any commercially available ready-made broth microdilution panels that are “FDA approved” for clinical diagnostic use. The only commercially available ready-made panel is produced by Sensititre (Trek Diagnostic Systems); it is designated for research purposes only and is not FDA approved for diagnostic testing. Thus, a clinical laboratory would have the option of either using Etest (which is FDA approved), using noncommercial panels with CLSI-approved methodology (thus, FDA approval would not be relevant), or sending the isolates to a commercial or reference laboratory for testing.
Testing in a Research or Reference Laboratory
Agar dilution.
Agar dilution involves the incorporation of different concentrations of the antimicrobial agent into a nutrient agar medium followed by the application of a standardized number of bacterial cells to the surface of the agar plate (Fig. 1). Plates are read after ∼48 h of growth by visually comparing the growths of different strains in the series, and the MIC is designated the lowest antimicrobial concentration that inhibits growth. The CLSI method specifies the use of control strains including B. fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Clostridium difficile ATCC 700057.
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 a different 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 be considered the MIC.
Broth microdilution.
In the broth microdilution assay, a polystyrene tray (usually containing 96 wells) is filled with small volumes of serial 2-fold dilutions of different antibiotics (Fig. 2). If made in-house, trays can be tailored to the particular needs of the laboratory, using the drugs and concentration ranges needed. The panels can be prepared in advance, frozen, and used as needed. Details for this procedure are described in the CLSI manual (16).
Fig 2.
Broth microdilution. (Left) Sensititre pipette for filling microdilution plate. (Middle) Plate being inoculated with strains. (Right) Plate after growth of strains. 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.)
Etest.
For the Etest (AB Biodisk, bioMérieux) procedure, an individual isolate is suspended in broth or saline and swabbed onto a Brucella blood agar plate. The Etest is a plastic strip with a predetermined antimicrobial concentration gradient on one side and an interpretative MIC scale on the other. The MIC is read as the concentration where the elliptical zone of inhibition intersects the strip. In general, the Etest correlates well with the reference procedure, although for certain drugs, there are some discrepancies (39, 41). This has become the most popular test for testing of individual isolates (Fig. 3).
Fig 3.
Etest (AB Biodisk). (Courtesy of bioMérieux, reproduced with permission.)
Spiral gradient endpoint system.
The Autoplate 4000 (Advanced Instruments, Inc., Boston, MA) spiral gradient endpoint (SGE) system deposits a specific amount of antimicrobial stock solution in a spiral pattern on a 150-mm agar plate, producing a concentration gradient that decreases radially from the center of the plate (Fig. 4, left). After the antimicrobial agents are allowed to diffuse, the isolates are deposited onto the plate with an automated inoculator or manually streaked from the center to the edge of the plate. After incubation, endpoints of growth are marked, and the distance is measured in millimeters from the center of the plate to the point where growth stops (Fig. 4, middle). The data are then entered into a computer software program provided by the manufacturer, which determines the concentration of drug from the radius of growth and the molecular weight (i.e., diffusion characteristics) of the antimicrobial agent. Details of the procedure are described in the manufacturer's guidelines. Comparisons of 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 be easily determined (Fig. 4, right).
Fig 4.
Spiral gradient endpoint technique. (Left) Dye representing the gradient application of antimicrobial stock solution, decreasing from the center of the plate. (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 and translated into an MIC by a software program. (Right) Detail of the endpoint and observation of resistant colonies past the endpoint.
Commercially Available Testing
ThermoFisher Scientific (Cleveland, OH) now owns Remel, Oxoid, and Sensititre (Trek Diagnostic Systems). These manufacturers currently offer two ready-made panels. There is a dried anaerobic panel (AN02B; Sensititre [Trek Diagnostic Systems]) that includes 15 antimicrobials in a variety of dilution ranges, depending on the antibiotic (http://www.trekds.com/products/sensititre/c_pltformats.asp). The dried panels are stable at room temperature for 1 to 2 years. The panels are specifically designated for research purposes and not for diagnostic testing. Sensititre will also prepare custom panels which can be either frozen or air dried. There is also a frozen panel that was previously sold by Remel (ANA MIC panel, catalog number R8320100), now marketed through Oxoid; this can be ordered in the United States through ThermoFisher (it is shipped from the United Kingdom and requires a lead time of 4 to 6 weeks).
The question of whether these tests are FDA approved for diagnostic purposes is a bit confusing. FDA approval for a panel requires that all agents on the panel are FDA approved for use in anaerobic infections. If any agent does not meet this requirement, the panel is not FDA approved for diagnostic purposes, even though the testing method is, in fact, approved. In practice, most hospitals that use microbroth panels order the panels that reflect the needs of their physicians based on hospital formulary and drug used and not on FDA approval.
Specialty Laboratories (operated by Quest Diagnostics) provides testing services for 6 antimicrobials (cefoxitin, penicillin, clindamycin, piperacillin-tazobactam, metronidazole, and imipenem) using Etest methodology (anaerobic susceptibility panel 5711). Focus Diagnostics (also a subsidiary of Quest Diagnostics, Inc.) has discontinued the microdilution test for anaerobic testing and now offers routine testing using Etest (anaerobic susceptibility panel 51477). Six to nine drugs are offered routinely, depending on the organism being tested. For B. fragilis group organisms, ampicillin-sulbactam, clindamycin, imipenem, meropenem, metronidazole, and piperacillin-tazobactam are included in the routine panel. The addition of penicillin, cefoxitin, and cefotetan may be ordered for testing of Clostridium. Other drug tests can be custom ordered (depending on the availability of the Etest strip). Mayo Medical Laboratories (Rochester, MN) also uses Etest methodology.
β-Lactamase Test
Anaerobic organisms may be tested for the presence of the β-lactamase (BLA) enzyme by using a chromogenic cephalosporin test, such as nitrocefin disks. These are colorimetric tests that are very easy to perform, and results can be read quickly (5 to 30 min) and would be useful if penicillin or ampicillin therapy is being considered. The great majority of B. fragilis group isolates are β-lactamase producers; therefore, testing is generally not recommended for this group. Other isolates have less predictable patterns, and certain anaerobes (some Clostridium, Fusobacterium, and Prevotella species, for example) may be penicillin-ampicillin resistant due to β-lactamase. Isolates with a positive β-lactamase test should be considered resistant to penicillin and ampicillin. A negative test does not necessarily predict susceptibility to these drugs, as some anaerobes are resistant to β-lactam antimicrobial agents by other mechanisms.
Increased activity of efflux pumps or changes in penicillin-binding proteins (PBPs) have been shown to affect MICs of β-lactams for many Bacteroides isolates; systematic surveys of these mechanisms have not been conducted, so the percentage of strains that 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 for variability in reporting of MIC results. At that time, technical variability among laboratories was a major factor. Laboratories used different media and different inoculum sizes and may have read results after different incubation times. Since that time, the CLSI (formerly NCCLS) procedures have been extensively revised and have been adopted by virtually all testing laboratories in the United States and even worldwide. Therefore, the technical variability among laboratories has been greatly minimized. Differing breakpoints will also not influence the individual MIC results for a particular strain but will change the percentage of strains reported as susceptible or resistant in surveillance studies. Most studies conform to CLSI breakpoints, but certain EUCAST breakpoints are different. Many studies will acknowledge these differences and report results with both breakpoints.
Other factors that impact results reported in survey antibiograms include the particular makeup of the groups of strains included. The different species belonging to a particular genus may have markedly different susceptibility patterns. For example, within the B. fragilis group, MICs for B. thetaiotaomicron and B. ovatus are often higher than those for B. fragilis. Studies using different proportions of the various B. fragilis group species may reflect different antibiograms for the B. fragilis group as a whole, when in reality, the only difference is the proportion of the various species used. The source of isolates (i.e., stool, abscess, or appendiceal) may also influence the resistance profile of the species and should be considered when evaluating the survey results.
By far, the most prevalent cause of variability in MIC reports is the variation in interpreting what the MIC is in cases where endpoints are not very clear. CLSI reference protocol M11-A8 has included extensive discussion on breakpoint interpretation and included several sets of photographs to aid in reading of breakpoints.
There is a certain margin of error (usually ±1 2-fold dilution) for any of these techniques. With certain antimicrobial agents, the MICs for a large percentage of B. fragilis group strains cluster within one 2-fold dilution range of the breakpoint. Clustering around the breakpoint is a characteristic of the organism-drug interaction and is seen, to some degree, in all of the testing methods. When an MIC is near the breakpoint (e.g., in the case of the B. fragilis group and chloramphenicol as well for as many β-lactam agents), an organism called susceptible on one occasion may be retested and called resistant, all within the accepted variability of the technique. Therefore, in the case of single isolates, it is useful for the clinician to know the MIC of a drug for the strain as well as the established breakpoint rather than just the categorical determination. In cases where other factors that can cause variability are also involved (e.g., differences in techniques or media or different people reading results), it becomes clear that minor changes (e.g., less than 15%) in percent susceptibility may not be significant in reports of large groups of strains; still, survey studies provide useful information on trends and patterns in antimicrobial susceptibility of anaerobes.
On the other hand, changes in susceptibility that are known to be due to specific mechanisms (such as the presence of the nitroimidazole reductase gene [nim], which causes metronidazole resistance, or cfiA genes, which can result in carbapenem resistance) may begin to appear as relatively modest changes in resistance rates and then quickly increase as the resistance determinant becomes disseminated. Research laboratories that conduct large surveys, as well as pinpointing the mechanisms of resistance that may be relevant in the antibiograms, can help monitor, understand, and perhaps even control these shifts by making recommendations based on the molecular traits of the pathogen.
Detection of Resistance by Using Molecular Methods
Molecular methods are currently limited to research laboratories studying resistance mechanisms of anaerobic bacteria. The most commonly used molecular techniques are PCR amplifications to identify nim genes responsible for metronidazole resistance or cfiA-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-nitroimidazole (4- or 5-Ni, respectively) to 4- or 5-aminoimidazole (thus avoiding the formation of toxic nitroso radicals that are essential for antimicrobial activity). nim homologs were found in both Gram-positive and -negative genera of aerobic and anaerobic bacteria and Archaea, suggesting that the nim gene family is ancient and widespread. More significantly, nim genes are often found on mobilizable plasmids and therefore pose a significant threat to the continuing utility of 5-Ni drugs, including metronidazole, which is the most frequently prescribed drug for anaerobic infections worldwide (50).
PCR is used to detect the presence of the nim gene. Detection of nim genes was described in 1996, using the universal primers NIM-3 and NIM-5 (51), followed by restriction analysis to identify the specific nim type (52). Since then, nine nim genes were described for B. fragilis (nimA to nimI [nimA-I]), and an additional nimI gene was described for Prevotella (53, 54). However, increasing numbers of clinical metronidazole-resistant isolates that do not possess any of the nimA-H genes are being found. Also, metronidazole resistance could be induced in nim-negative strains by exposure to sub-MICs of metronidazole; the mechanisms behind the increased MICs are not clear (54, 55). However, it is clear that there is also a non-nim-based mechanism of resistance to metronidazole.
Clindamycin resistance is conveyed by a macrolide-lincosamide-streptogramin (MLS)-type 23S methylase, typically encoded by one of several erm genes that are regulated and expressed at high levels (56).
Carbapenem resistance in B. fragilis is associated with cfiA- or ccrA-encoded class B metallo-β-lactamase. Although not all cfiA-positive B. fragilis strains are resistant to carbapenems, they all have the possibility of becoming resistant to this group of antibiotics by acquisition of an appropriate insertion sequence (IS) element for full expression of the cfiA gene, leading to possible treatment failure. The presence of the cfiA gene, as well as associated IS elements, can be determined by a PCR technique (57–59). Two recent 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 to division II type strains (60, 61). Although this type of test is not currently appropriate for clinical settings, there is increasing interest in using this technology for clinical laboratory identification (58), and it is conceivable that it could be potentially developed for testing.
Five genes conferring ribosomal protection have been found in anaerobes; these are tet(Q) (the most common, found in 12 genera), tet(M) (found in nine genera), tet(W) (found in seven genera), tet(32), and tetB(P) (the ribosomal protection gene found in the P operon in Clostridium). Four tetracycline efflux genes have been found in anaerobes; these are tet(B) (Treponema), tet(K) (Eubacterium and Peptostreptococcus), tet(L) (Actinomyces, Peptostreptococcus, Veilonella, and Clostridium), and tetA(P) (the efflux gene in the P operon in Clostridium) (63). Three genes encoding enzymes that inactivate tetracycline, i.e., tet(X1), tet(X2), and tet(32), have been identified in Bacteroides (62, 63). Five genes conferring MLS resistance have been identified in anaerobes, including erm(B), erm(C), erm(F), erm(G), and erm(Q). In contrast, genes coding for MLS-resistant efflux proteins or inactivating enzymes are not generally described for anaerobic species (63), although homologs of the mefA efflux pump have been found on conjugative transposons in Bacteroides (154).
Is a Rapid Test on the Horizon?
It would be very appealing to wish that there could be a simple molecular test, or even a complex test, such as multiplex PCR, that would determine the actual or potential resistance of an organism to multiple antibiotics. One can envision a test that could, in fact, 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 parC genes). A multiplex PCR test that could detect multiple resistance determinants in B. fragilis isolates was recently described by Pumbwe et al. (64) and could be helpful to predict likely resistance patterns (Fig. 5). However, the presence of systems of multidrug efflux pumps may prove to be the confounding problem that will not permit a definitive determination of a resistance profile by molecular techniques. At least in aerobes, much of the multidrug resistance seen in the last several years is due to the action of multidrug efflux pumps, and we have indications that a similar phenomenon may be operative in anaerobes as well. In B. fragilis, 16 homologs of tripartite efflux pumps of the resistance nodulation division (RND) family have been described (Bacteroides multidrug efflux [Bme] pumps 1 to 16) and are apparently important in conferring multidrug resistance (23, 65, 68); pump activity related to resistance has also been described for Clostridium (69–72). Several multidrug-resistant isolates appear to have significantly increased efflux pump activity. Genes for efflux pumps are present in all strains of bacteria, so a PCR test to detect the gene would always be positive. It is likely that the levels of efflux pump genes transcribed and expressed are important. At this time, the only way to measure these genes in clinical isolates is to quantitatively identify and sequence RNA transcripts, which is not a practical solution.
Fig 5.
Multiplex PCR assay to detect common resistance determinants in B. fragilis. Amplification was done with a set of primers designed for detecting five resistance genes, including carbapenems (cfiA), cephalosporins (cepA), clindamycin (ermF), metronidazole (nimA-F), and tetracycline (tetQ), plus a set of primers for the B. fragilis 16S rRNA gene (positive control). Lane 0, negative control; 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 determinants present in either single- or multiple-strain samples.
ANTIMICROBIAL AGENTS EFFECTIVE AGAINST ANAEROBIC BACTERIA
Table 1 illustrates the antimicrobials effective against anaerobic bacteria and their efficacy against both aerobic and anaerobic bacteria. Many of the older antimicrobials do not have an FDA-approved indication for anaerobic infections, and many of the newer agents have only a limited number of indications for anaerobic infections (Table 2). Because of this, many of these agents are used for the treatment of anaerobic infections without an FDA indication. Table 3 illustrates the resistance of the B. fragilis group and other anaerobes to antimicrobial agents.
Table 1.
Antimicrobial agents effective against mixed infectiona
| Antimicrobial agent | Degree of activity |
|||
|---|---|---|---|---|
| Anaerobic bacteria |
Aerobic bacteria |
|||
| Beta-lactamase-producing AGNB | Other anaerobes | Gram-positive cocci | Enterobacteriaceae | |
| Penicillinb | 0 | +++ | + | 0 |
| Chloramphenicolb | +++ | +++ | + | + |
| Cephalothin | 0 | + | ++ | +/− |
| Cefoxitin | ++ | +++ | ++ | ++ |
| Carbapenems | +++ | +++ | +++ | +++ |
| Clindamycinb | ++ | +++ | +++ | 0 |
| Ticarcillin | + | +++ | + | ++ |
| Amoxicillin + clavulanateb | +++ | +++ | ++ | ++ |
| Piperacillin + tazobactam | +++ | +++ | ++ | ++ |
| Metronidazoleb | +++ | +++ | 0 | 0 |
| Moxifloxacin | ++ | ++ | ++ | +++ |
| Tigecycline | ++ | +++ | +++ | ++ |
Degrees of activity from 0 to +++.
Also available in an oral form.
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 postpartum endomyometritis, 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 by Bacteroides spp. including B. fragilis, B. distasonis, B. intermedius, and B. thetaiotaomicron, Fusobacterium spp., and Peptostreptococcus spp.; skin and skin structure infections, including abscesses, cellulitis, infected skin ulcers, and wound infections caused by and Bacteroides spp. including B. fragilis |
| Gynecological infections, including postpartum endomyometritis, caused by group D Streptococcus, Bacteroides intermedius, or Peptostreptococcus 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. fragilis | |
| Skin 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 group | |
| Central nervous system infections, including meningitis and brain abscess, caused by Bacteroides spp. including the B. fragilis group | |
| Lower respiratory tract infections, including pneumonia, empyema, and lung abscess, caused by Bacteroides spp. including the B. fragilis group | |
| Endocarditis caused by Bacteroides spp. including the B. fragilis group |
Table 3.
Percent resistance of Bacteroides fragilis group isolates and other anaerobes to antimicrobial agentsa,b
| Antimicrobial | MIC breakpoint (μg/ml) | % resistance to antimicrobial | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Susceptible | Resistant | B. fragilis | B. thetaiotaomicron | P. distasonis | B. ovatus | B. vulgatus | B. fragilis group | Prevotella spp. | Fusobacterium spp. | Clostridium spp. | Anaerobic Gram-positive cocci | |
| Ampicillin-sulbactam | ≤8/4 | ≥32/16 | 2.8–11 | 4.9–15 | 15–20.6 | 2–8 | 3–25 | 0 | 0 | 0 | ||
| Amoxicillin-clavulanate | ≤4/2 | ≥16/8 | 4–37 | 12–37 | 21 | 18 | 14 | 10–20 | 0–19 | 0–11 | 0–5 | 0–6 |
| Piperacillin-tazobactam | ≤32/4 | ≥128/4 | 0–5 | 0–12 | 0–14 | 0 | 1.1–7 | 0–8 | 0–1 | 0 | 0 | 0–3 |
| Cefoxitin | ≤16 | ≥64 | 4–25 | 6.8–68 | 11–60 | 18–59 | 11–20 | 17–33 | 0–3 | 0 | 16–35 | 0–2 |
| Ertapenem | ≤4 | ≥16 | 1.4–10 | 1.3–3 | 0–6 | 2–2.2 | 0–2 | 0 | 0 | 0–4 | 0 | |
| Imipenem | ≤4 | ≥16 | 0.3–7 | 0–7 | 0–1 | 0 | 0–7 | <1–1 | 0–6 | 4 | 15 | 0 |
| Meropenem | <4 | ≥16 | 1.2–22 | 0–3 | 0–1 | 0 | 0 | 8 | 0–5 | 0 | ||
| Doripenem | ≤4 | ≥16 | 1.3–12 | 0–3 | 0 | 0 | 0 | 0 | 0 | 0 | ||
| Clindamycin | ≤2 | ≥8 | 10–42 | 39.8–60 | 14.3–64 | 36–45.5 | 40–54 | 32–52 | 13–33 | 8–31 | 16–25 | 5–27 |
| Moxifloxacin | ≤2 | ≥8 | 10–41 | 13–75 | 12.5–52 | 8–87 | 21–74 | 14–57 | 11–42 | 10–25 | 7–53 | 4–36 |
| Tigecycline | ≤4 | ≥16 | 2–11 | 0–5.8 | 0–3.2 | 2–5.2 | 0–5 | 2–13 | 0 | 0 | 14 | 0 |
Including intermediate-resistant strains. Metronidazole is not included since >99% of Gram-negative strains are susceptible.
Adapted from reference 78 with permission from Elsevier.
β-Lactam Antibiotics
Penicillin G is the classical drug of choice when the infecting strains are susceptible to this drug in vitro. Most Clostridium strains (with the exception of some strains of Clostridium ramosum, Clostridium clostridioforme, and Clostridium innocuum) and Peptostreptococcus spp. remain susceptible to penicillin. Most clinical isolates of the B. fragilis group are resistant to penicillin G, and it should not be used for the treatment of infections caused by these organisms. Other strains that may show resistance to penicillins are growing numbers of AGNB, such as the pigmented Prevotella and Porphyromonas spp., Prevotella oralis, Prevotella bivia, Bacteroides disiens, strains of clostridia, Fusobacterium spp. (Fusobacterium varium and Fusobacterium mortiferum), and microaerophilic streptococci. Some of these strains show MICs of 8 to 32 units/ml of penicillin G. In these instances, administration of very high doses of penicillin G (for non-beta-lactamase producers) may eradicate the infection.
Clinical experience with penicillin G in the management of susceptible anaerobic bacterial infections has been good. Ampicillin, amoxicillin, and penicillin generally are equally active, but the semisynthetic penicillins are less active than the parent compound. Methicillin, nafcillin, and the isoxazolyl penicillins (oxacillin, cloxacillin, and dicloxacillin) are ineffective against the B. fragilis group, have unpredictable activity, and frequently are inferior to penicillin G against anaerobes (73).
Penicillin, ampicillin, and amoxicillin are of limited utility due to the production of beta-lactamases by many oral and most intra-abdominal anaerobes. Clavulanate, sulbactam, and tazobactam are beta-lactamase inhibitors that resemble the nucleus of penicillin but differ in several ways. They irreversibly inhibit beta-lactamase enzymes produced by some Enterobacteriaceae, staphylococci, and beta-lactamase-producing Fusobacterium spp. and AGNB (73–75). When used in combination with a beta-lactam antibiotic (such as ampicillin-sulbactam, amoxicillin-clavulanate, and piperacillin-tazobactam), they are effective in treating anaerobic infections caused by beta-lactamase-producing bacteria (BLPB).
Beta-lactam–beta-lactamase inhibitor combinations (BL-BLICs) are popular and appropriate choices for mixed aerobic-anaerobic infections. They have maintained good activity against the vast majority of anaerobes. While 89% of B. fragilis strains are susceptible to ampicillin-sulbactam, 98% are susceptible to piperacillin-tazobactam (17), compared to 86% and 92%, respectively, of B. thetaiotaomicron isolates. Recently, the Infectious Diseases Society of America (IDSA) has removed ampicillin-sulbactam from the recommended list of treatments for intra-abdominal infections due to increased Escherichia coli resistance worldwide, although it has maintained good activity against B. fragilis and other anaerobes (76). Amoxicillin-clavulanate remains the agent of choice for human and animal bite wound infections (77), especially when anaerobes may be involved. Piperacillin-tazobactam is also a frequently and appropriately prescribed agent for serious intra-abdominal infections. It has also maintained good activity against the vast majority of anaerobes (17).
The semisynthetic penicillins, the carboxypenicillins (carbenicillin and ticarcillin), and ureidopencillins (piperacillin, azlocillin, and mezlocillin) generally are administered in large quantities to achieve high serum concentrations. These drugs are effective against Enterobacteriaceae and have good activity against most anaerobes in these concentrations. However, up to 30% of strains of the B. fragilis group are resistant to these agents (78).
Many anaerobes possess cephalosporinases, and therefore, as a class, cephalosporins have very limited utility (41). The activity of cephalosporins against the beta-lactamase-producing AGNB varies. The antimicrobial spectrum of the narrow-spectrum cephalosporins against anaerobes is similar to that of penicillin G, although on a weight basis, they are less active. Most strains of the B. fragilis group and many Prevotella, Porphyromonas, and Fusobacterium spp. are resistant to these agents by virtue of cephalosporinase production (79). The enzyme has little or no hydrolytic activity for the second-generation antimicrobial cefoxitin (a cephamycin). Cefoxitin is therefore the most effective cephalosporin against the B. fragilis group. However, susceptibility may vary by geographic location and is generally related directly to its clinical use. Cefoxitin is relatively inactive against most species of Clostridium, including C. difficile, with the exception of Clostridium perfringens (6, 7, 79).
Studies done in the 1980s found cefoxitin to be effective in eradication of anaerobic infections (80–82). It has often been used for surgical prophylaxis at most body sites that are in proximity to mucus membranes. With the exception of moxalactam, the third-generation cephalosporins are not as active against B. fragilis as cefoxitin. However, these agents have improved activity against Enterobacteriaceae.
At present, approximately 85% of B. fragilis isolates are susceptible to cefoxitin, but the other B. fragilis group species are more resistant (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 Bacteroides spp. that were promoted to the genus level (Table 4) (83).
Table 4.
Members of the Bacteroides fragilis group
| Genus | Species |
|---|---|
| Bacteroides | B. acidifaciens |
| B. caccae | |
| B. coprocola | |
| B. coprosuis | |
| B. eggerthii | |
| B. finegoldii | |
| B. fragilis | |
| B. helcogenes | |
| B. intestinalis | |
| B. massiliensis | |
| B. nordii | |
| B. ovatus | |
| B. thetaiotaomicron | |
| B. vulgatus | |
| B. plebeius | |
| B. uniformis | |
| B. salyersai | |
| B. pyogenes | |
| B. dorei | |
| B. johnsonii | |
| Parabacteroides | P. distasonis |
| P. merdae | |
| P. goldsteinii | |
| P. chartae | |
| P. gordonii | |
| P. johnsonii |
Among the B. fragilis group, B. fragilis accounts for 40% to 54% of the Bacteroides isolates recovered from intra-abdominal as well as other infections (4, 84–86). Another important pathogen that belongs to the B. fragilis group is B. thetaiotaomicron, which accounts for 13% to 23% of the isolates. Other members of the B. fragilis group account for 33% to 39%. The antimicrobial susceptibilities of some members of the B. fragilis group vary, especially to the second- and third-generation cephalosporins. B. fragilis is generally the most susceptible, and B. thetaiotaomicron and Parabacteroides distasonis generally are more resistant (87, 88).
The cephamycins cefoxitin and cefotetan are often not used appropriately. This is because clinicians are not aware of their activity against the B. fragilis group locally and are unlikely to have knowledge of the specific antibiotic susceptibility of the isolate recovered from their patients. Sometimes, these agents are used for surgical prophylaxis for abdominal surgery and for the treatment of aspiration pneumonia. Recently, the IDSA has removed cefotetan from the recommended list of therapies for intra-abdominal infections due to poor B. fragilis group activity and resultant clinical failures (89–91).
The carbapenems (imipenem, meropenem, doripenem, and ertapenem) have excellent activity against anaerobes (92). Imipenem, a thienamycin, is a beta-lactam antibiotic that is effective against a wide variety of aerobic and anaerobic Gram-positive and Gram-negative organisms, including normally multiresistant species such as Pseudomonas aeruginosa, Serratia spp., Enterobacter spp., Acinetobacter spp., and enterococci (93, 94). It also possesses excellent activity against beta-lactamase-producing Bacteroides. It has low MICs for the B. fragilis group. Despite the emergence of carbapenemase-resistant Enterobacteriaceae, 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 poor absorption from the gastrointestinal tract, high plasma concentrations after intravenous administration, a small degree of systemic metabolism, and renal excretion. In the kidney, imipenem is metabolized by breakage of the beta-lactamase bond in the proximal tubular cells. The result is low-level urinary excretion of active imipenem, which may impair its ability to inhibit certain urinary pathogens. To overcome the problem of renal metabolism of imipenem, it is combined 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-life in serum. This agent is an effective single agent for the therapy of mixed aerobic-anaerobic infections.
Meropenem is a carbapenem antibiotic that has a very broad spectrum of activity against aerobic and anaerobic bacteria, similar to that of imipenem. Imipenem has more activity than meropenem against staphylococci and enterococci, but meropenem provides better coverage of aerobic and facultative Gram-negative bacteria such as Pseudomonas, Enterobacter, Klebsiella, Providencia, Morganella, Aeromonas, Alcaligenes, Moraxella, Kingella, Actinobacillus, Pasteurella, and Haemophilus spp. (96, 97). Meropenem has been effective in abdominal infections, meningitis in children and adults, community-acquired and nosocomial pneumonia, and neutropenic fever (98).
Ertapenem is a newer 1-beta-methyl carbapenem, stable to dehydropeptidase. It has a broad antibacterial spectrum for penicillin-susceptible Streptococcus pneumoniae, Streptococcus pyogenes, methicillin-sensitive Staphylococcus aureus, Haemophilus influenzae, Moraxella catarrhalis, Escherichia coli, Citrobacter spp., Klebsiella spp., Serratia spp., Proteus spp., C. perfringens, Fusobacterium spp., Peptostreptococcus spp., and AGNB (99). It is indicated for complicated intra-abdominal and skin structure infections, including diabetic foot infections without osteomyelitis, and acute pelvic infections, including postpartum endomyometritis, septic abortion, and postsurgical gynecological infections. In comparison to other available carbapenems, ertapenem has a long half-life of 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 carbapenem to be commercially released. Its antimicrobial spectrum more closely resembles those of meropenem and imipenem than that of ertapenem (94). Thus, it has significant in vitro activity against streptococci, methicillin-susceptible staphylococci, Enterobacteriaceae (including extended-spectrum-beta-lactamase-producing strains), P. aeruginosa, Acinetobacter spp., and the B. fragilis group. Doripenem does not have activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, and the majority of Gram-negative bacilli that are resistant to meropenem or imipenem (94). In vitro, resistant P. aeruginosa mutants appear to be more difficult to select with doripenem than with other carbapenems. Doripenem has been approved in the United States for use in treatment of complicated intra-abdominal infection and complicated urinary tract infection.
In general, clinicians recognize the generally good activity of carbapenems against anaerobes and prescribe them appropriately. Consequently, they are employed in more serious anaerobic infections, such as intra-abdominal and skin and soft tissue infections (89–91). Two recent reports have noted the development of some carbapenem resistance among anaerobes (22), ranging from 1.1 to 2.5% in a multicenter U.S. survey but with a higher rate for a small number of isolates from Taiwan (33).
Resistance to β-lactam antibiotics.
Anaerobes manifest three major mechanisms of resistance to β-lactam antibiotics: inactivating enzymes, mainly beta-lactamases (BLAs), which include penicillinases and cephalosporinases; low-affinity penicillin-binding proteins (PBPs); and decreased permeability through alterations in the porin channel (35). The production of BLAs is the most common mechanism of resistance to β-lactam antibiotics in anaerobes, especially among the B. fragilis group and Prevotella spp. (100) The cephalosporinases are most often of the 2e class type and can be inhibited by three beta-lactamase inhibitors, clavulanic acid, sulbactam, and tazobactam. Each individual cephalosporin may have either a class or specific inhibitor enzyme that is able to inactivate it.
BLA hydrolyzes the cyclic amide bond of the penicillin or cephalosporin nucleus, causing its inactivation. There are a variety of BLAs which are produced by different organisms. These enzymes can be exoenzymes, inducible or constitutive, and genetically, they can be of either chromosomal or plasmid origin (101). There are different classifications of the enzymes. A classification based on amino acid sequence was created by Ambler (102), and a classification based upon substrate-of-inhibition profiles, molecular weight, and isoelectric points was proposed by Richmond and Sykes (103).
Most B. fragilis group strains produce constitutive BLs that are primarily cephalosporinases (104). Over 97% of Bacteroides isolates 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 primarily penicillinases (107).
Carbapenemases are active against the carbapenems as well as all β-lactam antibiotics. Although these enzymes are generally chromosomally mediated, a plasmid-mediated metallo-BL has been reported in Japan (108). Carbapenem resistance occurs in <1% of U.S. isolates, and up to 3% of Bacteroides strains harbor one of the genes that is expressed at a very low level. BLA inhibitors cannot inactivate the carbapenemases, which are zinc metalloenzymes encoded by either ccrA or cfiA genes of the B. fragilis group (109).
An evaluation of the molecular characterization of 15 strains of imipenem-resistant, cfiA-positive B. fragilis strains (109) noted that the cfiA genes of 10 of the strains were upregulated by insertion sequence (IS) elements, while 5 others did not harbor an IS but produced carbapenemase. These findings suggest that some isolates possessed novel inactivation mechanisms, suggesting that more than one mechanism of inactivation exists. A recent report from Taiwan noted increased carbapenem resistance in B. fragilis and other B. fragilis group species as well as some Prevotella species strains (33).
With some exceptions among some Clostridium spp., strains of Clostridium, Porphyromonas, and Fusobacterium have also been found to express resistance by one or more of the BLAs. BLA-producing Fusobacterium and Clostridium spp. express enzymes that are generally inhibited by clavulanic acid (110). Resistance to β-lactam antibiotics through changes in the outer membrane protein (OMP)/porin channels, decreased PBP affinity, and efflux pumps (111) is less well studied.
B. fragilis group species are generally resistant to penicillins (average, 90%), piperacillin (25%), cefoxitin (25%), cefotetan (30 to 85%), and third-generation cephalosporins (27, 88).
The combinations of BL-BLICs and carbapenems have maintained their excellent antibacterial activity. The combination agents of ampicillin-sulbactam, amoxicillin-clavulanate, ticarcillin-clavulanate, and piperacillin-tazobactam are generally very active against members of the B. fragilis group (27). However, species-to-species variations in susceptibility occur, and many non-BLA-producing P. distasonis strains have elevated MICs at or approaching the susceptible breakpoint (112). B. fragilis group resistance 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 rates continued to be low for the other B. fragilis group species.
The carbapenems (imipenem, meropenem, doripenem, and ertapenem) are very effective against all members of the B. fragilis group, and resistance is rare, at <0.1% (27, 112, 113). Geometric mean MICs for imipenem and meropenem for P. distasonis, B. thetaiotaomicron, and Bacteroides ovatus have been reported to be 1-fold dilution lower than those for ertapenem (27) in 2004. Goldstein et al. (80) reported that all Bacteroides isolates recovered from pediatric intra-abdominal infections were beta-lactamase producers and susceptible to carbapenems and BL-BLICs. However, cefoxitin has poor activity against B. thetaiotaomicron isolates.
β-Lactams are generally effective against non-B. fragilis group species, and resistance to them is generally low, except that more than half of Prevotella species isolates may also produce BLAs. A multicenter survey (93) found penicillin resistance for Fusobacterium spp., Porphyromonas spp., and Peptostreptococcus spp. at rates of 9%, 21%, and 6%, respectively. No resistance to cefoxitin, cefotetan, β-lactam–BLA inhibitor combinations, and carbapenems was found in that survey, with the exception of Peptostreptococcus spp. and Porphyromonas spp. (4% and 5% resistance to ampicillin-sulbactam, respectively). Beta-lactamases were identified in several Prevotella and Porphyromonas species strains recovered from pediatric intra-abdominal infections.
Chloramphenicol
Chloramphenicol, a bacteriostatic agent, is active against most anaerobic bacteria but is rarely used in the United States (3, 79). Resistance to this drug is rare, although it has been reported for some Bacteroides spp. (113). One must be aware that MICs of chloramphenicol often cluster around the susceptibility breakpoint. 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 of anaerobic infections. Chloramphenicol was regarded in the past as the drug of choice for treatment of serious anaerobic infections when the nature and susceptibility of the infecting organisms are unknown and of infections of the central nervous system (CNS). However, the drug has potential significant toxicity. The risk of fatal aplastic anemia with chloramphenicol is estimated to be approximately 1 per 25,000 to 40,000 patients treated. This serious complication is unrelated to the reversible, dosage-dependent leukopenia. Other side effects include the production of the potentially fatal “gray baby syndrome” when given to neonates, hemolytic anemia in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency, and optic neuritis in those who take the drug for a prolonged time.
Serum level measurements are often advocated for infants, young children, and occasionally adults, owing to their wide variations (115). The usual objective is therapeutic levels of 10 to 25 μg/ml. Levels exceeding 25 μg/ml are commonly considered potentially toxic in terms of reversible bone marrow suppression, and levels of 40 to 200 μg/ml have been associated with gray syndrome 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 solubility to permit penetration across lipid barriers. A consistent observation is the high concentrations achieved in the CNS, even in the absence of inflammation. Levels in the cerebrospinal fluid, with or without meningitis, usually are one-third to three-fourths of the serum concentrations. Levels in brain tissue may be substantially higher than serum levels (116).
The Macrolides: Erythromycin, Azithromycin, and Clarithromycin
The macrolides, which possess low human or animal toxicity, have moderate to good in vitro activity against anaerobic bacteria other than B. fragilis group strains and fusobacteria (79). Macrolides are active against pigmented Prevotella and Porphyromonas spp. and microaerophilic streptococci, Gram-positive non-spore-forming anaerobic bacilli, and certain clostridia. They are less effective against Fusobacterium and Peptostreptococcus spp. (117). They show relatively good activity against C. perfringens and poor or inconsistent activity against AGNB.
Clarithromycin is the most active macrolide against Gram-positive oral cavity anaerobes, including Actinomyces spp., Propionibacterium spp., Lactobacillus spp., and Bifidobacterium dentium. Azithromycin is slightly less active than erythromycin against these species (117). Azithromycin is, in general, the most active macrolide against AGNB such as Fusobacterium spp., Bacteroides spp., Wolinella spp., and Actinobacillus actinomycetemcomitans, including strains resistant to erythromycin. Clarithromycin showed similar activity to that of erythromycin against most AGNB (118).
Emergence of erythromycin-resistant organisms during therapy has been documented (119, 120). Erythromycin is effective in the treatment of mild to moderately severe anaerobic soft tissue and pleuropulmonary infections when combined with adequate debridement or drainage of infected tissue. Phlebitis is reported to develop in one-third of patients receiving intravenous erythromycin, but the oral preparation is well tolerated.
Clindamycin
Clindamycin has a broad range of activity against anaerobic organisms and has proven its efficacy in clinical trials. It is used for dental infections, especially in patients who are allergic to penicillin, and for aspiration pneumonia. Clindamycin hydrochloride is rapidly and virtually completely absorbed from the gastrointestinal tract (121–123). It rapidly penetrates into body tissues and fluids, including saliva, sputum, respiratory tissue, pleural fluid, soft tissues, prostate, semen, bones, and joints (124), as well as into fetal blood and tissues. Clindamycin does not cross the blood-brain barrier or eye efficiently and should not be administered in CNS infections.
The side effect of most concern is C. difficile-associated colitis (125, 126). Colitis has also been associated with a number of other antimicrobials, such as ampicillin, cephalosporins, and quinolones, and occasionally also in the absence of previous antimicrobial therapy.
Clindamycin resistance.
Although the patterns differ by region, B. fragilis resistance to clindamycin is increasing worldwide. This is why it is no longer recommended as empirical therapy for intra-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. thetaiotaomicron, and 35.6% of B. vulgatus isolates were clindamycin resistant. This is a significant increase compared to only 3% clindamycin resistance in 1987 (88). A study of pediatric intra-abdominal isolates revealed clindamycin resistance in only 6% of B. fragilis isolates, compared to 80% for B. thetaiotaomicron and 45% for other B. fragilis group isolates (80).
Resistance has also increased for many non-Bacteroides anaerobes. Up to 10% resistance was noted for Prevotella spp., Fusobacterium spp., Porphyromonas spp., and Peptostreptococcus spp., with higher rates for some Clostridium spp. (especially C. difficile) (93). Propionibacterium acnes isolates have also become more resistant to clindamycin, and this has been associated with prior therapy for acne (127).
Clindamycin has lost some of its activity against anaerobic Gram-positive cocci (Finegoldia magna [30% resistant] and Peptoniphilus spp., etc.) and Prevotella spp. (P. bivia [70% resistant], P. oralis, and P. melaninogenica [both 40% resistant]), although its activity against Fusobacterium and Porphyromonas spp. remains good.
Among the other resistant anaerobes are various species of clostridia, especially C. difficile. Approximately 20% of Clostridium ramosum strains are resistant to clindamycin, as are a smaller number of C. perfringens strains.
Metronidazole and Tinidazole
The nitroimidazoles metronidazole and tinidazole have similar in vitro efficacy against anaerobic bacteria. Metronidazole has excellent in vitro activity against most obligate anaerobic bacteria, such as the B. fragilis group, other species of Bacteroides, fusobacteria, and clostridia (99). Only six strains of the B. fragilis group were ever reported to be clinically resistant and associated with therapeutic failure (2).
Resistance of anaerobic Gram-positive cocci is rare, and resistance of nonsporulating bacilli is common. Microaerophilic streptococci, P. acnes, and Actinomyces spp. are almost uniformly resistant (128). Aerobic and facultative anaerobes, such as coliforms, are usually highly resistant. Over 90% of obligate anaerobes are susceptible to less than 2 μg/ml metronidazole (79).
Some clinicians may not appreciate metronidazole's limited activity against anaerobic and microaerophilic Gram-positive cocci, especially the microaerophilic streptococci, which are often lumped together with anaerobes. Because of metronidazole's lack of activity against aerobic bacteria, an antimicrobial effective against these organisms (e.g., a cephalosporin or a fluoroquinolone) needs to be added when treating a polymicrobial infection. There are occasions when clinicians are unsure of a specific drug's activity against anaerobes and may use redundant coverage with metronidazole, such as a carbapenem or a BL-BLIC. Antibiotic stewardship personnel should review this practice.
Adverse reactions to metronidazole therapy are rare and include CNS toxicity, such as ataxia, vertigo, headaches, and convulsions, and peripheral neuropathy. Peripheral neuropathy is associated with prolonged metronidazole use. Gastrointestinal side effects are common and include nausea, vomiting, metallic taste, anorexia, and diarrhea. Tinidazole may be better tolerated in patients with gastrointestinal side effects due to metronidazole. Other adverse reactions include reversible neutropenia, phlebitis at intravenous infusion sites, and drug fever. The tolerance of metronidazole in patients is generally very good.
Some studies in mice (129) have shown possible mutagenic activity associated with administration of large doses of this drug. It should be noted that in these animal toxicity studies, the drug has generally been administered for the lifetime of the animal, a situation that may not be relevant for humans. Other experiments (130) have shown that administration of metronidazole to rats and hamsters does not induce any pathology. Furthermore, evidence of mutagenicity was never found in humans despite metronidazole use for over 2 decades for other diseases (131). Because of safety concerns, the FDA approved the use of metronidazole for the treatment of serious anaerobic infections only in adults.
Clinical experiences of adults (132) illustrated metronidazole's efficacy in the treatment of infections caused by anaerobes, including CNS infections (133). Available data on the safety of the drug during pregnancy are contradictory, and valid data on the safety of metronidazole in pregnancy are still needed. The nonteratogenicity of metronidazole is difficult to prove, but the existing available data indicate no major risks and no indication for the termination of pregnancies (134).
Metronidazole resistance.
Although rare, resistance to metronidazole among B. fragilis group isolates has been observed worldwide (33, 135). Resistant B. fragilis group isolates carry one of nine known nim genes (nimA-I) on either the chromosome or a mobilizable plasmid that seems to encode a nitroimidazole reductase, which converts 4- or 5-Ni to 4- or 5-aminoimidazole, preventing the formation of toxic nitroso residues necessary for the agent's activity. These nim genes were found in 50/206 (24%) Bacteroides species isolates and resulted in MICs of 1.5 to >256 μg/ml for metronidazole, including 16 isolates with MICs of ≥32 μg/ml (54). These findings suggested incomplete mobilization of nim gene-associated resistance. Gal and Brazier (54) speculated that other mechanisms of resistance can occur and that prolonged exposure to metronidazole may select them. The mechanism of metronidazole resistance for non-Bacteroides anaerobes is unknown. Resistance among Gram-positive organisms that are not strict anaerobes is frequent, especially for P. acnes and Actinomyces spp.
Tetracyclines
Tetracycline, once the drug of choice for anaerobic infections, is presently of limited usefulness because of the development of resistance to it by virtually all types of anaerobes, including Bacteroides and Prevotella spp. Resistance to P. acnes has been related to previous use (127). Only about 45% of all B. fragilis strains are presently susceptible to this drug (79). The newer tetracycline analogs doxycycline and minocycline are more active than the parent compound. Because of the significant resistance to these drugs, they are useful only when susceptibility tests can be performed or in less severe infections in which a therapeutic trial is feasible. The use of tetracycline is not recommended for patients less than 8 years of age because of the adverse effect on teeth.
Tigecycline is the first antibiotic approved in a new class called glycylcyclines. Glycylcyclines are tetracycline antibiotics containing a glycylamido moiety attached to the 9-position of a tetracycline ring; tigecycline is a direct analog of minocycline with a 9-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, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant enterococci, Acinetobacter baumannii, beta-lactamase-producing strains of H. influenzae and M. catarrhalis, and extended-spectrum-beta-lactamase-producing strains of E. coli and Klebsiella pneumoniae. In contrast, MICs for Pseudomonas and Proteus spp. are markedly elevated. It is active against the Streptococcus anginosus group (including S. anginosus, S. intermedius, and S. constellatus), B. fragilis, B. thetaiotaomicron, Bacteroides uniformis, Bacteroides 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 complicated skin and soft tissue infections, including those due to B. fragilis (153), and intra-abdominal infections, including those due to B. fragilis, B. thetaiotaomicron, B. uniformis, B. vulgatus, C. perfringens, and Ps. micros (138). In a study that investigated its efficacy in the treatment of abdominal infections, tigecycline was compared to imipenem-cilastatin. Six tigecycline-treated patients compared to two imipenem-treated patients developed sepsis/shock, again lending caution to its clinical use.
Tetracycline resistance.
Tetracycline resistance is prevalent among Bacteroides species, Prevotella species, and many other anaerobic bacteria, limiting its clinical use (139). Several tetracycline resistance genes have been described among several anaerobes, which encode protective proteins leading to protection of the ribosomes. Tetracycline resistance and the inducible transfer of resistance determinants can occur after exposure to low levels of these agents. The appearance of tetracycline resistance in P. acnes has been correlated with previous tetracycline therapy (127). Other antimicrobials, such as doxycycline and minocycline, are more 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% of 831 B. fragilis group isolates were susceptible to ≤8 μg/ml of tigecycline and that P. distasonis isolates were the most resistant. Snydman 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 all Gram-positive anaerobes and 228/232 Gram-negative anaerobes to be susceptible to ≤1 μg/ml of tigecycline.
Fluoroquinolones
The earlier fluoroquinolones, such as ciprofloxacin and ofloxacin, are inactive against most anaerobic bacteria. However, some broad-spectrum quinolones have significant antianaerobic activity. Quinolones with low activity against anaerobes include ciprofloxacin, ofloxacin, levofloxacin, fleroxacin, pefloxacin, enoxacin, and lomefloxacin. Compounds with intermediate antianaerobic activity include sparfloxacin and grepafloxacin (141). Trovafloxacin, gatifloxacin, and moxifloxacin yield low MICs against most groups of anaerobes (40). The use of trovafloxacin has been limited because of hepatotoxicity. Quinolones with the greatest in vitro activity against anaerobes include clinafloxacin and sitafloxacin (142).
Moxifloxacin has been studied and approved by the FDA as monotherapy in intra-abdominal infections in adults (76, 90) and has shown activity against intra-abdominal anaerobic isolates (143, 144). However, concern over increasing fluoroquinolone resistance in both E. coli and B. fragilis group species has made clinicians cautious in its use in intra-abdominal infections (76). A variety of studies (19, 22, 144) have reported increases in B. fragilis resistance to moxifloxacin.
A recent study (143) reported a pooled analysis of 4 randomized clinical trials (2000 to 2010) that assessed the comparative efficacy of moxifloxacin in complicated intra-abdominal infections of 745 microbiologically evaluable cases and focused on its efficacy against 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 resistant at ≥8 mg/liter. Moxifloxacin achieved similar clinical success rates against all anaerobes, including those isolated from patients infected 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 all anaerobes was 82.3%. For all anaerobes combined, the clinical success rates were 83.1% (466 of 561 patients) for an MIC of ≤2 mg/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 an MIC of ≥32 mg/liter. This suggests that moxifloxacin can be cautiously used for anaerobic intra-abdominal infections provided that the patient has mild or moderate disease and has not been exposed to a fluoroquinolone recently. It may be an alternative for highly penicillin-allergic patients.
The use of the quinolones is restricted in growing children because of their possible adverse effects on cartilage. The major concerns with expanding the use of fluoroquinolones to treat anaerobic infections have been reports of increasing resistance in strains of the B. fragilis group as well as anaerobic Gram-positive cocci and the impact of these antibiotics on the growing incidence of C. difficile-associated disease (142).
Fluoroquinolone resistance.
Bacteroides species resistance to fluoroquinolones has been attributed to either an alteration in efflux of the antibiotic or a mutation in the quinolone resistance-determining region (QRDR) of the gyrase A gene (gyrA) from single or multiple mutations (144). High-level resistance can be caused by both mechanisms.
A study (145) of 4,434 B. fragilis group isolates recovered from 12 U.S. medical centers between 1994 and 2001 illustrated that fluoroquinolone resistance was dependent on species and source of isolation, with B. vulgatus isolates from decubitus ulcers being the most resistant (71%). Moxifloxacin resistance rates varied from 17% for B. fragilis strains isolated from the female genitourinary tract to 52% for all blood culture isolates (moxifloxacin MIC breakpoint, 4 μg/ml). The most recent national survey (27) reported that 27% of B. fragilis, 26% of B. thetaiotaomicron, 38% of B. ovatus, and 55% of B. vulgatus isolates were resistant to moxifloxacin. A study of strains isolated from intra-abdominal infections (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. fragilis group isolates and 417/450 isolates of all other anaerobic genera and species, including Fusobacterium, Prevotella, Porphyromonas, C. perfringens, Eubacterium, and Peptostreptococcus spp., were susceptible to ≤2 μg/ml of moxifloxacin. Wexler et al. (146), who studied 179 respiratory tract anaerobes, identified a single resistant strain of C. clostridioforme. A study by Edmiston et al. (40), who evaluated 550 anaerobes recovered from intra-abdominal and diabetic foot infections, reported that 97% were susceptible to moxifloxacin. Liu et al. (33) observed that 90% of B. fragilis isolates recovered from nosocomial infections and bacteremias in Taiwan were susceptible to moxifloxacin. In contrast, a study from Europe (147) found an overall fluoroquinolone resistance rate of 15%, with geographic variations from 7% in southern Europe to 30% in northern Europe. Factors that could account for these variations include differences in susceptibility that depend on 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 infrequently used in children.
Fusobacterium canifelinum, recovered from cat and dog bite wound infections, is intrinsically resistant to fluoroquinolones because of Ser79 replacement with leucine and Gly83 replacement with arginine on gyrA (148).
Moxifloxacin has been approved by the FDA for the treatment of complicated skin and skin structure infections, including those due to B. fragilis, and for mixed intra-abdominal infections caused by B. fragilis, B. thetaiotaomicron, Peptostreptococcus spp., and C. perfringens.
Other Agents
Bacitracin was active in vitro against pigmented Prevotella and Porphyromonas spp. but is inactive against B. fragilis and Fusobacterium nucleatum (79). Vancomycin and daptomycin are effective against all Gram-positive anaerobes but are inactive against AGNB (149). Quinupristin-dalfopristin shows antibacterial activity against anaerobic organisms including C. perfringens, Lactobacillus spp., and Peptostreptococcus spp. (150). Linezolid is active against Fusobacterium nucleatum, other Fusobacterium spp., Porphyromonas spp., Prevotella spp., and Peptostreptococcus spp. (117). However, little clinical experience been gained in the treatment of anaerobic bacteria using these agents.
GENERAL CONSIDERATION OF ANTIMICROBIAL SELECTION
Because anaerobic infection is often polymicrobial and is caused by aerobic and anaerobic organisms, antimicrobials that are effective against both components of the infection should be administered. When such therapy is not given, the infection may persist, and serious complications may occur (2, 3, 151). A number of factors should be considered when choosing appropriate antimicrobial agents: they should be effective against all target organisms, induce little or no resistance, achieve sufficient levels in the infected site, have minimal toxicity, and have maximum stability and longevity.
When selecting antimicrobials for the therapy of mixed infections, their aerobic and anaerobic antibacterial spectrum and their availability in oral or parenteral form should be considered (Table 1). Some antimicrobials have a limited range of activity. For example, metronidazole is active against only anaerobic bacteria and therefore cannot be administered as a single agent for the therapy of mixed infections. Other antimicrobials, such as carbapenems, tigecycline, and combinations of a beta-lactam and a beta-lactamase inhibitor, possess a broader spectrum of activity against aerobic and anaerobic bacteria.
Selection of antimicrobial agents is simplified when a reliable culture result is available. However, this may be particularly difficult in anaerobic infections because of the difficulties in obtaining appropriate specimens. For this reason, many patients are treated empirically on the basis of suspected, rather than established, pathogens. Fortunately, the types of anaerobes involved in many anaerobic infections and their antimicrobial susceptibility patterns tend to be predictable (2, 3). However, some anaerobic bacteria have become resistant to antimicrobial agents, and many can develop resistance while a patient is receiving therapy (111, 152).
Anaerobic bacteria have always been resistant to aminoglycosides and trimethoprim-sulfamethoxazole. Resistance among some anaerobes has increased significantly over the past 3 decades. The potential for growing resistance of anaerobes to antimicrobials is especially noted with penicillins, fluoroquinolones, clindamycin, and cephalosporins. Chloramphenicol is rarely used in the United States, and resistance is very rare; when present, it is due to chloramphenicol's inactivation by acetyltransferase.
Aside from susceptibility patterns, other factors influencing the choice of antimicrobial therapy include the pharmacological characteristics of the various drugs, their toxicity, their effect on the normal flora, and their bactericidal activity (2, 3). Although identification of the infecting organisms and their antimicrobial susceptibility may be needed for selection of optimal therapy, the clinical setting and Gram stain preparation of the specimen may indicate the types of anaerobes present in the infection as well as the nature of the infectious process.
Antimicrobial therapy for anaerobic infections usually should be given for prolonged periods because of their tendency to relapse. This period may range from 3 weeks to 3 months, depending on the site and severity of the infection.
Because anaerobic bacteria generally are recovered mixed with aerobic organisms, selection of proper therapy becomes more complicated. In the treatment of mixed infection, the choice of the appropriate antimicrobial agents should provide for adequate coverage of most of the pathogens, aerobic and anaerobic. Some broad-spectrum antibacterial agents possess such qualities, while for some organisms, additional agents should be added to the therapeutic regimen.
CURRENT PRACTICE OF SELECTION OF ANTIBIOTIC FOR ANAEROBIC BACTERIA
In our personal experience, clinicians are generally aware of the importance of anaerobic bacteria in a wide variety of infections. These include mainly diabetic foot and intra-abdominal infections and aspiration pneumonia. Clinicians are less aware of all the taxonomic changes that have occurred and the names of new species. To most clinicians, B. fragilis group species are recognized as the major anaerobic pathogen, but the individual B. fragilis group subspecies are less readily recognized. For many, there is only a vague familiarity with Prevotella and Porphyromonas spp. and other Gram-negative organisms as anaerobic pathogens. Among the Gram-positive anaerobes, clinicians are also aware of Clostridium species, especially C. perfringens and Clostridium botulinum and, more recently, the great importance of C. difficile. If asked, clinicians will know that anaerobic Gram-positive cocci exist but would be unlikely to know either their current nomenclature or their involvement in specific infections. Consequently, when asked, most clinicians will consider the presence and role of B. fragilis in an infectious process and will likely lump all other anaerobes into the single category of “other anaerobes.” This narrowed view is also a result of current schemas of limited laboratory identification and reporting of anaerobes. Hence, the clinician is likely to consider only the in vitro activity of specific agents against B. fragilis and assume that all the other anaerobes will likely be susceptible as well.
In addition, clinicians may be less aware of the variability between laboratories in their capabilities and interest in anaerobic bacteriology and the specific level of performance and capability of their own clinical microbiology laboratories to both isolate anaerobes and perform susceptibility testing (20, 36, 37). Clinicians are likely to be unaware of recent changes in CLSI breakpoints for any specific bacterium-drug combination (17). Coupled with this dilemma is the often unappreciated need for proper specimen collection and transport media to facilitate the viability of anaerobic pathogens (2–4).
Controversy even about the importance of obtaining anaerobic blood cultures has reemerged (12). This occurred after the decline in rates anaerobic bacteremia in the 1970s and 1980s, which led many medical centers to discontinue obtaining blood cultures for anaerobic bacteria. However, with the reemergence of anaerobic bacteremia in the 1990s, many centers resumed processing of blood cultures for anaerobes. Because many medical centers do not process anaerobic cultures or do so in an inappropriate manner, results of anaerobic isolation and susceptibility testing for the individual patient remain virtually inaccessible to clinicians, who must then rely on the occasional and periodic published surveys from a small cadre of scattered research institutions (22, 27, 78). All of these factors affect the clinician's cognitive choices in the selection of an antimicrobial agent to treat anaerobic infections.
A U.S. national survey on anaerobic susceptibility testing (20) found that drugs tested by hospital laboratories that performed testing were as follows: metronidazole (89% of laboratories), penicillin and clindamycin (83%), cefotetan and ampicillin-sulbactam (67%), cefoxitin (50%), imipenem (44%), piperacillin-tazobactam (39%), and all other drugs (<34%). The agar dilution method seemed reserved for “centers of excellence” or research centers.
All this suggests that clinicians rely on “FDA indications, information from the manufacturers supplied by drug reps, published study/survey data or just make an educated guess at the appropriate empirical or directed therapy” (20).
The limited medical education that students and residents receive about anaerobes and the limitations of microbiology laboratory culture, with delays in identification and anaerobic susceptibility testing, suggest that at best, the choice of an agent for a serious anaerobic infection is an empirical and educated guess (38). The general principles are limited to questions of whether anaerobes are involved in this type of infection and if the agent chosen has activity against B. fragilis. Even though the CLSI proposed that laboratories perform anaerobic susceptibility testing against certain major anaerobic pathogens (17), these recommendations are rarely followed and are performed at only a limited number of medical centers. Anaerobes (including the B. fragilis group) are conspicuous in their absence from the microbiological reports of most hospitals (17, 27, 78).
For the treatment of anaerobic infections, the clinician will likely choose an antimicrobial therapy empirically and continue that agent as definitive therapy because specific anaerobic microbiological data will not be forthcoming. The choice will be based on published literature and surveys and local or specific patient data. The CLSI has published an appendix to its recent document that reports the cumulative susceptibilities for B. fragilis group species collected from three U.S. medical centers from 2007 to 2009 (17). Consequently, the most likely agents to cover the vast majority of anaerobes encountered in mixed infections will be either a carbapenem or a BL-BLIC such as piperacillin-tazobactam. Metronidazole can be used but in combination with another agent. Should rapid diagnostics (such as MALDI-TOF MS) become available for anaerobes in the future, it is likely that this paradigm would change.
CONCLUDING REMARKS
This review describes current methods for antimicrobial testing in research or reference laboratories. These methods include the use of agar dilution, broth microdilution, Etest, and the spiral gradient endpoint (SGE) system.
Antimicrobial resistance among anaerobes has consistently increased in the past decades, and the susceptibility of anaerobic bacteria to antimicrobial agents has become less predictable. In the last few decades, the need for testing of anaerobic isolates has been increasingly recognized, and the testing methodologies used have been standardized.
Performance of susceptibility testing for anaerobic bacterial isolates recovered from selected cases can provide important information that can influence the choice of antimicrobial therapy. Susceptibility testing should be performed on isolates recovered from sterile body sites, those that are isolated in pure culture, or those that are clinically important and have a variable or unique susceptibility. The CLSI has standardized many laboratory procedures, including anaerobic susceptibility testing, and has published documents for anaerobic susceptibility testing (commonly called M11) (16). The standardization of testing methods by the CLSI allows for comparison of resistance trends among various laboratories (15–17). Organisms that should be considered for individual isolate testing include highly virulent pathogens for which susceptibility cannot be predicted, such as Bacteroides, Prevotella, Fusobacterium, and Clostridium spp.; Bilophila wadsworthia; and Sutterella wadsworthensis.
The decline in the number of hospital laboratories performing anaerobic susceptibility tests during exactly the same time period that more multidrug-resistant strains of anaerobes are being found in serious infections is problematic. The performance of susceptibility tests for individual isolates when indicated and obtaining local surveillance testing to monitor regional trends have been recommended by the CLSI. Most hospitals that send strains out for susceptibility testing are getting test results by using Etest methodology, which is within the expertise available in most hospitals. If these tests are done in-house, they might yield faster results (results can typically be read after 48 h, while most test results from a commercial laboratory take up to 7 days) and would be clinically useful in therapeutic decisions. Testing that does not demand quick turnarounds, such as surveillance or batch testing or to monitor trends, could be done by reference laboratories.
Biographies
Itzhak Brook, M.D., is an Adjunct Professor of Pediatrics at Georgetown University, Washington, DC. He is the past chairman of the Anti-Infective Drug Advisory Committee of the Food and Drug Administration. He has done extensive research on anaerobic and respiratory tract infections, anthrax, and infections following exposure to ionizing radiation. He is the author of 6 medical textbooks, 128 book chapters, and over 700 scientific publications. He is an editor, associate editor, and member of the editorial board 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 the American Academy of Otolaryngology-Head and Neck Surgery.
Hannah M. Wexler, Ph.D., is a research microbiologist at the Greater Los Angeles Veterans Administration Healthcare System (GLAVAHCS) and Adjunct Professor of Medicine at the UCLA School of Medicine. Dr. Wexler is currently Councilor of Division A (Antimicrobial Agents and Chemotherapy) of the American Society for Microbiology after serving as Chair of the Division. She began her career with anaerobic bacteria as Director of the Wadsworth Anaerobe Laboratory at the GLAVAHCS from 1981 to 2008. Dr. Wexler has been active in the Working Group for Anaerobes for the Subcommittee on Antimicrobial Susceptibility Testing of the Clinical and Laboratory Standards Institute (CLSI) and was one of the primary authors of the M11 Standard for determination of susceptibility for anaerobic bacteria. She is currently a Career Scientist at the GLAVAHCS, focusing on regulation of efflux pump activity as an important factor in the development of antibiotic resistance in Bacteroides. Her work currently includes comparative genomic and transcriptomic studies of multidrug-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 at the David Geffen School of Medicine, UCLA; Director of the R. M. Alden Research Laboratory; and in private practice in Santa Monica, CA. He has received the IDSA Clinician of the Year Award and has over 380 publications. His interests include the diagnosis, pathogenesis, and therapy of anaerobic infections, including intra-abdominal infections, diabetic foot infections, C. difficile, human and animal bites, and the in vitro susceptibility of anaerobic bacteria to new antimicrobial agents. He is active in the Anaerobe Society of the Americas, the IDSA, ASM, and the Surgical Infection Society. He founded, and served as President of, the Infectious Diseases Association of California and the Anaerobe Society of the Americas. He is currently a Section Editor for Clinical Infectious Diseases and chair of the publications committee of Anaerobe. In the past, he has served as an Associate Editor for Clinical Infectious Diseases and the Journal of Medical Microbiology.
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