Journal of Pediatric Infectious Diseases 4 (2009) 5365 DOI 10.

3233/JPI-2009-0151 IOS Press

53

Review Article

Choosing antimicrobials for anaerobic infections

Ellie J. C. Goldsteina, and Gary E. Steinb
a b

UCLA School of Medicine and R.M. Alden Research Laboratory, Santa Monica, CA, USA Department of Medicine, Michigan State University, East Lansing, MI, USA

Received 11 May 2008 Revised 21 May 2008 Accepted 25 May 2008

Abstract. Anaerobes are opportunistic pathogens that are the predominant indigenous normal ora of the skin and mucous membranes in humans. Anaerobic infections are common, often found in proximity to their anatomical locations, and may be difcult to treat. Anaerobes are usually found in mixed culture along with aerobes. This article reviews the activity of various classes of antimicrobials used against anaerobes, along with their pharmacodynamics, and the susceptibility patterns and resistance mechanisms of important anaerobic pathogens such as Bacteroides fragilis. It is hoped that the reader will be able to use this information and synthesize it into an approach on how to select antimicrobials for anaerobic therapy. Keywords: Anaerobic bacteria, B. fragilis, carbapenems, antimicrobial resistance, pharmacokinetics

1. Introduction Anaerobes are secret and recondite pathogens, despite being the predominant normal ora of humans. In association with aerobic bacteria, anaerobes are involved in many serious infections, including sinusitis, aspiration pneumonia, appendicitis, and abscesses [1,2]. Despite advances in medical microbiology, the clinical diagnosis of anaerobic infections is underappreciated, their isolation by clinical laboratories requires substantial improvement, and their susceptibility patterns are under-reported and often unappreciated. Each patient is an experiment, and therapeutic success or failure depends on the happenstance of human immune defenses, surgical skill and an antimicrobial that is empirically selected [2,3].
Ellie J.C. Goldstein, MD, R.M. Alden Research Laboratory, 2021 Santa Monica Boulevard, Suite 740 E, Santa Monica, CA 90404, USA. Tel.: +1 310 315 1511; Fax: +1 310 315 3663; E-mail: ejcgmd@aol.com.
Correspondence:

The clinical consequences of anaerobic infection, especially those that are missed, include prolonged hospital stay, increased costs and increased morbidity and mortality. The specic site of infection will determine the type of complication encountered. When milder infections are unrecognized and left untreated, the infections will linger causing increased malaise and discomfort. The more serious infections may provoke fevers, repeated hospitalizations, and additional surgical procedures for abscess formation and tissue debridement. Failure to recognize the mixed aerobic/anaerobic nature of necrotizing fasciitis can lead to disguring surgery and even death. Failure to treat the anaerobic component of an intra-abdominal infection often requires broader spectrum antibiotics and leads to re-operation and prolonged hospital stay. There is also an associated increased risk of development of resistant isolates, both aerobic and anaerobic [2,3]. The dilemma of selecting an appropriate antimicrobial agent to treat anaerobic infections is predicated upon the slow growth of anaerobes as compared to aer-

1305-7707/09/$17.00 2009 IOS Press and the authors. All rights reserved

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E.J.C. Goldstein and G.E. Stein / Antimicrobials in anaerobic infections

obes and the resultant lack of specic culture data for directed therapy. Many hospital clinical microbiology laboratories are unfamiliar with anaerobes or may not have the expertise or facilities to isolate them. Consequently, clinicians are forced to rely upon original governmental regulatory drug indications which often list efcacy (either in vitro or in vivo) of a very few genera and species for a limited number of indications. This extrapolation may not be appropriate to the clinical situation facing the medical care team. Published antimicrobial susceptibility surveys may not reect the clonality of local isolates and their unique susceptibility patterns and may therefore lead one astray as to selecting appropriate specic therapy [4]. This review acts as a basis for understanding how to select an antimicrobial for anaerobic infections in children and adults. It highlights the example of mixed aerobic/anaerobic intra-abdominal infections and their infectious burden, reviews the categories of potentially useful individual antimicrobial agents including their in vitro activity, pharmacokinetics and pharmacodynamics properties, the problem of obtaining accurate susceptibility results due to various discrepancies of testing methods and culminates to synthesize how one may attempt to select an appropriate antimicrobial agent. Anaerobic bacteria were rst described by Pasteur and were shown to be human pathogens by Prevot in 1898 [5]. Although the term anaerobe suggests that they grow in the absence of oxygen, they range from those that are extremely oxygen sensitive (which are usually isolated only in normal ora studies) to those that can survive on the surface of a fresh agar plate in the presence of atmospheric oxygen, such as Bacteroides fragilis. Anaerobes require an environment with a low oxidation-reduction potential (eH gradient), which is associated with low pH, tissue destruction, byproducts from aerobic bacterial metabolism and low oxygen content. They are categorized by Gram stain reaction as well as biochemical reactions. Recent taxonomic advances have led to the reclassication and renaming of many anaerobic species, causing confusion by many clinicians and laboratorians [6].

most always in combination with aerobic bacteria. Diseases associated with mixed aerobic/anaerobic infections are seen in Fig. 1. On the skin, Propionibacterium acnes, in interaction with the sebaceous secretions, cause the common bane of teenagers, acne. In the oral cavity, anaerobes outnumber aerobes by 10 to 1 and are involved in the most common of human infections, gingivitis and periodontitis. When the oral ora is aspirated into the lung, anaerobes are involved in causing aspiration pneumonia, lung abscesses and empyema. In the colon, anaerobes outnumber aerobes by up to 1,000 to 1 with the average human colon contains between 10 and 100 trillion organisms [7,8]. Colon anaerobes are involved in appendicitis (the most frequent cause for abdominal surgery), diverticulitis, abdominal abscess and peritonitis. Anaerobes can also cause skin and soft tissue infections in proximity to the fecal ora-such as gas gangrene and Fourniers gangrene of the scrotum and in the feet of diabetic patients (fetid foot) [9]. As the predominant bioora of the vagina [10], they help maintain vaginal health and pH; but when that balance is upset, the result is bacterial vaginosis, which occurs in 50% of all women, in their lifetimes. The ora of these infections is complex, and communities of bacteria communicate with each other (quorum sensing) [11] and help direct the individual organisms, via transmitter substances, to reproduce or express pathogenicity factors. Anaerobic bacteria possess a variety of virulence factors [12] that facilitate adhesion (mbriae, pili, hemagglutinins, lectins), inhibit phagocytosis (capsule, lipopolysaccharise), assist invasion and tissue destruction (proteases, neuraminidases, heparinases, exotoxins, hemolysins), or act as toxins (endotoxins and enterotoxins). These factors differ between the species.

3. The intra-abdominal infection model Intra-abdominal infections encompass a spectrum of specic infections (such as appendicitis, fecal peritonitis and cholecystitis) that are grouped together by anatomical coincidence. Intra-abdominal infections are commonly recognized as the quintessential prototype for mixed aerobic anaerobic infections. Treatment of these infections often requires a multidisciplinary approach with co-operation between, primary care doctors of pediatrics or internal medicine, infectious diseases consultants, surgeons and radiologists [3]. An animal model of intra-abdominal abscess [13 15] demonstrated that intra-abdominal infections have

2. The burden of infection from anaerobic bacteria As the predominant indigenous normal ora of the human cutaneous and mucous membrane surfaces (Fig. 1), anaerobes perform many benecial functions. However, they are also consummate opportunistic pathogens that cause serious and lethal infection, al-

E.J.C. Goldstein and G.E. Stein / Antimicrobials in anaerobic infections

Figure 1 Skin Oral cavity and upper respiratory Propionibacterium acnes Predominant anaerobes of the normal flora and associated diseases. Adapted from Finegold passages Peptostreptococcus SM, Sutter VL: Diagnosis and Management of Anaerobic Infections. Kalamazoo, MI, Upjohn, Prevotella, Porphyromonas 1976. Copyright Dr. Finegold.) Fusobacterium nucleatum Chronic sinusitis Peptostreptococcus Chronic otitis media Actinomyces Necrobacillosis Eubacterium Peritonsilar abscess Gingivitis Periodontitis Dental abscess Post extraction infection Peritonitis Hepatic abscess Appendicitis Chronic cholecystitis Post-operative infection/abscess

55

Bacteremia B. fragilis Clostridia Peptostreptococci Lungs Aspiration pneumonia Empyema Lung abscess

Female genital tract Lactobacillus Peptostreptococcus Pigmented Bacteroides Diabetic foot Gas gangrene Necrotizing fasciitis

Endometritis Salpingitis Bacterial vaginosis Post-abortal sepsis Fournier s gangrene Vulvar abscess

Colon Bacteroides fragilis group Prevotella Peptostreptococcus Clostridium sp Eubacterium Bifidobacterium Diarrhea C difficile Toxogenic B fragilis

Fig. 1. Predominant anaerobes of the normal ora and associated diseases (Adapted from Finegold SM, Sutter VL. Diagnosis and Management of Anaerobic Infections. Kalamazoo, MI, Upjohn, 1976. Copyright Dr. Finegold).

a biphasic nature. The initial stage of peritonitis was associated with Escherichia coli bacteremia and 43% mortality [15]. All animals surviving the acute peritonitis stage developed localized intra-abdominal abscesses by the 7th postoperative day [15]. In a subsequent experiment, individual pathogens and all possible dual combinations of E. coli, enterococci, B. fragilis and Fusobacterium varium were implanted into the peritoneal cavities of Wistar rats. Acute mortality was associated only with E. coli and was associated with the size of the inoculum. An inoculum of 5 10 7 was associated with 100% mortality within two days, whereas a 50% reduction of the size of the inoculum resulted in 65% mortality. There was no mortality in animals that received single isolates of enterococci, B. fragilis or F. varium. There was no abscess formation found on autopsy in any of the 80 animals that received

single isolate challenges. Abscesses occurred in 89 100% of animals that received a combination of one facultative and one anaerobic bacterium; the highest rate of abscess formation was caused by combination of E. coli and B. fragilis [15]. Intra-abdominal infections exemplify the biphasic nature of infections that result from fecal contamination of the peritoneal cavity and highlight the importance of the synergistic interactions of aerobic and anaerobic bacteria. The specic bacteriology of an intra-abdominal infection depends on which organ is involved and its specic ora [16,17]. The upper gastrointestinal tract, stomach, duodenum and upper small intestines contain a sparse amount of bacteria, and resultant infections are generally mild and made up of fewer organisms. From the terminal ileum, through the entire colon and until the rectum, the intensity of

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E.J.C. Goldstein and G.E. Stein / Antimicrobials in anaerobic infections

microorganisms increases dramatically, with between 10100 trillion colony forming units in the large intestine or about 10 12 cfu/g of feces [7,8]. Figure 2 lists the relative frequency of various aerobic and anaerobic bacteria found in intra-abdominal infections. In a variety of studies [16,17] aerobes account for 48% and anaerobes for 52% of isolates. The isolation of Pseudomonas aeruginosa occurs in varying rates, often 5%, but at some centers [18] as high as a 15% rate of isolation. Microbiological studies [1922] have isolated more than 21 genera and 40 species from patients with perforated appendicitis and recovered 2.7 anaerobes and 7.4 anaerobes per specimen. Benion et al. [19] studied gangrenous appendicitis and he and colleagues (20,21) studied perforated appendicitis and in a small number of patients found differences in the bacteriology. These differences included E. coli in 70% of patients with gangrenous appendicitis and 77% in perforated appendicitis, viridans streptococci, 19% vs. 43%, and P. aeruginosa 11% vs. 18%, respectively. Pediatric studies have yielded generally similar results except that Pseudomonas can occur in up to 35% of cases [3,2325].

PBP2, etc., but when one encounters the term PBP1 this may not be the same PBP1 found in B. fragilis as in E. coli despite an identical number. PBP numbering is specic to a genus/species and may be the same or unique in individual species. Each PBP drug-binding complex may have a different effect on the cell structure and resultant mechanism of cell injury [26]. 4.1.1. Penicillins Penicillins, which have a thiozolidine ring and side chains in addition to a beta-lactam ring, block cell wall synthesis by adhering to PBPs. They adhere to different PBPs in different bacteria. They are active against many anaerobes from the normal ora [26]. Resistance to -lactam antibiotics occurs by betalactamase-inactivating enzymes, which include penicillinases (most commonly found in B. fragilis group species and Prevotella species), target-site alteration (which can create low-afnity PBPs), and permeability barriers (porin channel alterations). Penicillinaseresistant penicillins, such as oxacillin, are useful against many strains of penicillinase producing Staphylococcus aureus, but are less active than penicillin against anaerobes. Ureidopenicillins, such as piperacillin, are not appreciably more active against anaerobes. Production of beta-lactamase enzymes by anaerobic bacteria other than B. fragilis has been less well studied, but strains of Clostridium, Porphyromonas and Fusobacterium can express resistance. Penicillinase-producing Fusobacterium species and Clostridium species express penicillinases that are typically inhibited by clavulanic acid, although some exceptions in Clostridium spp. occur [27,28]. 4.1.2. Beta-lactam/beta lactamase inhibitor combinations Combinations of a parent beta-lactam compound (such as ampicillin, amoxicillin and piperacillin) with a beta-lactamase inhibitor (such as clavulanic acid, sulbactam, tazobactam) can also prove effective. Betalactamase inhibitors restore activity of the associated compound by acting as suicide inhibitors; the bacterial beta-lactamase-inactivating enzymes have a greater afnity to these beta-lactamase inhibitors than to the parent beta-lactam agents. Thus, the parent beta-lactam agent can run the gauntlet and bind to the PBPs. Resistance to these agents is infrequent, allowing these agents to have good activity against B. fragilis and many other beta-lactamase-producing anaerobes. Beta-lactamase inhibitors may not inactivate carbapenem resistance [26].

4. Antibiotics against anaerobes Antimicrobials are divided into classes based on their chemical structures and derivation. While there are often class characteristics, each individual agent has a unique prole, advantages, and disadvantages. Some antimicrobials, such as monobactams, aminoglycosides, macrolides and sulfamethoxazole-trimethoprim, have no appreciable anti-anaerobe activity. Below we discuss the main compounds with anti-anaerobe activity. The in vitro activity of various antimicrobial agents is summarized in Table 1. While antibiotic therapy is often instituted empirically, once culture data is available, then specic therapy should be instituted. Recently, there has been an updated recommendation regarding therapy for both children and adults [3]. 4.1. Beta-lactams This class includes penicillins, cephalosporins, betalactam-betalactamase inhibitor combinations, and carbapenems. All share a beta-lactam ring as a component of the structure. All are bactericidal, exhibit timedependent killing, and inhibit cell wall synthesis by binding to penicillin binding proteins (PBPs). Each organism has its own PBPs that are numbered, e.g. PBP1,

E.J.C. Goldstein and G.E. Stein / Antimicrobials in anaerobic infections

Escherichia coli
14% 24% 2% 3% 5% 5%

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Enterococcus faecalis Proteus spp. Klebsiella spp Enterobacter spp Pseudomonas aeruginosa Staphylococcus aureus Other streptococci Other aerobes

Aerobes 48%

8% 24% 15%

Anaerobes 52%
9% 4%

Bacteroides fragilis
6%

Other Bacteroides species Clostridia

38%

5% 5%

Peptostreptococci Fusobacteria Peptococci

10%

Propionibacteria
11% 12%

Veillonella Other

Fig. 2. Distribution of aerobic and anaerobic bacteria isolated from intra-abdominal infections (Adapted from refs 1519).

4.1.3. Cephalosporins Cephalosporins are bicyclic agents with a betalactam ring that is associated with a six-membered dihydrothiazine ring. Cephalosporins have differences of activity that are due to various substitutions on the C-3 and C-7 position, as well as a hydroxyl-methyl on the C-6 position. Bacterial cephalinosporinases of the 2e class inactivate most cephalosporins against anaerobes. First generation cephalosporins (such as cefazolin) and third generation cephalosporins (such as cefotaxime, ceftizoxime and cefepime) do not have reliable anaerobic activity especially against B. fragilis group species. The second generation cephalosporin cefoxitin, a cephamycin due to its methoxy group on the C-6 position, has activity against anaerobes, including B. fragilis. However, resistance to cefoxitin has become fairly widespread, relegating this drug to second line therapy. Each cephalosporin may have either a class or a specic individual agent enzyme that will inactive it, such as the cefoxitin-inactivating enzymes that occur in many B. fragilis group species [29].

4.1.4. Carbapenems Carbapenems, such as ertapenem, meropenem, imipenem and doripenem, are semi-synthetic bicyclic structures with a four-membered beta-lactam ring associated with a ve-membered carbapenem ring. They are stable against many beta-lactamases, including those of most anaerobes. Carbapenems are grouped by classes on the basis of their aerobic activity. Class 1 agents include ertapenem and are active against most aerobic Gram-positives (except for enterococci) and fermentative Gram-negative aerobes but not Acinetobacter spp. and P. aeruginosa. Class 2 agents include doripenem, imipenem and meropenem and also have activity against enterococci, Acinetobacter spp. and P. aeruginosa. Class 3 agents, not yet clinically available, have activity against MRSA. Rarely, anaerobic isolates are resistant to carbapenems owing to efux mechanisms or zinc metallo-beta-lactamases. The zinc carbapenemases are encoded by either ccrA or cA genes of B. fragilis group [30] and inactivate all -lactam antibiotics. Beta-lactamase inhibitors do not inactivate carbapenemases. These enzymes are expressed in <

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E.J.C. Goldstein and G.E. Stein / Antimicrobials in anaerobic infections Table 1 Antimicrobial susceptibility patterns for anaerobic bacteria

Drugs Moxioxacin Clindamycin Metronidazole Penicillin Beta-lactamase Cefoxitin Tigecycline Carbapenems inhibitors Bacteroides fragilis ++ + + ++ + V ++ Bacteroides thetaiotaomicron ++ V + + V V ++ Other Bacteroides fragilis group ++ V + + + V ++ Prevotella species V ++ + ++ + + ++ Fusobacterium nucleatum V ++ + ++ V + ++ Fusobacterium necrophorum + ++ + ++ V + ++ Porphyromonas species + ++ + ++ + + ++ Peptostreptococci + ++ + ++ V + Propionibacterium acnes + + + ++ + + V Veillonella + + + ++ + + ++ Actinomyces + + + ++ + + Clostridia + + V + + V V +
Based

Bacteria

on sundry in vitro susceptibility studies from various laboratories and using various source clinical isolates. inhibitor--lactam combination (e.g., ampicillin/sulbactam, amoxicillin clavulanate, piperacillin/tazobactam). Carbapenems: Imipenem, meropenem, ertapenem, doripenem. + = Susceptible; = Resistant; V = Variable activity.
-Lactamase

1% of B. fragilis isolates in the United States, but may be present in up to 3% of Bacteroides strains, which can carry ccrA or cA. A study of 15 strains of imipenemresistant, cifA-positive B. fragilis from the US, Hungary and Kuwait suggested that more than one mechanism of activation exists [31]. 4.2. Clindamycin Clindamycin is a bacteriostatic agent that inhibits protein synthesis by binding to the 50S ribosome. Its activity against anaerobes is variable and varies geographically. Approximately 30% of B. fragilis and Bacteroides thetaiotaomicron species are resistant [22,32] to clindamycin, although most oral anaerobes remain susceptible. Intra-abdominal pediatric B. thetaiotaomicron isolates have shown 80% clindamycin resistance in one small study [33]. Side effects of clindamycin include Clostridium difcile associated diarrhea [34]. 4.3. Tetracyclines Tetracyclines are bacteriostatic agents that bind to the 30S ribosome and have a broad spectrum of activity. Doxycycline and minocycline are more active than tetracycline against aerobic bacteria, but have limited anti-anaerobic activity. Tigecycline, a parenteral glyclcycline that is derived from minocycline, exhibits a broad spectrum of activity against anaerobic bacteria, including B. fragilis [35]. It is not affected by beta-lactamases and is primarily excreted in the feces as unchanged drug. This class of agent is not used in children whose teeth are still growing [36].

4.4. Vancomycin Vancomycin is the prototype glycopeptide antibiotics and inhibits the synthesis of peptidogylcans for the cell wall. Vancomycin is active against most Grampositive anaerobes. An oral formulation is useful for the treatment of C. difcile diarrhea, but acts locally and is not absorbed systemically [34]. 4.5. Daptomycin Daptomycin is a novel lipopeptide agent that is active against some Gram-positive aerobic and anaerobic bacteria, such as Clostridium species, Propionibacterium species, Eubacterium species and peptostreptococci. However, daptomycin has limited activity against lactobacilli, and is inactive against Gram-negative bacteria (as lipopeptides cannot cross the membranes of Gramnegative bacteria). It has calcium-dependent binding to the cell membrane, causing depolarization and disruption of the membrane, and is rapidly bactericidal [37]. 4.6. Oxazolidinones These are synthetic, heterocyclic agents with a ve membered ring that includes a nitrogen and an oxygen molecule, and are bridged with a carbonyl group. The only available oxazolidinone, linezolid, is active against Gram-positive anaerobes and has shown some Gram-negative anti-anaerobic activity [38].

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4.7. Nitroimidazoles Metronidazole, tinidazole, and ornidazole are the members of the Nitroimidazole class of agents. They are active against almost all anaerobes, except those that are microaerophilic (such as Actinomyces and P. acnes). To date, only ve clinical cases of B. fragilis resistance to metronidazole have been reported [26]. Nitroimidazoles have no aerobic activity and are used in combination with agents that are active against common aerobic pathogens [2,3]. 4.8. Fluoroquinolones These are synthetic agents with bicyclic, heteroaromatic rings that bind to topoisomerase II (also known as DNA gyrase) and thereby inhibits DNA replication. These agents are generally not used in children unless a resistant Gram-negative rod such as Serratia or Pseudomonas is present and there are no other therapeutic options [3,39]. Several of the newer uoroquinolone agents, such as gemioxacin and moxioxacin, also inactivate topoisomerase IV, which enhances their activity against Gram-positive organisms. Substitutions on the 8 position improved anaerobic activity and pharmacokinetic properties as well [40,41]. Moxioxacin has improved activity against anaerobic Gram-positive and Gram-negative organisms, but the development of resistance, especially by B. fragilis group species and peptostreptococci is of concern [4, 26,35,41]. Fluoroquinolone resistance among Bacteroides spp. has been attributed to either a mutation in the quinolone resistance-determining region of the gyrase A gene (gyrA), or from an efux pump mechanism [4244]. Moxioxacin was recently approved for therapy against mixed aerobic and anaerobic intraabdominal, skin and skin-structure infections in adults but not children [45]. 4.9. Chloramphenicol Chloramphenicol is an inexpensive synthetic agent with broad-spectrum aerobic and anaerobic activity. It is active against most Gram-positive and Gramnegative anaerobes, including B. fragilis group species. However, its minimal inhibitory concentrations (MICs) often cluster near the MIC breakpoint. It is also active against Rickettsia, Mycoplasma and Chlamydia, but is not active against Enterobacter and Pseudomonas species. While not frequently used in the developed world due to potential marrow suppression and aplastic

anemia, it is widely used in the developing world because it is cheap. A bacteriostatic agent inhibits protein synthesis by directly inhibiting the 50S ribosomal subunit and preventing peptide bond formation. Resistance may occur by reduced membrane permeability, ribosomal mutation or enzymatic inactivation by an acetyltransferase. Plasmid-mediated chloramphenicol resistance may also encode for multidrug resistance [26].

5. Antibiotic pharmacodynamics The MIC has long been used clinically for choosing an antimicrobial, although several limitations of this laboratory method exist [46]. The MIC is based on static drug concentrations in serum and does not represent free drug levels at the site of infection. Moreover, the infection inoculum and pH may not be the same as those that are used in the laboratory when determining the MIC for an antibiotic. Pharmacodynamics, on the other hand, describes the relationship between antibiotic concentrations in serum or at the infection site and biological effect [47]. Pharmacodynamic parameters integrate both pharmacokinetic and MIC data (Fig. 3). Such parameters include the time (t) for which antibiotic concentration remains above the MIC (t > MIC), the ratio between the peak (C max ) concentration and MIC (Cmax /MIC); and the ratio between the area under the serum concentration-time curve (AUC) and the MIC (AUC/MIC) [48]. To optimize antibiotic activity, it is important to know whether the drug kills in a concentration-dependent or independent fashion. Concentration-dependent (or time-independent) antibiotics kill at a greater rate and to a greater extent with increasing antibiotic concentrations, whereas concentration-independent (time-dependent) agents kill bacteria at the same rate and to the same extent once an appropriate antibiotic concentration has been achieved. There are very few differences between these considerations in adults and children [49,50]. 5.1. -lactam antibiotics lactams primarily exhibit concentration-independent activity, and the focus has been on T > MIC as a predictive parameter. Most pharmacodynamic studies have found that a continuous infusion of -lactams is at least as effective as more conventional intermittent dosing, and that very high doses do not increase efcacy. Recommendations suggest that serum levels should

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Fig. 3. Pharmacokinetics of antimicrobial dosing relative to the minimal inhibitory concentration (MIC) of an organism. This diagram shows the concentration of an antimicrobial given in intermittent bolus dosing (y axis) which reaches a C max or peak level after attaining the volume of distribution in the body and then declines over time (x axis). The MIC is the minimal inhibitory concentration that a drug needs to achieve to inhibit bacterial growth (this does not mean that it kills the organism but inhibits its growth in an in vitro system). There are two different ways in which antimicrobials are thought to be effective in inhibiting bacterial grow. One scenario is concentration dependent killing in which the higher the C max compared to the MIC means increased and enhanced killing of a microorganism. Fluoroquinolones and aminoglycosides kill by this method. The other scenario is time dependent killing as occurs with beta-lactam agents such as penicllins, cephalosporins and carbapenems. Time dependent killing suggest that killing occurs during the time the drug concentration is higher than the MIC of the bacteria. For anaerobic bacteria it is thought that if the time the drug concentration is greater than the MIC (minimum >50% of the time) is associated with effective killing. Some feel that it is the size of the area under the curve compared to the MIC that is important in killing efcacy. These parameters are studied for new drugs in order to help predict efcacy and efcient dosing regimens and schedules.

exceed the MIC by 24 multiples for between 30% and 100% of the dosage interval [51]. For -lactams that are highly protein bound (> 90%), the time free drug is kept above the MIC should be maximized. Recent pharmacodynamic studies have shown that a prolonged infusion (3 hr) can improve bactericidal exposure compared to 30-min infusions and is comparable to continuous infusion strategies [52]. 5.2. Fluoroquinolones The pharmacodynamic measure that best correlates with bactericidal activity of uoroquinolones is the AUC/MIC ratio. In contrast to studies with aerobic bacteria, time-kill experiments found that low (AUC/MIC > 11) serum levels of uoroquinolones can produce a signicant killing against anaerobes [53]. In ex vivo studies, both high-dose levooxacin (750 mg) and moxioxacin (400 mg) have exhibited prolonged serum bactericidal activity against anaerobes such as C. perfringens, B. fragilis, Fusobacterium nucleatum, Peptostreptococcus magnus, and Prevotella melaninogenica [53]. In experimental intraabdominal abscesses caused by B. fragilis and E. coli, uoroquinolones (such as trovaoxacin) were able to signicantly reduce colonies of B. fragilis and produce survival rates comparable to those resulting from treatment with clindamycin plus gentamicin [54].

5.3. Clindamycin Clindamycin exhibits concentration-independent bactericidal characteristics against anaerobic bacteria [55]. Time-kill data suggests that the antimicrobial activity of clindamycin is maximized near the MIC of the organism. Therefore, the goal of clindamycin dosing should be to achieve a drug concentration at the site of infection that is close to the MIC for an extended period [56]. 5.4. Metronidazole This antibiotic provides concentration-dependent killing and has a long half-life, which enables manipulation of metronidazole dosing regimens. In time-kill experiments, metronidazole reduced colony counts of anaerobic bacteria at least 3 log cfu/mL regardless of whether dosed thrice daily or once daily [57]. The rates of killing were also similar for a given isolate-dosing regimen combination. In an ex vivo study in healthy adult subjects, different serum levels of metronidazole were found to have similar bactericidal activity against various anaerobes [58]. 5.5. Tigecycline Similar to tetracyclines, tigecycline has demonstrated time-dependent killing. In animal models, the ratio of AUC/MIC is the index that best correlates to bacterial eradication [59]. This agent is not indicated for children.

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6. Selecting an appropriate empirical therapy To select an empirical therapy, doctors must select the agent/s that is/are active against the most likely pathogens to be encountered at a site of infection. Knowledge of the usual ora at the location of infection is an indispensable guide in selecting and instituting empirical antimicrobial therapy [8]. There are various clinical clues to the involvement of anaerobes in a clinical infection [60], which include infection in proximity of a mucous membrane surface where anaerobes are predominant normal ora, or oral ora contaminated wounds such as human or animal bites, foul odor to a discharge or wound, crepitus or gas in tissues, infections associated with necrotic tissue, including tumors and Gram stain with many organisms, which do not grow on routine aerobic cultures. Once it is clear that anaerobes are involved, and prior to instituting antibiotic therapy, cultures should be collected (pus and aspirates of pus are best, swabs are subject to sampling error) from an acceptable specimen source (specimens contaminated by normal ora, such as sputum, are not acceptable) and stored in a non-nutritive transport media. These samples should be analyzed, and the empirical antibiotics that have been selected should be adjusted to cover the specic pathogens isolated once this information is clinically available. Just as in aerobic bacteriology obtaining specic samples for culture is the basis to guide therapy; this is equally true for anaerobic infections [2,3]. Empirical selection of an antibiotic should take into account local patterns of susceptibility and resistance. Patterns of resistance will vary by country and by city, and even by hospital [22,61]. For anaerobic bacteria, clinicians must often rely on published studies, as many laboratories do not perform anaerobic susceptibility testing. It is of paramount importance that doctors know what antibiotics a patient has recently taken. If an agent has failed to treat a specic infection prior to the visit, it is unwise to continue it and another agent or class of agents should be used.

Recommendations by Clinical and Laboratory Standards Institute [65] and the American Society for Microbiology [66] suggest that hospitals should test individual patient isolates when: 1) it is critical for disease management, 2) for long-term therapy, 3) isolates were recovered from the blood, brain, bone, or joint, 4) when therapy has failed and 5) when organisms with unpredictable susceptibility patterns are suspected. They also suggest that hospitals should periodically perform surveys for inclusion in the hospital antibiogram for the B. fragilis group member species, Prevotella spp., Fusobacterium spp., and Clostridium spp. The antimicrobial agents that are tested against clinical isolates should be those, which are available to the clinician; since there may be restricted access to specic antibiotics on the hospitals formulary, or patients health plan, knowing the susceptibility of the isolate to available agents will help clinicians in their selection of empirical therapy [2,3]. A recent survey showed that 76% of hospital labs now perform some anaerobic bacteriology, such as blood, sterile site and wound cultures [64]. However, only 21% of hospital labs perform anaerobic susceptibility testing in house [64]. These rates have declined from 70% in 1990 [62] and 33% in 1993 [63]. When susceptibility testing was done, the selection of the antimicrobials tested was dependent on the agents from commercial panels that were available to laboratories. No hospital lab tested for tigecycline, doxycycline, moxioxacin or ertapenem susceptibility. The most frequently tested antimicrobial against anaerobes is metronidazole (16%); agents with less frequent testing included penicillin/ ampicillin (15%), clindamycin (15%), piperacillin/tazobactam (7%), cefoxitin (9%), imipenem (8%), and chloramphenicol (5%) [55]. Since specic data is limited, one must assume that clinicians must be using information from the manufacturer, the Food and Drug Administration indications, published study/survey data or just guessing at appropriate empirical as well as directed therapy. Consequently, wrong guesses may lead to adverse outcomes A variety of susceptibility testing methods is available: a. Etest (AB Biodisk, Solna, Sweden) [67], a plastic strip with an interpretative MIC scale on one side and predetermined antimicrobial concentration gradient on the other side and which is suitable for performing tests on individual isolates. b. Microbroth dilution method (reserved only for the B. fragilis group). This is commercially available.

7. The limitations of susceptibility testing Limitations of susceptibility testing occur because clinical laboratories vary in their capabilities and interest in the isolation and identication of anaerobes and in their performance of anaerobic susceptibility testing [6267].

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c. Agar dilution method, which is reference method but is cumbersome and expensive and therefore done only by research centers. d. Beta-lactamase testing provides very limited data, but can be useful if penicillin therapy is being considered. e. Broth disk elution and disk diffusion tests should not be performed for anaerobic bacteria, as their susceptibility results do not correlate with the agar dilution reference method [65,66]. Quality control strains that should be used for anaerobic bacteria are B. fragilis ATCC 25285 and B. thetaiotaomicron ATCC 29741 and Eubacterium lentum ATCC 43055 limited to use with the agar dilution or ETest methods [65,66].

8. Susceptibility surveys Since most clinicians do not have access to specic isolate susceptibility data for their patients, published surveys may act as an imperfect guide for empirical antimicrobial selection. Only a few reference labs scattered throughout the world do these surveys. It is important to note the susceptibility results may be different and vary greatly depending on the clinical source of the isolates (that is, intra-abdominal, skin and soft tissues, blood and so on), the year of isolation, technical and methodological factors, isolate distribution, and differences in geographic patterns [22,33,41,68]. A recent US survey by Snydman et al. [32] examined 5.225 B. fragilis group isolates (clinical sources not reported) from 10 geographically representative medical centers from 1997 to 2004. The survey reported that B. fragilis accounted for 52% of isolates, B. thetaiotaomicron for 18.7%, Bacteroides ovatus for 10.4% and each of the other species (B. Bacteroides distasonis, Bacteroides uniformis, Bacteroides vulgatus and other species) for 36% [32]. B. fragilis was generally the most susceptible to antimicrobials, and approximately 1% were resistant to carbapenems, 1.7% to ampicillinsulbactam, 0.4% to piperacillin tazobactam, 5% to both cefoxitin, and tigecycline. Clindamycin resistance was found in 20% of B. fragilis isolates, and moxioxacin was found in 27% of isolates. By contrast, a study by Goldstein et al. [4] of 923 intra-abdominal isolates found 13% resistance to moxioxacin (with MICs of 2 ug/mL). Overall, Snydman et al. [32] reported that carbapenems, piperacillin-tazobactam, metronidazole and

chloramphenicol (MIC < 16 ug/mL) were active against all isolates. Only one metronidazole-resistant isolate from 2002 was found. B. thetaiotaomicron isolates were very susceptible to carbapenems and piperacillin-tazobactam (99.6%), ampicillin-sulbactam (97.9%) and tigecycline (96.4%). Susceptibility to other agents varied as follows: cefoxitin, 83.2%; clindamycin, 66.7%; and moxioxacin 73.6%. Again, Goldstein et al. [4] reported a different susceptibility to moxioxacin: 85.5% susceptible at 2 ug/mL. B. distasonis isolates were generally less susceptible to ampicillin-sulbactam (83.2%), cefoxitin (70.1%) and clindamycin (70.8%). Clindamycin resistance (MIC > 8 ug/mL) was frequent (19.3% to 35.3%), precluding its empirical use against most species. These disparities between survey by Snydman [32] and by Goldstein [4] demonstrate that caution must be observed in interpreting any one surveys results; local and specic data can be very useful. A paucity of recent data exists about pediatric intra-abdominal isolates and their susceptibilities.

9. How to select an anti-anaerobic agent The selection of an anti-anaerobic agent for both empirical and specic therapy is complicated by the frequent lack of local data to predicate empirical selection. As many labs do not perform anaerobic susceptibility testing (and when performed these results are often delayed), therapy is maintained and evaluated by clinical response. In part, the severity of the infection and anatomical location will dictate appropriate choices, especially empirically. There is no single agent that can be assigned as an answer to the question of what is the best therapy for . . . ? as resistance has occurred to all available agents [69]. Reliable broad-spectrum activity for almost all isolates can be achieved individually with the carbapenems, piperacillin-tazobactam and metronidazole (in combination). Ampicillin-sulbactam and amoxicillin clavulanate are also very active against anaerobes, but local patterns of E. coli resistance often limit their use. Chloramphenicol is also generally very active, but potential side effects limit its use. Publication of its pivotal studies and further surveillance data will indicate whether tigecycline can be used, but so far, it appears to be an active agent [59]. Agents with good, but variable, activity include ampicillin-sulbactam, cefoxitin and moxioxacin [53] and their use is situation dependent. Clindamycin is active against most respiratory isolates (aspiration pneumonia) and is used

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63

for these situations empirically, but resistance patterns vary geographically for intra-abdominal and diabetic foot infections, and unless the specic susceptibility of cultured isolates are known, these agents should be relegated for specic therapy. Penicillin and ampicillin are unreliable empirical agents owing to the prevalence of beta-lactamase activity of both many oral and intraabdominal anaerobic isolates.

[9]

[10]

[11]

[12]

10. Conclusion
[13]

Anaerobes are involved in various infections that occur in proximity to the mucous membranes, where anaerobes predominate as normal ora. Diagnosis is often based on clinical suspicion, as many laboratories are unable or unwilling (due to perceived cost considerations) to culture for anaerobes. Therapy is often empirical and based presumably on information from published surveys or supplied to clinicians by the pharmaceutical industry. Hopefully, newer technologies such as real-time polymerase chain reaction, microarray assays and uorescent techniques, will allow for more rapid diagnosis and timely susceptibility test results. These would aide clinicians in the decision of which antimicrobial to use and would result in better therapeutic outcomes.

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[15]

[16]

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