Sydney M. Finegold

Lessons Learned from the Anaerobe Survey: Historical Perspective and Review of the Most Recent Data (2005–2007)

BACKGROUNDnThe rationale and lessons learned through the evolution of the National Survey for the Susceptibility of Bacteroides fragilis Group from its initiation in 1981 through 2007 are reviewed here. The survey was conceived in 1980 to track emerging antimicrobial resistance in Bacteroides species.nnnMETHODSnData from the last 11 years of the survey (1997-2007), including 6574 isolates from 13 medical centers, were analyzed for in vitro antimicrobial resistance to both frequently used and newly developed anti-anaerobic agents. The minimum inhibitory concentrations of the antibiotics were determined using agar dilution in accordance with Clinical and Laboratory Standards Institute recommendations.nnnRESULTSnThe analyses revealed that the carbapenems (imipenem, meropenem, ertapenem, and doripenem) and piperacillin-tazobactam were the most active agents against these pathogens, with resistance rates of 0.9%-2.3%. In the most recent 3 years of the survey (2005-2007), resistance to some agents was shown to depend on the species, such as ampicillin-sulbactam against Bacteroides distasonis (20.6%) and tigecycline against Bacteroides uniformis and Bacteroides eggerthii ( approximately 7%). Very high resistance rates (>50%) were noted for moxifloxacin and trovafloxacin, particularly against Bacteroides vulgatus. During that period of study, non-B. fragilis Bacteroides species had >40% resistance to clindamycin. Metronidazole-resistant Bacteroides strains were also first reported during that period.nnnCONCLUSIONSnIn summary, resistance to antibiotics was greater among non-B. fragilis Bacteroides species than among B. fragilis and was especially greater among species with a low frequency of isolation, such as Bacteroides caccae and B. uniformis. The emergence of resistance among the non-B. fragilis Bacteroides species underscores the need for speciation of B. fragilis group isolates and for clinicians to be aware of associations between species and drug resistance.

Toxigenicity and antimicrobial susceptibility ofClostridium difficile, a cause of antimicrobial agent-associated colitis

Fourteen of 16 strains ofClostridium difficile, a recently recognized cause of antimicrobial agent-associated ileocolitis in laboratory animals and colitis in man, were found to be toxigenic. The susceptibility of these isolates to a variety of antimicrobial agents provides information that may be of value in assessing the means by whichC. difficile may produce colitis, in developing selective bacteriologic media for diagnosis, and in selecting appropriate antimicrobial therapy for colitis.

Comparative In Vitro Activity of New Beta-Lactam Antibiotics Against Anaerobic Bacteria

Several new beta-lactam antimicrobial agents have been introduced in the last few years. In this investigation, the in vitro activities of several recently introduced cephalosporins (cefoperazone, cefotaxime, ceftazidime, and ceftizoxime), moxalactam, and N-formimidoyl thienamycin were compared with those of cefoxitin, clindamycin, and metronidazole against 203 strains of anaerobic bacteria. At achievable serum levels, all of the antimicrobial agents were active against essentially 100% of the strains of anaerobic gram-positive cocci, Clostridium perfringens, Leptotrichia buccalis, and species of Selenomonas, Veillonella, and Eubacterium. Clindamycin, metronidazole, and N-formimidoyl thienamycin were the most active agents against the Bacteroides fragilis group, inhibiting all strains at concentrations which can be achieved in serum. Of the remaining agents tested against the B. fragilis group, cefoxitin (which required 64 μg/ml to inhibit 90% of the strains) was the most active, followed by cefoperazone (128 μg/ml), cefotaxime (128 μg/ml), moxalactam (128 μg/ml), ceftizoxime (256 μg/ml), and ceftazidime (>256 μg/ml). Important differences in cephalosporin susceptibility among species of the B. fragilis group were observed. Metronidazole and N-formimidoyl thienamycin were the most active drugs against species of clostridia other than C. perfringens; the other antibiotics displayed poor activity, although this is partly due to inclusion of a relatively large number of strains of Clostridium difficile which were very resistant to several of the cephalosporins. Only metronidazole was active against all species of Fusobacterium. Clindamycin and N-formimidoyl thienamycin displayed excellent activity against gram-positive, non-spore-forming bacilli, requiring ≤8 μg/ml to inhibit 100% of the strains. Ceftazidime, cefoperazone, and moxalactam were bactericidal for 25 strains of B. fragilis at concentrations equal or close to those required for inhibition. On the basis of its activity in vitro, N-formimidoyl thienamycin appears to be the most promising of the new beta-lactam antibiotics for the treatment of infections involving anaerobic bacteria.

State of the art; microbiology in health and disease. Intestinal bacterial flora in autism

Autism of the regressive variety is selected as an example of the importance of intestinal bacterial microflora in disease other than classical infection. Our studies have indicated that intestinal bacteria play a role in this disease since it responds to oral vancomycin, a drug that is not absorbed from the GI tract. Pyrosequencing studies document an abnormal gut microflora in regressive autism subjects as compared to controls. Finally, we present preliminary evidence suggesting that Desulfovibrio may play a key role in this disease.

Aerobic and anaerobic bacteriology of acute otitis media in children

Tympanocentesis and aerobic and anaerobic cultivation of the middle-ear fluid obtained was performed through one or both tympanic membranes of 62 children with acute otitis media. Aerobic bacteria alone, predominantely pneumococcus and Hemophilus influenzae, were isolated from 57% of patients; anaerobic organisms alone, most commonly Propionibacterium acnes and Peptococcus, from 15%. Thirteen percent yielded mixtures of aerobes and anaerobes, and several had multiple aerobic or anaerobic agents. The isolation of only anaerobic bacteria, supported in some patients by gram-stain observations of middle-ear fluid, suggest that these bacteria, especially Petococcus, may occasionally play a direct role in acute otitis media.

Lower Respiratory Tract Infection

The most important lower respiratory infection is pneumonia, the fourth leading cause of death. Most cases of bronchitis are of viral etiology and are not major problems. Empyema can present an important problem in management. Although the diagnosis of pneumonia is usually relatively straightforward, the specific etiologic diagnosis remains a major problem. Availability of empyema fluid or a positive blood culture result can be helpful in making the etiologic diagnosis, but these are unavailable in most patients. Screening of sputum Gram stains under 100 X magnification is very important; there should be fewer than 10 squamous epithelial cells, more than 25 polymorphonuclear leukocytes, or both per field of this size. The major causes of pneumonia are Streptococcus pneumoniae, Mycoplasma pneumoniae, anaerobic bacteria, Staphylococcus aureus, various gram-negative aerobic or facultative bacilli and Legionella. However, many other organisms are capable of causing pneumonia, even in the immunocompetent host. Further adding to the problem is the fact that a number of different organisms are manifesting increasing resistance to antimicrobial agents. Our study with ticarcillin plus clavulanic acid included seven patients with pneumonia, one with empyema, and one with purulent tracheobronchitis. Organisms recovered from pleural fluid, transtracheal aspiration and sputum or tracheostomy aspirate included multiple anaerobes, pneumococci, S. aureus, Hemophilus influenzae, Klebsiella pneumoniae, K. ozaenae, Pseudomonas aeruginosa, Acinetobacter, Enterobacter cloacae, Proteus mirabilis, beta-hemolytic streptococci, Neisseria meningitidis and Branhamella catarrhalis. Several of the organisms were ticarcillin resistant. Eight of the patients had cures and the other patient showed improvement. Only minor side-effects were encountered--Coombs positivity (without hemolysis), eosinophilia, drug fever and one case of questionable neutropenia.

Effect of beta-lactamase inhibitors on the activities of various beta-lactam agents against anaerobic bacteria.

The in vitro activities of several new beta-lactam-beta-lactamase inhibitor combinations (piperacillin plus tazobactam, ceftizoxime and cefonicid with sulbactam and clavulanic acid, and ampicillin plus 8 micrograms of sulbactam per ml) were tested with anaerobic bacteria and compared with known beta-lactam-beta-lactamase inhibitor combinations and other potent antianaerobe agents. All the combinations tested (except for the cefonicid-inhibitor combinations) were active against almost all strains of the Bacteroides fragilis group. This report indicates that beta-lactamase inhibitors may improve the activity of beta-lactam agents with marginal activity against the B. fragilis group.

Impact of imipenem/cilastatin therapy on normal fecal flora

The impact of parenteral imipenem/cilastatin therapy on the bowel flora of six patients was evaluated. Stool samples were collected before and during therapy and qualitative and quantitative bacteriologic studies were performed. Imipenem had no effect on total microorganism counts. Two patients acquired Candida albicans during therapy, and three patients acquired Proteus species. Pseudomonas species in one patient acquired resistance. Imipenem appears to have a relatively modest effect on the bowel flora and apparently does not readily induce resistance in the resident flora as compared with other agents.

Transformation of 4-androsten-3,17-dione by growing cultures and cell extracts of Clostridium paraputrificum

Growing cultures of Clostridium paraputrificum transformed 4-androsten-3,17-dione to 3 alpha-hydroxy-5 beta-androstan-17-one in a sequential manner with 5 beta-androstan-3,17-dione as an intermediate. The addition of 1.5 mM menadione to log-phase cultures which had formed 5 beta-androstan-3,17-dione resulted in a partial reoxidation of this steroid to 4-androsten-3,17-dione. However, this treatment also resulted in transient inhibition of culture growth. Resumption of growth was accompanied by complete reduction of 4-androsten-3,17-dione to 5 beta-androstan-3,17-dione. Cell extracts of C. paraputrificum were capable of carrying out these reductive transformations in the absence of added cofactors. However, Sephadex G-25 treated extracts required NADH or NADPH for these reactions. A flavin nucleotide, either FAD (plus NADH or NADPH) or FMN (plus NADH) was highly stimulatory for 4-androsten-3,17-dione reduction to 5 beta-androstan-3,17-dione. NADH was the preferred reduced pyridine nucleotide for reduction of the C4-C5 double bond, while time-course measurements suggested that NADPH was the preferred donor for reduction of the 3-keto group.

Susceptibility testing of anaerobic bacteria

With the significant increase in resistance of anaerobic bacteria to antimicrobial agents in recent years, susceptibility testing of these organisms becomes very important. In addition to survey studies, hospitals should be determining their local patterns, and therapy of at least the more serious infections should be guided by susceptibility tests. Of the various tests available, those most suitable for smaller hospitals for guidance of patient management are the broth disc elution procedure and the micro broth dilution tray test.