Clinical Infectious Diseases Advance Access published May 27, 2014 1
Antimicrobial Resistance and Susceptibility Testing of Anaerobic Bacteria
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Audrey N. Schuetz
Associate Director, Clinical Microbiology Laboratory, Assistant Professor, Department of
Pathology and Laboratory Medicine, Assistant Professor, Department of Internal Medicine, Weill Cornell Medical College/ NewYork-Presbyterian Hospital, 525 East 68th Street, Starr
Corresponding Author: Phone: 212-746-0833, Fax: 212-746-8945, E-mail:
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[email protected]
Alternate Corresponding Author: Yelena Lavrova, NewYork-Presbyterian Hospital, 525 East
[email protected]
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68th Street, Starr 737, New York, NY 10065, Phone: 212-746-2408, Fax: 212-746-8945, E-mail:
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Summary: Resistance of anaerobic bacteria to certain classes of antibiotics is increasing globally. Resistance trends vary by geographic region and often vary between species. Susceptibility
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testing of anaerobes is important in directing appropriate antibiotic therapy.
© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e‐mail: [email protected].
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737C, New York, NY 10065
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ABSTRACT
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Infections due to anaerobic bacteria can be severe and life-threatening. Susceptibility
testing of anaerobes is not frequently performed in laboratories, but such testing is important to
direct appropriate therapy. Anaerobic resistance is increasing globally, and resistance trends vary by geographic region. An overview of a variety of susceptibility testing methods for anaerobes is
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situations warranting anaerobic susceptibility testing are discussed.
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covered, and the advantages and disadvantages of each method are reviewed. Specific clinical
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INTRODUCTION
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Anaerobic infections can be severe and life-threatening, and their increasing incidence is due in part to the high numbers of patients with complex underlying diseases [1]. Studies
demonstrate that failure to direct appropriate anaerobic therapy leads to poor clinical response
[2]. Anaerobic collection, transport and manipulation of culture isolates can be time-consuming
infections has often been instituted long before results of susceptibility testing are available.
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However, antimicrobial bacterial resistance among anaerobic bacteria is increasing globally, as demonstrated by numerous surveys in Europe, the United States, Canada, and New Zealand [3-6]. Differences in resistance patterns may be due to the variety of susceptibility
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testing methodology used, the selective antibiotic pressures associated with antimicrobial usage, and the lack of uniformity in adoption of interpretive breakpoints. This article covers typical
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resistance patterns and recent changes in resistance of certain organisms over time. Anaerobic susceptibility testing methods and their challenges are reviewed. RESISTANCE PATTERNS OF ANAEROBIC BACTERIA Bacteroides fragilis Group
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The members of the B. fragilis group are among some least susceptible anaerobes to antibiotics. Resistance of Bacteroides species is linked to outcome, even in the presence of
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mixed infections [4, 7]. The B. fragilis group consists of 24 species including Bacteroides fragilis, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides caccae, and Parabacteroides distasonis. Fewer than 3% of Bacteroides isolates are susceptible to penicillin and ampicillin [8].
Penicillin- and cephalosporin-resistance is mediated by cepA and cfxA genes. The chromosomal
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and difficult to perform correctly. Empirical broad-spectrum antimicrobial therapy for anaerobic
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cepA gene is a cephalosporinase encoding for resistance to cephalosporins and aminopenicillins but not piperacillin or β-lactam-β-lactamase inhibitor combinations (BLBLIs). The cfxA gene
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encodes for high level resistance to cefoxitin and other β-lactams [9]. Although β-lactamases are the principal mechanism of resistance to penicillins, the expression of altered penicillin-binding proteins can also lead to resistance [10]. Membrane modifications, such as porin losses, may further increase the minimal inhibitory concentrations (MICs) to β-lactams and BLBLIs.
fragilis group [3, 11, 12]. Carbapenems are generally active against the B. fragilis group, and
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most U.S.-based studies report susceptibility rates of 98.5-99% [4, 13, 14]. Carbapenem resistance is usually mediated by a chromosomal zinc metallo-β-lactamase enzyme encoded by
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the cfiA gene [15]. Although cephamycins were used in the past for anaerobic coverage, the Infectious Diseases Society of America (IDSA) recently stated that cefotetan and clindamycin are no longer recommended as therapy for community-acquired intra-abdominal infections in
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adults due to increasing resistance among the B. fragilis group [16]. Clindamycin resistance is mediated by erm genes located on transferable plasmids which can also carry tetracycline resistance genes [17]. Worldwide, clindamycin resistance is increasing and approaches 60% [3, 4, 12, 18].
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Although rare, metronidazole-resistant strains of B. fragilis have been reported and are
associated with the nim gene [3, 11, 15]. However, the presence of this gene does not invariably
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lead to resistance, and MICs must be obtained. Of 206 B. fragilis isolates in one study, the nim gene was detected in 7.3%, but metronidazole MICs ranged from 1.5 (susceptible) to >256 mg/L (resistant) [19]. Susceptibility rates to moxifloxacin are highly variable across studies, and isolates are acquiring resistance rapidly. Moxifloxacin susceptibility rates range from 30 to 60%
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Currently, ticarcillin-clavulanate and piperacillin-tazobactam are active against >90% of the B.
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for B. fragilis group, and such resistance is mediated by gyrA mutations, efflux pumps, or topoisomerase gene modifications [12]. Tigecycline MICs are relatively low in surveillance
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studies, but there have been reports of resistance [6].
Susceptibility within the B. fragilis group varies by species, with B. fragilis being the most susceptible. Parabacteroides distasonis demonstrates high MICs to β-lactams, and B. thetaiotaomicron also demonstrates higher resistance rates to various antimicrobials in
isolates collected from 10 U.S. medical centers confirmed that susceptibility rates differed
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widely by species [4]. In this series, B. ovatus was more resistant to the carbapenems; B. vulgatus was more resistant to piperacillin-tazobactam and showed the highest resistance rates to
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moxifloxacin (54%); P. distasonis was more resistant to ampicillin-sulbactam and cefoxitin; and B. ovatus and B. uniformis showed higher resistance rates to moxifloxacin (39% and 41%, respectively). However, a 2008 practice survey of anaerobes across the U.S. reported that 33% of
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reference laboratories did not identify B. fragilis group isolates to the species level [20]. Susceptibility rates among B. fragilis group are decreasing worldwide. Snydman et al. reported a decrease in susceptibility of approximately 25% over 11 years to ampicillin-sulbactam in P. distasonis and B. vulgatus [4].
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Prevotella, Porphyromonas, and Other Anaerobic Gram-negative Rods Porphyromonas spp. are generally susceptible to β-lactams , clindamycin, and
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metronidazole [21]. One-fourth to one-third of Porphyromonas produce β-lactamases, and clindamycin resistance has been observed in a minority of strains [22]. Compared to Porphyromonas, approximately 95% of Prevotella spp. is resistant to penicillin and ampicillin [22]. A recent Belgian study showed that clindamycin susceptibility in Prevotella spp. has been
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comparison to other members of the B. fragilis group. A survey of 6,574 B. fragilis group
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decreasing from 91% in 1993-1994 to 69% in 2012 [23]. Susceptibility to moxifloxacin, metronidazole, carbapenems, and amoxicillin-clavulanate is typically 90% or greater. Penicillin
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resistance in Fusobacterium spp. ranges from 4 to 15% and is generally due to the production of β-lactamases [22]. Fusobacterium varium, Fusobacterium mortiferum, and Fusobacterium
nucleatum are most often reported to produce β-lactamases, while over 90% of Fusobacterium necrophorum are susceptible to cephalosporins and cephamycins [14,24]. Fusobacterium spp.
clindamycin.
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Gram-positive Non-Spore-forming Rods
Actinomyces, Propionibacterium, Bifidobacterium, and the “Eubacterium” group are
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usually susceptible to β-lactams and BLBLIs [25, 26]. Propionibacterium spp., Actinomyces spp., Bifidobacterium spp., and Lactobacillus spp. are usually resistant to metronidazole. Clindamycin shows moderate activity against this group [27]. Decreasing clindamycin
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susceptibility of P. acnes is associated with prior topical acne therapy [28]. Vancomycin shows some activity against certain species of Lactobacillus; however, MICs >256 µg/mL are common for the most frequently isolated lactobacilli from human specimens - Lactobacillus casei and Lactobacillus rhamnosus [29]. Telavancin has shown good activity against some Lactobacillus
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spp. [33]. Newer antimicrobial agents such as linezolid, daptomycin, and dalbavancin exhibit excellent in vitro activity against most anaerobic gram-positive species [31, 32].
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Gram-positive Spore-forming Rods Of the clostridial species, Clostridium perfringens is one of the most susceptible to
penicillin, but clindamycin resistance is increasing, with up to 14% of blood isolates of C.
perfringens in one study [5, 26, 33]. Tetracycline resistance (defined as MIC >2 µg/mL) has
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are typically susceptible to metronidazole, BLBLIs, cephalosporins, carbapenems, and
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been documented in up to 75% of C. perfringens isolates obtained from commercial poultry, but there are little data in humans [34, 35]. The activities of tetracycline against other clostridial
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species and of doxycycline against clostridia in general have not been well documented [35].
Drugs which maintain activity against non-perfringens Clostridium spp. include piperacillin, BLBLIs, carbapenems, metronidazole, and vancomycin [36]. Antimicrobial agents lacking
significant activity include ampicillin, aminoglycosides, trimethoprim-sulfamethoxazole, and
tertium, Clostridium difficile, and the “RIC” group of clostridia – namely, Clostridium ramosum,
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Clostridium innocuum and Clostridium clostridioforme. In a study of 540 clinical isolates of Clostridium spp., tigecycline was highly active, with an MIC90 of 0.25 µg/mL [37].
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Clostridium difficile is usually susceptible to metronidazole and vancomycin but resistant to β-lactams, fluoroquinolones, and clindamycin [38]. Outbreaks of fluoroquinolone-resistant C. difficile strains have been associated with increased mortality [39]. Strains resistant to rifampin
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or rifaximin associated with rpoB mutations have also been linked to clinical failure [38]. Metronidazole-resistant strains are rare but have been documented [40]. A recent surveillance study of C. difficile isolates collected from 14 European countries reported that over one-half of isolates were multidrug resistant, with the majority resistant to moxifloxacin, clindamycin,
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erythromycin, and rifampicin [41]. Fidaxomicin shows excellent activity against C. difficile, but reduced susceptibility due to drug target modifications has been documented in vitro [42].
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Anaerobic Cocci
The anaerobic gram-positive cocci (GPC) have undergone major taxonomic changes
within the past 40 years; therefore, susceptibility data have been difficult to interpret. Greater than 90% of anaerobic GPC are susceptible to penicillin, including most Peptostreptococcus spp.
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clindamycin. Resistance to clindamycin is typical for many species, including Clostridium
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[35]. Anaerobic GPC are also generally susceptible to β-lactams, BLBLIs, cephalosporins, and carbapenems [24]. Alterations in penicillin-binding proteins account for the majority of β-lactam
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resistance [43]. Clindamycin resistance rates among anaerobic GPC range from 7 to 20%, and
this resistance is rising especially in Finegoldia magna and Peptoniphilus spp. [35, 14]. 90-95% of GPC are susceptible to metronidazole, but rare nimB-positive, metronidazole-resistant strains of F. magna and Parvimonas micra have been described [35, 44]. Strains of streptococci which
misidentified as anaerobic GPC and will test resistant to metronidazole, so metronidazole
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resistance in anaerobic GPC should be confirmed.
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ANAEROBIC SUSCEPTIBILITY TESTING (AST)
Susceptibility testing is performed to obtain information on the predicted response of the bacteria to antibiotics in the form of an MIC, which is defined as the lowest concentration of the
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antibiotic that inhibits growth of organisms. Drugs are typically tested at two-fold doubling (log2) serial dilutions (i.e., 2 µg/mL, 4 µg/mL, 8 µg/mL, etc.). MICs may be obtained by utilizing the CLSI-defined agar or broth microdilution methods or by using a variety of commercially available methods. Commercial methods include: Etest® strips (bioMérieux, Durham, NC),
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M.I.C.Evaluator strips (M.I.C.E.™) (Thermo Fisher Scientific, Basingstoke, United Kingdom), and Sensititre panels (Trek Diagnostics Systems, Thermo Fisher Scientific, Cleveland, OH).
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Etests and M.I.C.Evaluator strips are gradient diffusion methods. Agar and broth microdilution methods are usually employed by research or reference laboratories. Advantages and disadvantages of AST methods are listed in Table 1.
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grow well anaerobically, such as the Streptococcus anginosus group, may initially be
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In a 2008 practice survey of anaerobic culture and AST in U.S. hospitals, Goldstein and colleagues reported that only 21% (21/98) of hospital laboratories performed anaerobic AST in-
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house (as opposed to outsourcing to a reference laboratory) [20]. Twenty hospital laboratories reported sending their isolates out for susceptibility testing, and the remaining 60% (58/98) of
laboratories did not offer any AST. In 1990, 70% of hospital laboratories performed anaerobic AST, but only 33% of hospital labs did so in 1993. This decline may be explained by the fact
susceptibility testing in 1993, are no longer considered appropriate for susceptibility testing. In
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the 2008 survey, 65% of hospital laboratories which performed AST used Etest methodology [20]. The remainder of hospital laboratories used microbroth dilution, and none used agar
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dilution.
Procedural guidelines for anaerobic AST have been published by CLSI, but the Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing
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(EUCAST) also develop breakpoints [45, 46, 47]. Occasionally, there are differences in interpretive criteria between organizations, which may be explained by differences in dosages, administration intervals, inoculum size, or test media. It is important for clinicians to be aware of which breakpoints are being employed. Antimicrobial susceptibility testing in anaerobes relies
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primarily on phenotypic characterization, although a few molecular assays exist (e.g., nim gene for metronidazole resistance and cfiA for carbapenem resistance) [48]. As molecular data on
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anaerobic susceptibility are gathered, it is possible that additional molecular assays could become available.
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that the disk diffusion and broth disk elution methods, which were the predominant methods of
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Agar Dilution The CLSI agar dilution procedure is the gold standard reference method for anaerobic
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AST [45]. Different concentrations of antimicrobial agents are prepared in two-fold dilutions,
added to molten agar, poured into Petri dishes and allowed to solidify. A standardized inoculum of bacterial cells is spotted onto the surface of each plate with an inoculator, replicator, or pipette. After 48 hours of incubation, the plates are examined visually for the lowest
time, labor and expertise and is not intended for testing of single isolates. Thus, it is not a
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technique which many clinical laboratories employ. Broth Microdilution
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The broth microdilution method is less labor-intensive than agar dilution. In this assay, drugs in serial two-fold dilutions are added to small volumes of broth liquid media pipetted in wells of plastic microdilution trays or plates. A standard inoculum of bacterial cells is added into
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each well. MICs are read after 48 hours of incubation. Multiple antibiotics (ten or more) can be tested at one time, and the laboratory can design its own drug panels. However, this technique is limited by poor growth of certain oxygen-sensitive anaerobes, and is currently validated by CLSI for use only with B. fragilis group [46].
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MIC Gradient Diffusion Method
The Etest® and M.I.C.E.™ strips are thin plastic strips which are impregnated on one
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face with an increasing gradient concentration of an antimicrobial agent (Figure 1). The other face of the strip is marked with an MIC scale with increasing antibiotic concentrations, including increments between doubling dilutions (i.e., 1 µg/mL, 1.5 µg/mL, 1.75 µg/mL, 2 µg/mL, etc.). A Brucella blood agar plate supplemented with hemin and vitamin K1 is pre-reduced in an
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concentration of antibiotic which inhibits growth. Agar dilution testing requires considerable
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anaerobic environment to rid the media of toxic oxygen metabolites. A standard inoculum of bacterial cells is swabbed onto the plate. The Etest or M.I.C.E.™ strip is placed antibiotic face
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down. The MIC is read at the intersection of the lower part of the ellipse with the corresponding
number on the test strip. Results are generally available at 48 hours; however, clostridial species and B. fragilis group isolates usually grow rapidly enough to be read at 24 hours. MIC gradient diffusion methods are easier to use in routine clinical laboratory practice than agar dilution. A
quantitative and precise MIC. Additionally, several drugs can be tested at one time, and
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laboratories can tailor testing to their needs. However, Etest strips cost approximately $2 to $3 U.S. dollars apiece. Etest results generally correlate well with agar dilution, but the MIC gradient
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diffusion method can overestimate metronidazole resistance if anaerobiosis is not adequate [49]. Since the activity of metronidazole is dependent upon an active intermediate which requires the reduced atmosphere of anaerobiosis, it is important to utilize anaerobic control organisms when
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setting up such testing.
Commercially Available Broth Microdilution Panels Two commercially available panels for anaerobic susceptibility testing are available. The Anaerobe Sensititre panel (ANO2, Thermo Fisher) is a panel of antimicrobials of varying two-
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fold dilutions (Figure 2) [50]. A second anaerobic susceptibility panel is available through Oxoid (Thermo Fisher) [51].
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Rapid β-Lactamase Disk Testing This disk test evaluates for the presence of β-lactamase enzyme. Bacterial colonies are
smeared onto a disk embedded with a chromogenic cephalosporin. If the organism expresses β-
lactamase, the disk will change colors after reagent is added. Although most reactions occur
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significant advantage of the MIC gradient diffusion method is the ability to generate a
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within five to 10 minutes, some β-lactamase-positive strains may react more slowly (up to 30 minutes. If the disk is positive, the organism is considered to be resistant to penicillin, ampicillin,
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and amoxicillin. Since β-lactamases are not the sole mediator of resistance in anaerobes, a
negative test does not rule out penicillin or ampicillin resistance resulting from alterations of
penicillin-binding proteins [52]. Therefore, a negative result by the rapid β-lactamase disk test
must be followed by an MIC test for accurate prediction of beta-lactam susceptibility. Also, the
are presumed to be resistant due to almost universal presence of β-lactamases.
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Disk Diffusion
Disk diffusion (Kirby Bauer) tests should not be performed for anaerobic bacteria for the
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purpose of obtaining susceptibility results, as the results are inaccurate and do not correlate with the agar dilution method [46]. Special-potency antibiotic disks of vancomycin (5µg), kanamycin (1000 µg), and colistin (10 µg) are occasionally used in the laboratory as an aid in preliminary
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identification of some anaerobes based on patterns of resistance [53]. However, due to the high antibiotic concentrations in these disks, these disks are not intended for use in determining antibiotic susceptibility for therapeutic purposes.
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STRATEGIES FOR TESTING AND REPORTING OF SUSCEPTIBILITY DATA Antimicrobial Testing Guidelines
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For individual patient management, AST should be performed when: 1) selection of an
active agent is critical for disease management; 2) long-term therapy is being considered; 3) anaerobes are recovered from sterile body sites; or, 4) the infection persists despite adequate therapy with an appropriate therapeutic regimen (Table 2). In cases of a single recovered
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β-lactamase disk testing should not be performed on members of the B. fragilis group, since they
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anaerobic pathogen from culture, AST should be performed. CLSI suggests certain antimicrobial agents to be considered for testing and reporting on anaerobic organisms (Table 3) [46]. For
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gram-negative anaerobes, penicillin or ampicillin may be tested and/or reported selectively for all except the B. fragilis group which are almost uniformly resistant. For non-spore-forming
gram-positive anaerobes, resistance to metronidazole is common, and ampicillin and penicillin
are recommended for primary testing. Although CLSI MIC breakpoints for amoxicillin have not
[46]. The remaining supplemental antimicrobials are helpful to test when strains are resistant to
Antibiograms
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primary drugs or for use in patients who are allergic to primary drugs.
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Establishment of an antibiogram outlining patterns of resistance on a periodic basis is helpful in guiding antimicrobial therapy. The CLSI suggests gathering susceptibility data from at least 30 isolates of the same genus or species collected over the period of a year in order to
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obtain a reasonable number of isolates upon which to estimate susceptibilities [54]. Examples of cumulative antimicrobial susceptibility reports for B. fragilis group and other anaerobes from across the U.S. are included in the CLSI documents M100 and M11 [45, 46].
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CONCLUSIONS
Susceptibility among anaerobes differs from species to species and also from region to
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region. Surveillance studies have shown that susceptibility trends of anaerobes to certain antimicrobial agents are changing, with more resistance recognized in many parts of the world. Despite the difficulties inherent in anaerobic AST, testing should be performed and observed for trends over time.
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been established for anaerobes, amoxicillin breakpoints are considered equivalent to ampicillin
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ACKNOWLEDEMENTS
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The work in this paper was not supported financially. The author has no conflict of interest to
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declare.
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blood isolates. Clin Infect Dis, 2006; 42: e35-e44.
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30. Goldstein EJ, Citron DM, Merriam CV, Warren YA, Tyrrell KL, Fernandez HT. In vitro activities of the new semisynthetic glycopeptide telavancin (TD-6424), vancomycin, daptomycin, linezolid, and four comparator agents against anaerobic gram-positive
31. Citron DM, Merriam CV, Tyrrell KL, Warren YA, Fernandez H, Goldstein EJ. In vitro
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activities of ramoplanin, teicoplanin, vancomycin, linezolid, bacitracin, and four other antimicrobials against intestinal anaerobic bacteria. Antimicrob Agents Chemother, 2003;
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32. Goldstein EJ, Citron DM, Merriam CV, et al. Comparative in vitro activities of XRP 2868, pristinamycin, quinupristin-dalfopristin, vancomycin, daptomycin, linezolid,
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clarithromycin, telithromycin, clindamycin, and ampicillin against anaerobic grampositive species, actinomycetes, and lactobacilli. Antimicrob Agents Chemother, 2005; 49: 408-413.
33. Leal J, Gregson DB, Ross T, Church DL, Laupland KB. Epidemiology of Clostridium
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34. Johansson A, Greko C, Engström BE, Karlsson M. Antimicrobial susceptibility of
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Swedish, Norwegian and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes. Vet Microbiol, 2004; 99: 251-257.
35. Hecht DW. Anaerobes: antibiotic resistance, clinical significance, and the role of susceptibility testing. Anaerobe, 2006; 12: 115-121.
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species and Corynebacterium spp. Antimicrob Agents Chemother, 2004; 48: 2149-2152.
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36. Stevens DL, Bryant AE, Berger A, von Eichel-Streiber C. Clostridium. In: Versalovic J, Carroll KC, Funke G, Jorgensen JH, Landry ML, Warnock DW. Manual of Clinical
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Microbiology. Vol 1. 10th edition. Washington, DC, USA: ASM Press; 2011: 834-857. 37. Hawser SP. Activity of tigecycline against multidrug-resistant clinical isolates of Clostridium spp. from Europe. Int J Antimicrob Agents, 2010; 35: 310-311.
38. Hecht DW, Galang MA, Sambol SP, Osmolski JR, Johnson S, Gerding DN. In vitro
isolates collected from 1983 to 2004. Antimicrob Agents Chemother, 2007; 51: 2716-
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39. Labbé AC, Poirier L, Maccannell D, et al. Clostridium difficile infections in a Canadian
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tertiary care hospital before and during a regional epidemic associated with the BI/NAP1/027 strain. Antimicrob Agents Chemother, 2008; 52: 3180-3187. 40. Kunishima H, Chiba J, Saito M, Honda Y, Kaku M. Antimicrobial susceptibilities of
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Clostridium difficile isolated in Japan. J Infect Chemother, 2013; 19: 360-362. 41. Spigaglia P, Barbanti F, Mastrantonio P; European Study Group on Clostridium difficile (ESGCD). Multidrug resistance in European Clostridium difficile clinical isolates. J Antimicrob Chemother, 2011; 66: 2227-2234.
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42. Leeds JA, Sachdeva M, Mullin S, Barnes SW, Ruzin A. In vitro selection, via serial passage, of Clostridium difficile mutants with reduced susceptibility to fidaxomicin or
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vancomycin. J Antimicrob Chemother, 2013; [Epub ahead of print]. PMID: 23887866.
43. Nagy E. Anaerobic infections: update on treatment considerations. Drugs, 2010; 70: 841858.
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activities of 15 antimicrobial agents against 110 toxigenic Clostridium difficile clinical
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44. Veloo AC, Welling GW, Degener JE. Antimicrobial susceptibility of clinically relevant
Antimicrob Agents Chemother, 2011; 55: 1199-1203.
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gram-positive anaerobic cocci collected over a three-year period in the Netherlands.
45. CLSI. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria;
Approved Standard – Eighth edition. CLSI document M11-A8. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
Informational Supplement. CLSI document M100-S23. Wayne, PA: Clinical and
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Laboratory Standards Institute; 2013.
47. European Committee on Antimicrobial Susceptibility Testing, EUCAST. Clinical
Accessed 9 May 2014.
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breakpoints for bacteria. Available at: http://www.eucast.org/clinical_breakpoints/.
48. Toprak NU, Uzunkaya OD, Sóki J, Soyletir G. Susceptibility profiles and resistance
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genes for carbapenems (cfiA) and metronidazole (nim) among Bacteroides species in a Turkish University Hospital. Anaerobe, 2012; 18: 169-171. 49. Cormican MG, Erwin ME, Jones RN. False resistance to metronidazole by E-test among anaerobic bacteria investigations of contributing test conditions and medium quality.
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Diagn Microbiol Infect Dis, 1996; 24: 117-119.
50. Sensititre TREK Diagnostic Systems Anaerobe MIC Plate. Thermo Fisher Scientific.
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Available at: http://www.trekds.com/products/sensititre/c_anaerobe.asp. Accessed 9 May 2014.
51. Thermo Scientific™ Oxoid ANA MIC Panels. http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=15648363&gr
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46. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third
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oupType=PRODUCT&searchType=0&storeId=11152&from=search. Accessed 9 May 2014.
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52. Wexler HM. Bacteroides: the good, the bad, and the nitty-gritty. Clin Microbiol Rev, 2007; 20: 593-621.
53. Summanen P. Identification by Using Special-potency Disks, p 4.6.5.1-4.6.5.2. In Garcia LS (ed), Clinical Microbiology Procedures Handbook, 3rd ed, vol 1. ASM Press,
54. CLSI. Analysis and Presentation of Cumulative Antimicrobial Susceptibility Testing
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Data; Approved Guideline – Third Edition. CLSI document M39-A3. Wayne, PA:
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Clinical and Laboratory Standards Institute; 2008.
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Washington, DC. 2010.
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Figure 1. Penicillin Etest® (bioMérieux, Durham, NC) of Bacteroides fragilis on Brucella blood
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agar plate supplemented with hemin and vitamin K1 (Anaerobe Systems, Morgan Hill, CA). Minimal inhibitory concentration (MIC) of this isolate equals 32 µg/mL.
Figure 2. Anaerobic susceptibility testing panel ANO2 (Sensititre, Thermo Fisher Scientific,
Waltham, MA) inoculated with Bacteroides fragilis American Type Culture Collection (ATCC)
plate. Growth of the bacteria in a well is indicated by a dot of organismal growth in the bottom of
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the well. In a series of increasing concentration of a single antibiotic, the first empty (no growth) well is considered the minimal inhibitory concentration (MIC) of that antibiotic. For example,
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clindamycin is present in the fourth row from the top, beginning at a concentration of 0.2 µg/mL (fourth row, first well) and extending to 8 µg/mL (fourth row, sixth well from the left). Thus, the MIC for clindamycin is 2 µg/mL (fourth row, fourth well from the left), which is susceptible.
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Photo is courtesy of Thermo Fisher Scientific. Copying is prohibited.”
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25285. Various antimicrobial agents in increasing two-fold concentrations are present in the
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Table 1. Advantages and Disadvantages of Antimicrobial Susceptibility Testing Methods for Anaerobic Bacteria Disadvantages Labor-intensive Expertise required for performance Considerable time is involved in setting up testing Not amenable/ appropriate for testing small numbers of organisms Broth Multiple antimicrobials Recommended by Microdilution (10 or more) can be CLSI only for tested per isolate Bacteroides fragilis group organisms Commercial panels are available Shelf life of frozen panels may be Laboratories can limited customize their own panel of antibiotics to Poor growth by some test strains has been shown MIC Gradient Ease of use Expensive for surveillance Precise MIC value is Diffusion purposes obtained Metronidazole Convenient for testing of Method resistance can be a few individual isolates overestimated if Multiple drugs can be anaerobiosis is tested at one time inadequate Rapid β Rapid (at most 30 Only tests for βminutes to results) lactamases Lactamase Can be used on a limited number of Disk Testing bacterial species Negative result must be followed up with an MIC test for accurate prediction of beta-lactam susceptibility CLSI= Clinical and Laboratory Standards Institute; MIC= minimal inhibitory concentration
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Advantages Reference method against which other methods are compared
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Method Agar Dilution
Table 2. Indications for Susceptibility Testing of Anaerobic Bacteria* Indication
Examples Any anaerobe
Particular species of bacteria is implicated in
Bacteroides fragilis
disease
Prevotella spp.
Clostridium spp. other than Clostridium
therapy with an appropriate therapeutic
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perfringens
Pivotal role of antimicrobial agent in clinical outcome
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Infections of specific body sites
Bilophila wadsworthia
Fusobacterium spp.
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Long-term therapy needed
Sutterella wadsworthensis
Anaerobic organisms involved in osteomyelitis, endocarditis, or brain abscess
B. fragilis group implicated in osteomyelitis or joint infection
Brain abscess
Endocarditis
Prosthetic devices or graft infections
Bacteremia
* The above suggestions are examples only and are not intended to be used as all-inclusive lists.
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regimen
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Persistence of infection despite adequate
Table 3. Suggested Antimicrobial Agents for Testing and Reporting on Anaerobic Organisms*
Bacteroides fragilis Group and Other Gram-
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Antimicrobials for Primary Testing and Reporting
Gram-positive Anaerobes
negative Anaerobes
Penicillin
Piperacillin-tazobactam
Clindamycin Doripenem
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Ertapenem
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Ticarcillin-clavulanate
Ampicillin-sulbactam Piperacillin-tazobactam
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Ampicillin-sulbactam
Amoxicillin-clavulanate
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Amoxicillin-clavulanate
Ticarcillin-clavulanate
Clindamycin Doripenem Ertapenem Imipenem
Meropenem
Meropenem
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Imipenem
Metronidazole
Metronidazole
Supplemental Antimicrobials for Selective Testing and Reporting
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Ampicillin
Bacteroides fragilis Group and Other Gram-
Gram-positive Anaerobes
Penicillin
Penicillin
Ceftizoxime
Ceftizoxime
Ceftriaxone
Ceftriaxone
Cefotetan
Cefotetan
Cefoxitin
Cefoxitin
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Ampicillin
Piperacillin
Piperacillin Ticarcillin
Moxifloxacin
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Chloramphenicol
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Ticarcillin
Tetracycline
Moxifloxacin
*Only one of the antimicrobial agents in each small box needs to be tested under usual
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circumstances, as antibiotics which are listed together demonstrate similar clinical efficacy and interpretive results Table adapted with permission. Performance Standards for Antimicrobial
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Susceptibility Testing, Twenty-third Informational Supplement. CLSI document M100-S23. Wayne, Pa, USA: Clinical and Laboratory Standards Institute; 2013 www.clsi.org [46].
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Ampicillin
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negative Anaerobes
Antimicrobial Resistance and Susceptibility Testing of Anaerobic Bacteria
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Audrey N. Schuetz
Associate Director, Clinical Microbiology Laboratory, Assistant Professor, Department of
Pathology and Laboratory Medicine, Assistant Professor, Department of Internal Medicine, Weill Cornell Medical College/ NewYork-Presbyterian Hospital, 525 East 68th Street, Starr
Corresponding Author: Phone: 212-746-0833, Fax: 212-746-8945, E-mail:
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[email protected]
Alternate Corresponding Author: Yelena Lavrova, NewYork-Presbyterian Hospital, 525 East
[email protected]
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68th Street, Starr 737, New York, NY 10065, Phone: 212-746-2408, Fax: 212-746-8945, E-mail:
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Summary: Resistance of anaerobic bacteria to certain classes of antibiotics is increasing globally. Resistance trends vary by geographic region and often vary between species. Susceptibility
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testing of anaerobes is important in directing appropriate antibiotic therapy.
© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e‐mail: [email protected].
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737C, New York, NY 10065
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ABSTRACT
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Infections due to anaerobic bacteria can be severe and life-threatening. Susceptibility
testing of anaerobes is not frequently performed in laboratories, but such testing is important to
direct appropriate therapy. Anaerobic resistance is increasing globally, and resistance trends vary by geographic region. An overview of a variety of susceptibility testing methods for anaerobes is
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situations warranting anaerobic susceptibility testing are discussed.
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covered, and the advantages and disadvantages of each method are reviewed. Specific clinical
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INTRODUCTION
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Anaerobic infections can be severe and life-threatening, and their increasing incidence is due in part to the high numbers of patients with complex underlying diseases [1]. Studies
demonstrate that failure to direct appropriate anaerobic therapy leads to poor clinical response
[2]. Anaerobic collection, transport and manipulation of culture isolates can be time-consuming
infections has often been instituted long before results of susceptibility testing are available.
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However, antimicrobial bacterial resistance among anaerobic bacteria is increasing globally, as demonstrated by numerous surveys in Europe, the United States, Canada, and New Zealand [3-6]. Differences in resistance patterns may be due to the variety of susceptibility
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testing methodology used, the selective antibiotic pressures associated with antimicrobial usage, and the lack of uniformity in adoption of interpretive breakpoints. This article covers typical
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resistance patterns and recent changes in resistance of certain organisms over time. Anaerobic susceptibility testing methods and their challenges are reviewed. RESISTANCE PATTERNS OF ANAEROBIC BACTERIA Bacteroides fragilis Group
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The members of the B. fragilis group are among some least susceptible anaerobes to antibiotics. Resistance of Bacteroides species is linked to outcome, even in the presence of
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mixed infections [4, 7]. The B. fragilis group consists of 24 species including Bacteroides fragilis, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides caccae, and Parabacteroides distasonis. Fewer than 3% of Bacteroides isolates are susceptible to penicillin and ampicillin [8].
Penicillin- and cephalosporin-resistance is mediated by cepA and cfxA genes. The chromosomal
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and difficult to perform correctly. Empirical broad-spectrum antimicrobial therapy for anaerobic
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cepA gene is a cephalosporinase encoding for resistance to cephalosporins and aminopenicillins but not piperacillin or β-lactam-β-lactamase inhibitor combinations (BLBLIs). The cfxA gene
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encodes for high level resistance to cefoxitin and other β-lactams [9]. Although β-lactamases are the principal mechanism of resistance to penicillins, the expression of altered penicillin-binding proteins can also lead to resistance [10]. Membrane modifications, such as porin losses, may further increase the minimal inhibitory concentrations (MICs) to β-lactams and BLBLIs.
fragilis group [3, 11, 12]. Carbapenems are generally active against the B. fragilis group, and
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most U.S.-based studies report susceptibility rates of 98.5-99% [4, 13, 14]. Carbapenem resistance is usually mediated by a chromosomal zinc metallo-β-lactamase enzyme encoded by
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the cfiA gene [15]. Although cephamycins were used in the past for anaerobic coverage, the Infectious Diseases Society of America (IDSA) recently stated that cefotetan and clindamycin are no longer recommended as therapy for community-acquired intra-abdominal infections in
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adults due to increasing resistance among the B. fragilis group [16]. Clindamycin resistance is mediated by erm genes located on transferable plasmids which can also carry tetracycline resistance genes [17]. Worldwide, clindamycin resistance is increasing and approaches 60% [3, 4, 12, 18].
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Although rare, metronidazole-resistant strains of B. fragilis have been reported and are
associated with the nim gene [3, 11, 15]. However, the presence of this gene does not invariably
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lead to resistance, and MICs must be obtained. Of 206 B. fragilis isolates in one study, the nim gene was detected in 7.3%, but metronidazole MICs ranged from 1.5 (susceptible) to >256 mg/L (resistant) [19]. Susceptibility rates to moxifloxacin are highly variable across studies, and isolates are acquiring resistance rapidly. Moxifloxacin susceptibility rates range from 30 to 60%
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Currently, ticarcillin-clavulanate and piperacillin-tazobactam are active against >90% of the B.
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for B. fragilis group, and such resistance is mediated by gyrA mutations, efflux pumps, or topoisomerase gene modifications [12]. Tigecycline MICs are relatively low in surveillance
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studies, but there have been reports of resistance [6].
Susceptibility within the B. fragilis group varies by species, with B. fragilis being the most susceptible. Parabacteroides distasonis demonstrates high MICs to β-lactams, and B. thetaiotaomicron also demonstrates higher resistance rates to various antimicrobials in
isolates collected from 10 U.S. medical centers confirmed that susceptibility rates differed
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widely by species [4]. In this series, B. ovatus was more resistant to the carbapenems; B. vulgatus was more resistant to piperacillin-tazobactam and showed the highest resistance rates to
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moxifloxacin (54%); P. distasonis was more resistant to ampicillin-sulbactam and cefoxitin; and B. ovatus and B. uniformis showed higher resistance rates to moxifloxacin (39% and 41%, respectively). However, a 2008 practice survey of anaerobes across the U.S. reported that 33% of
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reference laboratories did not identify B. fragilis group isolates to the species level [20]. Susceptibility rates among B. fragilis group are decreasing worldwide. Snydman et al. reported a decrease in susceptibility of approximately 25% over 11 years to ampicillin-sulbactam in P. distasonis and B. vulgatus [4].
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Prevotella, Porphyromonas, and Other Anaerobic Gram-negative Rods Porphyromonas spp. are generally susceptible to β-lactams , clindamycin, and
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metronidazole [21]. One-fourth to one-third of Porphyromonas produce β-lactamases, and clindamycin resistance has been observed in a minority of strains [22]. Compared to Porphyromonas, approximately 95% of Prevotella spp. is resistant to penicillin and ampicillin [22]. A recent Belgian study showed that clindamycin susceptibility in Prevotella spp. has been
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comparison to other members of the B. fragilis group. A survey of 6,574 B. fragilis group
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decreasing from 91% in 1993-1994 to 69% in 2012 [23]. Susceptibility to moxifloxacin, metronidazole, carbapenems, and amoxicillin-clavulanate is typically 90% or greater. Penicillin
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resistance in Fusobacterium spp. ranges from 4 to 15% and is generally due to the production of β-lactamases [22]. Fusobacterium varium, Fusobacterium mortiferum, and Fusobacterium
nucleatum are most often reported to produce β-lactamases, while over 90% of Fusobacterium necrophorum are susceptible to cephalosporins and cephamycins [14,24]. Fusobacterium spp.
clindamycin.
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Gram-positive Non-Spore-forming Rods
Actinomyces, Propionibacterium, Bifidobacterium, and the “Eubacterium” group are
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usually susceptible to β-lactams and BLBLIs [25, 26]. Propionibacterium spp., Actinomyces spp., Bifidobacterium spp., and Lactobacillus spp. are usually resistant to metronidazole. Clindamycin shows moderate activity against this group [27]. Decreasing clindamycin
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susceptibility of P. acnes is associated with prior topical acne therapy [28]. Vancomycin shows some activity against certain species of Lactobacillus; however, MICs >256 µg/mL are common for the most frequently isolated lactobacilli from human specimens - Lactobacillus casei and Lactobacillus rhamnosus [29]. Telavancin has shown good activity against some Lactobacillus
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spp. [33]. Newer antimicrobial agents such as linezolid, daptomycin, and dalbavancin exhibit excellent in vitro activity against most anaerobic gram-positive species [31, 32].
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Gram-positive Spore-forming Rods Of the clostridial species, Clostridium perfringens is one of the most susceptible to
penicillin, but clindamycin resistance is increasing, with up to 14% of blood isolates of C.
perfringens in one study [5, 26, 33]. Tetracycline resistance (defined as MIC >2 µg/mL) has
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are typically susceptible to metronidazole, BLBLIs, cephalosporins, carbapenems, and
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been documented in up to 75% of C. perfringens isolates obtained from commercial poultry, but there are little data in humans [34, 35]. The activities of tetracycline against other clostridial
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species and of doxycycline against clostridia in general have not been well documented [35].
Drugs which maintain activity against non-perfringens Clostridium spp. include piperacillin, BLBLIs, carbapenems, metronidazole, and vancomycin [36]. Antimicrobial agents lacking
significant activity include ampicillin, aminoglycosides, trimethoprim-sulfamethoxazole, and
tertium, Clostridium difficile, and the “RIC” group of clostridia – namely, Clostridium ramosum,
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Clostridium innocuum and Clostridium clostridioforme. In a study of 540 clinical isolates of Clostridium spp., tigecycline was highly active, with an MIC90 of 0.25 µg/mL [37].
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Clostridium difficile is usually susceptible to metronidazole and vancomycin but resistant to β-lactams, fluoroquinolones, and clindamycin [38]. Outbreaks of fluoroquinolone-resistant C. difficile strains have been associated with increased mortality [39]. Strains resistant to rifampin
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or rifaximin associated with rpoB mutations have also been linked to clinical failure [38]. Metronidazole-resistant strains are rare but have been documented [40]. A recent surveillance study of C. difficile isolates collected from 14 European countries reported that over one-half of isolates were multidrug resistant, with the majority resistant to moxifloxacin, clindamycin,
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erythromycin, and rifampicin [41]. Fidaxomicin shows excellent activity against C. difficile, but reduced susceptibility due to drug target modifications has been documented in vitro [42].
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Anaerobic Cocci
The anaerobic gram-positive cocci (GPC) have undergone major taxonomic changes
within the past 40 years; therefore, susceptibility data have been difficult to interpret. Greater than 90% of anaerobic GPC are susceptible to penicillin, including most Peptostreptococcus spp.
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clindamycin. Resistance to clindamycin is typical for many species, including Clostridium
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[35]. Anaerobic GPC are also generally susceptible to β-lactams, BLBLIs, cephalosporins, and carbapenems [24]. Alterations in penicillin-binding proteins account for the majority of β-lactam
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resistance [43]. Clindamycin resistance rates among anaerobic GPC range from 7 to 20%, and
this resistance is rising especially in Finegoldia magna and Peptoniphilus spp. [35, 14]. 90-95% of GPC are susceptible to metronidazole, but rare nimB-positive, metronidazole-resistant strains of F. magna and Parvimonas micra have been described [35, 44]. Strains of streptococci which
misidentified as anaerobic GPC and will test resistant to metronidazole, so metronidazole
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resistance in anaerobic GPC should be confirmed.
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ANAEROBIC SUSCEPTIBILITY TESTING (AST)
Susceptibility testing is performed to obtain information on the predicted response of the bacteria to antibiotics in the form of an MIC, which is defined as the lowest concentration of the
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antibiotic that inhibits growth of organisms. Drugs are typically tested at two-fold doubling (log2) serial dilutions (i.e., 2 µg/mL, 4 µg/mL, 8 µg/mL, etc.). MICs may be obtained by utilizing the CLSI-defined agar or broth microdilution methods or by using a variety of commercially available methods. Commercial methods include: Etest® strips (bioMérieux, Durham, NC),
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M.I.C.Evaluator strips (M.I.C.E.™) (Thermo Fisher Scientific, Basingstoke, United Kingdom), and Sensititre panels (Trek Diagnostics Systems, Thermo Fisher Scientific, Cleveland, OH).
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Etests and M.I.C.Evaluator strips are gradient diffusion methods. Agar and broth microdilution methods are usually employed by research or reference laboratories. Advantages and disadvantages of AST methods are listed in Table 1.
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grow well anaerobically, such as the Streptococcus anginosus group, may initially be
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In a 2008 practice survey of anaerobic culture and AST in U.S. hospitals, Goldstein and colleagues reported that only 21% (21/98) of hospital laboratories performed anaerobic AST in-
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house (as opposed to outsourcing to a reference laboratory) [20]. Twenty hospital laboratories reported sending their isolates out for susceptibility testing, and the remaining 60% (58/98) of
laboratories did not offer any AST. In 1990, 70% of hospital laboratories performed anaerobic AST, but only 33% of hospital labs did so in 1993. This decline may be explained by the fact
susceptibility testing in 1993, are no longer considered appropriate for susceptibility testing. In
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the 2008 survey, 65% of hospital laboratories which performed AST used Etest methodology [20]. The remainder of hospital laboratories used microbroth dilution, and none used agar
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dilution.
Procedural guidelines for anaerobic AST have been published by CLSI, but the Food and Drug Administration (FDA) and European Committee on Antimicrobial Susceptibility Testing
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(EUCAST) also develop breakpoints [45, 46, 47]. Occasionally, there are differences in interpretive criteria between organizations, which may be explained by differences in dosages, administration intervals, inoculum size, or test media. It is important for clinicians to be aware of which breakpoints are being employed. Antimicrobial susceptibility testing in anaerobes relies
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primarily on phenotypic characterization, although a few molecular assays exist (e.g., nim gene for metronidazole resistance and cfiA for carbapenem resistance) [48]. As molecular data on
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anaerobic susceptibility are gathered, it is possible that additional molecular assays could become available.
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that the disk diffusion and broth disk elution methods, which were the predominant methods of
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Agar Dilution The CLSI agar dilution procedure is the gold standard reference method for anaerobic
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AST [45]. Different concentrations of antimicrobial agents are prepared in two-fold dilutions,
added to molten agar, poured into Petri dishes and allowed to solidify. A standardized inoculum of bacterial cells is spotted onto the surface of each plate with an inoculator, replicator, or pipette. After 48 hours of incubation, the plates are examined visually for the lowest
time, labor and expertise and is not intended for testing of single isolates. Thus, it is not a
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technique which many clinical laboratories employ. Broth Microdilution
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The broth microdilution method is less labor-intensive than agar dilution. In this assay, drugs in serial two-fold dilutions are added to small volumes of broth liquid media pipetted in wells of plastic microdilution trays or plates. A standard inoculum of bacterial cells is added into
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each well. MICs are read after 48 hours of incubation. Multiple antibiotics (ten or more) can be tested at one time, and the laboratory can design its own drug panels. However, this technique is limited by poor growth of certain oxygen-sensitive anaerobes, and is currently validated by CLSI for use only with B. fragilis group [46].
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MIC Gradient Diffusion Method
The Etest® and M.I.C.E.™ strips are thin plastic strips which are impregnated on one
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face with an increasing gradient concentration of an antimicrobial agent (Figure 1). The other face of the strip is marked with an MIC scale with increasing antibiotic concentrations, including increments between doubling dilutions (i.e., 1 µg/mL, 1.5 µg/mL, 1.75 µg/mL, 2 µg/mL, etc.). A Brucella blood agar plate supplemented with hemin and vitamin K1 is pre-reduced in an
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concentration of antibiotic which inhibits growth. Agar dilution testing requires considerable
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anaerobic environment to rid the media of toxic oxygen metabolites. A standard inoculum of bacterial cells is swabbed onto the plate. The Etest or M.I.C.E.™ strip is placed antibiotic face
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down. The MIC is read at the intersection of the lower part of the ellipse with the corresponding
number on the test strip. Results are generally available at 48 hours; however, clostridial species and B. fragilis group isolates usually grow rapidly enough to be read at 24 hours. MIC gradient diffusion methods are easier to use in routine clinical laboratory practice than agar dilution. A
quantitative and precise MIC. Additionally, several drugs can be tested at one time, and
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laboratories can tailor testing to their needs. However, Etest strips cost approximately $2 to $3 U.S. dollars apiece. Etest results generally correlate well with agar dilution, but the MIC gradient
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diffusion method can overestimate metronidazole resistance if anaerobiosis is not adequate [49]. Since the activity of metronidazole is dependent upon an active intermediate which requires the reduced atmosphere of anaerobiosis, it is important to utilize anaerobic control organisms when
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setting up such testing.
Commercially Available Broth Microdilution Panels Two commercially available panels for anaerobic susceptibility testing are available. The Anaerobe Sensititre panel (ANO2, Thermo Fisher) is a panel of antimicrobials of varying two-
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fold dilutions (Figure 2) [50]. A second anaerobic susceptibility panel is available through Oxoid (Thermo Fisher) [51].
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Rapid β-Lactamase Disk Testing This disk test evaluates for the presence of β-lactamase enzyme. Bacterial colonies are
smeared onto a disk embedded with a chromogenic cephalosporin. If the organism expresses β-
lactamase, the disk will change colors after reagent is added. Although most reactions occur
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significant advantage of the MIC gradient diffusion method is the ability to generate a
12
within five to 10 minutes, some β-lactamase-positive strains may react more slowly (up to 30 minutes. If the disk is positive, the organism is considered to be resistant to penicillin, ampicillin,
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and amoxicillin. Since β-lactamases are not the sole mediator of resistance in anaerobes, a
negative test does not rule out penicillin or ampicillin resistance resulting from alterations of
penicillin-binding proteins [52]. Therefore, a negative result by the rapid β-lactamase disk test
must be followed by an MIC test for accurate prediction of beta-lactam susceptibility. Also, the
are presumed to be resistant due to almost universal presence of β-lactamases.
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Disk Diffusion
Disk diffusion (Kirby Bauer) tests should not be performed for anaerobic bacteria for the
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purpose of obtaining susceptibility results, as the results are inaccurate and do not correlate with the agar dilution method [46]. Special-potency antibiotic disks of vancomycin (5µg), kanamycin (1000 µg), and colistin (10 µg) are occasionally used in the laboratory as an aid in preliminary
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identification of some anaerobes based on patterns of resistance [53]. However, due to the high antibiotic concentrations in these disks, these disks are not intended for use in determining antibiotic susceptibility for therapeutic purposes.
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STRATEGIES FOR TESTING AND REPORTING OF SUSCEPTIBILITY DATA Antimicrobial Testing Guidelines
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For individual patient management, AST should be performed when: 1) selection of an
active agent is critical for disease management; 2) long-term therapy is being considered; 3) anaerobes are recovered from sterile body sites; or, 4) the infection persists despite adequate therapy with an appropriate therapeutic regimen (Table 2). In cases of a single recovered
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β-lactamase disk testing should not be performed on members of the B. fragilis group, since they
13
anaerobic pathogen from culture, AST should be performed. CLSI suggests certain antimicrobial agents to be considered for testing and reporting on anaerobic organisms (Table 3) [46]. For
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gram-negative anaerobes, penicillin or ampicillin may be tested and/or reported selectively for all except the B. fragilis group which are almost uniformly resistant. For non-spore-forming
gram-positive anaerobes, resistance to metronidazole is common, and ampicillin and penicillin
are recommended for primary testing. Although CLSI MIC breakpoints for amoxicillin have not
[46]. The remaining supplemental antimicrobials are helpful to test when strains are resistant to
Antibiograms
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primary drugs or for use in patients who are allergic to primary drugs.
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Establishment of an antibiogram outlining patterns of resistance on a periodic basis is helpful in guiding antimicrobial therapy. The CLSI suggests gathering susceptibility data from at least 30 isolates of the same genus or species collected over the period of a year in order to
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obtain a reasonable number of isolates upon which to estimate susceptibilities [54]. Examples of cumulative antimicrobial susceptibility reports for B. fragilis group and other anaerobes from across the U.S. are included in the CLSI documents M100 and M11 [45, 46].
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CONCLUSIONS
Susceptibility among anaerobes differs from species to species and also from region to
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region. Surveillance studies have shown that susceptibility trends of anaerobes to certain antimicrobial agents are changing, with more resistance recognized in many parts of the world. Despite the difficulties inherent in anaerobic AST, testing should be performed and observed for trends over time.
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been established for anaerobes, amoxicillin breakpoints are considered equivalent to ampicillin
14
ACKNOWLEDEMENTS
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The work in this paper was not supported financially. The author has no conflict of interest to
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declare.
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51. Thermo Scientific™ Oxoid ANA MIC Panels. http://www.thermoscientific.com/ecomm/servlet/productsdetail?productId=15648363&gr
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46. CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third
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oupType=PRODUCT&searchType=0&storeId=11152&from=search. Accessed 9 May 2014.
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Clinical and Laboratory Standards Institute; 2008.
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Washington, DC. 2010.
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Figure 1. Penicillin Etest® (bioMérieux, Durham, NC) of Bacteroides fragilis on Brucella blood
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agar plate supplemented with hemin and vitamin K1 (Anaerobe Systems, Morgan Hill, CA). Minimal inhibitory concentration (MIC) of this isolate equals 32 µg/mL.
Figure 2. Anaerobic susceptibility testing panel ANO2 (Sensititre, Thermo Fisher Scientific,
Waltham, MA) inoculated with Bacteroides fragilis American Type Culture Collection (ATCC)
plate. Growth of the bacteria in a well is indicated by a dot of organismal growth in the bottom of
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the well. In a series of increasing concentration of a single antibiotic, the first empty (no growth) well is considered the minimal inhibitory concentration (MIC) of that antibiotic. For example,
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clindamycin is present in the fourth row from the top, beginning at a concentration of 0.2 µg/mL (fourth row, first well) and extending to 8 µg/mL (fourth row, sixth well from the left). Thus, the MIC for clindamycin is 2 µg/mL (fourth row, fourth well from the left), which is susceptible.
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Photo is courtesy of Thermo Fisher Scientific. Copying is prohibited.”
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25285. Various antimicrobial agents in increasing two-fold concentrations are present in the
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Table 1. Advantages and Disadvantages of Antimicrobial Susceptibility Testing Methods for Anaerobic Bacteria Disadvantages Labor-intensive Expertise required for performance Considerable time is involved in setting up testing Not amenable/ appropriate for testing small numbers of organisms Broth Multiple antimicrobials Recommended by Microdilution (10 or more) can be CLSI only for tested per isolate Bacteroides fragilis group organisms Commercial panels are available Shelf life of frozen panels may be Laboratories can limited customize their own panel of antibiotics to Poor growth by some test strains has been shown MIC Gradient Ease of use Expensive for surveillance Precise MIC value is Diffusion purposes obtained Metronidazole Convenient for testing of Method resistance can be a few individual isolates overestimated if Multiple drugs can be anaerobiosis is tested at one time inadequate Rapid β Rapid (at most 30 Only tests for βminutes to results) lactamases Lactamase Can be used on a limited number of Disk Testing bacterial species Negative result must be followed up with an MIC test for accurate prediction of beta-lactam susceptibility CLSI= Clinical and Laboratory Standards Institute; MIC= minimal inhibitory concentration
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Advantages Reference method against which other methods are compared
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Method Agar Dilution
Table 2. Indications for Susceptibility Testing of Anaerobic Bacteria* Indication
Examples Any anaerobe
Particular species of bacteria is implicated in
Bacteroides fragilis
disease
Prevotella spp.
Clostridium spp. other than Clostridium
therapy with an appropriate therapeutic
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perfringens
Pivotal role of antimicrobial agent in clinical outcome
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Infections of specific body sites
Bilophila wadsworthia
Fusobacterium spp.
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Long-term therapy needed
Sutterella wadsworthensis
Anaerobic organisms involved in osteomyelitis, endocarditis, or brain abscess
B. fragilis group implicated in osteomyelitis or joint infection
Brain abscess
Endocarditis
Prosthetic devices or graft infections
Bacteremia
* The above suggestions are examples only and are not intended to be used as all-inclusive lists.
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regimen
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Persistence of infection despite adequate
Table 3. Suggested Antimicrobial Agents for Testing and Reporting on Anaerobic Organisms*
Bacteroides fragilis Group and Other Gram-
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Antimicrobials for Primary Testing and Reporting
Gram-positive Anaerobes
negative Anaerobes
Penicillin
Piperacillin-tazobactam
Clindamycin Doripenem
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Ertapenem
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Ticarcillin-clavulanate
Ampicillin-sulbactam Piperacillin-tazobactam
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Ampicillin-sulbactam
Amoxicillin-clavulanate
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Amoxicillin-clavulanate
Ticarcillin-clavulanate
Clindamycin Doripenem Ertapenem Imipenem
Meropenem
Meropenem
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Imipenem
Metronidazole
Metronidazole
Supplemental Antimicrobials for Selective Testing and Reporting
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Ampicillin
Bacteroides fragilis Group and Other Gram-
Gram-positive Anaerobes
Penicillin
Penicillin
Ceftizoxime
Ceftizoxime
Ceftriaxone
Ceftriaxone
Cefotetan
Cefotetan
Cefoxitin
Cefoxitin
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Ampicillin
Piperacillin
Piperacillin Ticarcillin
Moxifloxacin
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Chloramphenicol
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Ticarcillin
Tetracycline
Moxifloxacin
*Only one of the antimicrobial agents in each small box needs to be tested under usual
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circumstances, as antibiotics which are listed together demonstrate similar clinical efficacy and interpretive results Table adapted with permission. Performance Standards for Antimicrobial
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Susceptibility Testing, Twenty-third Informational Supplement. CLSI document M100-S23. Wayne, Pa, USA: Clinical and Laboratory Standards Institute; 2013 www.clsi.org [46].
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Ampicillin
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negative Anaerobes
