Multicenter survey of the changing in vitro antimicrobial susceptibilities of clinical isolates of Bacteroides fragilis group, Prevotella, Fusobacterium, Porphyromonas, and Peptostreptococcus species.

In vitro surveys of antimicrobial resistance among clinically important anaerobes are an important source of information that can be used for clinical decisions in the choice of empiric antimicrobial therapy. This study surveyed the susceptibilities of 556 clinical anaerobic isolates from four large medical centers using a broth microdilution method. Piperacillin-tazobactam was the only antimicrobial agent to which all the isolates were susceptible. Similarly, imipenem, meropenem, and metronidazole were highly active (resistance, <0.5%), whereas the lowest susceptibility rates were noted for penicillin G, ciprofloxacin, and clindamycin. For most antibiotics, blood isolates were less susceptible than isolates from intra-abdominal, obstetric-gynecologic, and other sources. All isolates of the Bacteroides fragilis group were susceptible to piperacillin-tazobactam and metronidazole, while resistance to imipenem and meropenem was low (<2%). For these same isolates, resistance rates (intermediate and resistant MICs) to ampicillin-sulbactam, cefoxitin, trovafloxacin, and clindamycin were 11, 8, 7, and 29%, respectively. Among the individual species of the B. fragilis group, the highest resistance rates were noted among the following organism-drug combinations: for clindamycin, Bacteroides distasonis and Bacteroides ovatus; for cefoxitin, Bacteroides thetaiotaomicron, B. distasonis, and Bacteroides uniformis; for ampicillin-sulbactam, B. distasonis, B. ovatus, and B. uniformis; and for trovafloxacin, Bacteroides vulgatus. For the carbapenens, imipenem resistance was noted among B. fragilis and meropenem resistance was seen among B. fragilis, B. vulgatus, and B. uniformis. With few exceptions all antimicrobial agents were highly active against isolates of Prevotella, Fusobacterium, Porphyromonas, and Peptostreptococcus. These data further establish and confirm that clinically important anaerobes can vary widely in their antimicrobial susceptibilities. Fortunately most antimicrobial agents were active against the test isolates. However, concern is warranted for what appears to be a significant increases in resistance to ampicillin-sulbactam and clindamycin.

A multicenter study of the prevalence and susceptibility patterns of isolates of Streptococcus pneumoniae with reduced susceptibility to penicillin G in Louisiana.

Because of increasing reports of multiple-antibiotic-resistant strains of Streptococcus pneumoniae and associated clinical failures, this study was performed to determine the prevalence of multiresistance among strains from nine Louisiana medical centers. Using a National Committee for Laboratory Standards broth microdilution method, 481 strains were tested. Of these, 70% were penicillin-susceptible (PS), 23% had intermediate minimum inhibitory concentration values to penicillin (I), and 7% were fully resistant to penicillin (PR). The isolation rates (15% to 40% for I strains and 0% to 33% for PR strains) at the various medical centers varied appreciably. The prevalence of penicillin resistance was highest among upper respiratory isolates, while cross-resistance to other antimicrobials varied. The least cross-resistance was noted among PS strains. However, strains with reduced penicillin susceptibility had high levels of cross-resistance. Among I strains, the prevalence of cross-resistance (%) was noted for amoxicillin/clavulanate (6%), cefuroxime (71%), cefaclor (91%), ceftriaxone (13%), cefotaxime (34%), erythromycin (67%), azithromycin (32%), and clarithromycin (32%). For PR strains, the prevalence of cross-resistance was 97% for amoxicillin/clavulanate, cefuroxime, and cefaclor; 67% for ceftriaxone and erythromycin; 89% for cefotaxime; and 69% for azithromycin and clarithromycin. These data emphasize the high prevalence of multiple-antimicrobial-resistance among strains of S pneumoniae with reduced penicillin susceptibility in this geographic area.

Comparison of the in vitro activities of Bay 12-8039, a new quinolone, and other antimicrobials against clinically important anaerobes.

Bay 12-8039, a new 8-methoxy quinolone, was compared with other agents for activity against clinically relevant anaerobes. Bay 12-8039 inhibited 91 and 96% of the 410 test isolates at 2 and 4 micrograms/ml, respectively. Bay 12-8039 had activity comparable to that metronidazole and overall was at least 16-fold more active than ciprofloxacin, ofloxacin, and cefoxitin, 32-fold more active than cefotetan, and at least 128-fold more active than penicillin G.

A comparison of susceptibility results of the Bacteroides fragilis group and other anaerobes by traditional MIC results and statistical methods.

Antimicrobial susceptibility is compared by MIC values reflecting activity by weight and/or percentage of strains resistant. This study establishes these parameters in vitro in the traditional fashion and extends analysis statistically for various groups of anaerobic bacteria. Mode MIC, MIC50, MIC90 values and percentage of strains resistant indicated comparable activity for most of the beta-lactams. Geometric and arithmetic MICs suggested ceftizoxime to be the most active beta-lactam. Statistical analysis of all log2 values showed that metronidazole was the most active followed by clindamycin and that ceftizoxime was significantly more active than cefotaxime, piperacillin, ceftriaxone or cefoxitin.

Comparative in vitro activities of trovafloxacin (CP 99,219) and other antimicrobials against clinically significant anaerobes.

A total of 590 strains of clinically important anaerobes were tested to determine their susceptibility to trovafloxacin. Overall, trovafloxacin had a mode MIC of 0.25 micrograms/ml and a MIC at which 90% of the isolates were inhibited of 1 micrograms/ml and had activity comparable to that of metronidazole. Trovafloxacin was 8-, 8-, 16-, 32-, and 64-fold more active than ampicillin-sulbactam, clindamycin, ciprofloxacin, cefoxitin, and cefotetan, respectively. Of the Bacteroides fragilis group, 97% of the isolates were inhibited by trovafloxacin at 21 micrograms/ml, and trovafloxacin was more active than ciprofloxacin, cefoxitin, cefotetan, ampicillin-sulbactam, and clindamycin against Clostridium, Fusobacterium, Porphyromonas, and Prevotella strains.

Biomonitoring of possible human exposure to environmental genotoxic chemicals: lessons from a study following the wreck of the oil tanker Braer.

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.

The occurrence, virulence, and antimicrobial resistance of anaerobes in polymicrobial infections.

Polymicrobial aerobic/anaerobic bacterial infections occur frequently and have been documented in most anatomic sites in the body. The etiology of these infections is endogenous in that the normal microbial flora that colonize the various mucosal surfaces of the body can be isolated from these infections after trauma to these membranes allowing these organisms access to normally sterile sites. Subsequently, the organisms begin to proliferate, causing extensive tissue damage. These infections may spread to adjacent tissues or become loculated with abscess formation. In patients judged to have severe infection, surgery is often needed to debride the advancing spread of the infection, remove loculated pus, and reestablish sufficient blood flow to deliver appropriate antimicrobial agents to the infected site. Choice of antimicrobial agents should include agents with activity against both aerobes and anaerobes. Although a variety of anaerobes can be isolated from these infections, the Bacteroides fragilis group is the most clinically important group of anaerobes because of the production of virulence factors and the high incidence of beta-lactamase production. Against the various B. fragilis group species, metronidazole remains a very active agent, whereas resistance rates to clindamycin are high among the non-B. fragilis species. Because of the good activity of many cephalosporin/cephamycin agents against aerobic gram-negative bacteria and moderate to good activity against anaerobes, these compounds remain as broad-spectrum choices for use in therapy of mixed infections. The addition of beta-lactamase inhibitors (tazobactam, sulbactam, or clavulanate) to various beta-lactam agents has increased their antimicrobial spectrum against certain groups of aerobes, and particularly against beta-lactamase-producing anaerobes, including the B. fragilis group. The choice of single-agent therapy of mixed infections is ideally based on local data of susceptibility patterns of the various aerobes and anaerobes involved in these infections.

Cefotaxime in the treatment of staphylococcal infections. Comparison of in vitro and in vivo studies.

Staphylococcus aureus strains are well-established pathogens that may cause mild to serious life-threatening disease. Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, also have a pathogenic role in humans and cause infections primarily associated with prosthetic devices and indwelling catheters, whereas Staphylococcus saprophyticus usually causes urinary tract infections. Cefotaxime is a "third-generation" cephalosporin that is stable to the staphylococcal beta-lactamases. In vitro studies over the last 15 years have shown that this parenteral cephalosporin has remained highly active (MIC90 ranges of < or = 2-8 micrograms/ml) against oxacillin-susceptible staphylococci. Cefotaxime therapy of staphylococcal infections has resulted in clinical cure/improvement rates ranging from 78%-100% and bacteriologic eradication rates ranging from 85%-100% in a wide variety of infections. Contrary to contemporary dogma, this "third-generation" cephalosporin appears to be efficacious against staphylococcal infections from a review of 15 years of clinical experience.

In vitro killing of penicillin-susceptible, -intermediate, and -resistant strains of Streptococcus pneumoniae by cefotaxime, ceftriaxone, and ceftizoxime: a comparison of bactericidal and inhibitory activity with achievable CSF levels.

This study assessed total microbial killing of 30 penicillin-susceptible, -intermediate, and -resistant strains of Streptococcus pneumoniae by cefotaxime, ceftriaxone, and ceftizoxime and compared these values with MICs for each strain against each agent as determined by three different methods/media. The results confirm the appropriateness of recent NCCLS recommendations for MIC interpretive criteria for third generation cephalosporins in which < or = 0.25 microgram/ml = susceptible and > or = 2.0 micrograms/ml = resistant when these agents are used to treat pneumococcal meningitis and data from total microbial killing studies suggests that most isolates with MICs of 0.5 and 1.0 mcg/ml would respond to high dose therapy with all three agents. The study also confirmed the recently described two- to four-fold decrease in activity of ceftizoxime against S. pneumoniae as compared with either cefotaxime or ceftriaxone; but noted that current NCCLS MIC interpretive criteria for the therapy of meningitis remain valid for all three agents. Finally, the study found that MICs determined by the E test or by microdilution broth methods using supplemented Todd Hewitt broth predict susceptibility as well as the NCCLS reference method. The actual selection among these agents for the therapy of pneumococcal meningitis should also consider other parameters including protein binding, age groups of clinical use, maximum potency against all clinically relevant pathogens, and cost.

Comparative antimicrobial activity of piperacillin-tazobactam tested against more than 5000 recent clinical isolates from five medical centers. A reevaluation after five years.

Piperacillin combined with tazobactam at a fixed concentration (4 micrograms/ml) and a ratio (8:1) was tested against 5,029 aerobic isolates and 447 fastidious organisms, including anaerobes. Among the Enterobacteriaceae, > 95% inhibition was shared only by imipenem (99.1% at < or = 4 micrograms/ml), and some newer cephalosporins (95.1% - 99.8% at < or = 8 micrograms/ml), and piperacillin-tazobactam (95.8% at < or = 16/4 micrograms/ml). Piperacillin-tazobactam was the most active agent tested against nonenteric Gram-negative bacilli (93.5% at < or = 8 micrograms/ml). Ampicillin-sulbactam was the most active agent against staphylococci (95.0% at < or = 8 micrograms/ml), followed by imipenem (91.8%), piperacillin-tazobactam (89.3% at < or = 8/4 micrograms/ml), and cefepime (86.2% at < or = 8 micrograms/ml). Against the enterococci, only ampicillin (93.0% at < or = 8 micrograms/ml) with or without sulbactam, piperacillin (91.0% at < or = 16 micrograms/ml) with or without tazobactam, and imipenem (91.0%) had acceptable activity. Piperacillin-tazobactam and imipenem were the most active drugs tested against all aerobic isolates, inhibiting 93.5% of isolates each. Piperacillin-tazobactam inhibited all fastidious isolates tested, including Haemophilus influenzae (MIC90, 0.094/4 micrograms/ml), Moraxella catarrhalis (MIC90, 0.064/4 micrograms/ml), Neisseira gonorrhoeae (MIC90, < or = 0.016/4 micrograms/ml), and Streptococcus pneumoniae (all MICs, < or = 4/4 micrograms/ml). Against the anaerobic isolates, the most broad-spectrum antimicrobial agents tested were imipenem (100.0%), piperacillin-tazobactam (99.5% at < or = 32/4 micrograms/ml), metronidazole (98.4% at < or = 8 micrograms/ml), and ticarcillin-clavulanic acid (95.1% at < or = 32/2 micrograms/ml). These results are nearly identical to a previous study involving the same five medical centers in 1989. Piperacillin-tazobactam appears to remain a highly effective beta-lactamase inhibitor combination with a wide empiric spectrum and potency in teaching hospitals.

Major methodology-dependent discordant susceptibility results for Bacteroides fragilis group isolates but not other anaerobes.

Two standardized susceptibility test methods, a broth microdilution (BMD) and agar dilution (AD) method were performed on a total of 441 clinical isolates of anaerobes with ceftizoxime, cefotaxime, ceftriaxone, cefoxitin, piperacillin, and metronidazole. Against the 339 strains of the Bacteroides fragilis group BMD minimum inhibitory concentration (MIC) values were lower than those from AD testing for all the beta-lactams. Overall for the B. fragilis group and the beta-lactams, the mode MIC values were two- to 64-fold lower, and the MIC50 values two- to eightfold lower. Resistance rates were 11%-28% higher overall with AD results and were higher especially for non-B. fragilis species. For non-Bacteroides anaerobes no major discrepancies were noted for Prevotella species, Peptostreptococcus species, and Viellonella parvula. With Clostridium species and Eubacterium species, some differences were noted with ceftizoxime because of differences in cut-off points. These data illustrate the magnitude of differences in results produced by the two methods using essentially the same test medium for the B. fragilis group. Fortunately, such major discordant results were not widely noted with other groups of anaerobes.

Increased in vitro activity of ceftriaxone by addition of tazobactam against clinical isolates of anaerobes.

A total of 461 clinical strains of anaerobes were tested using a broth microdilution test to determine the activity of the combination of ceftriaxone and tazobactam and other antimicrobials against these isolates. Ceftriaxone was combined with tazobactam in ratios of 1:1, 2:1, 4:1, and 8:1 and twofold dilutions of ceftriaxone in constant concentrations to tazobactam of 2, 4, 8, 16, and 32 micrograms/ml. Against beta-lactamase-producing strains of the Bacteroides fragilis group, B. capillosus, and Prevotella species all combinations of ceftriaxone and tazobactam showed enhanced in vitro activity and were eight- to 2048-fold more active than ceftriaxone alone. By comparison ceftriaxone and tazobactam showed superior or equal activity to ampicillin and sulbactam, piperacillin and tazobactam, amoxicillin and clavulanate, ticarcillin and clavulanate, and metronidazole against these same strains. Against beta-lactamase nonproducing strains of Porphyromonas, Fusobacterium, Clostridium, Eubacterium, Peptostreptococcus, and Veillonella parvula the addition of tazobactam produced no appreciable enhanced ceftriaxone activity. Fixed concentrations of tazobactam at 2 and 4 micrograms/ml appear to be most suitable for susceptibility testing and are within the pharmacologic profile of this inhibitor. Pharmacologic and toxicity studies will be needed to define the role of ceftriaxone and tazobactam in infectious diseases.

Effect of diet and vitamin C on DNA strand breakage in freshly-isolated human white blood cells.

We have measured DNA strand breaks induced by ionising radiation in nucleated cells from freshly isolated whole blood from normal human subjects. Samples were taken after subjects had fasted overnight and again 1 h after they had eaten breakfast in combination with approximately 35 mg/kg vitamin C. Damage was measured by single cell gel electrophoresis (the 'comet' assay), in which DNA single strand breaks generate a comet tail streaming from the nucleus. In repeat experiments on 6 subjects a reduction in DNA damage, as indicated by a highly significant decrease in overall comet length, was observed following vitamin C ingestion, both in the unirradiated control blood samples and in the dose response to ionising radiation damage. In addition, consistent differences in dose response between individual subjects were found. The peak effect was 4 h after intake of food and vitamin C. An effect was also seen with vitamin C alone and after breakfast without additional vitamin C. Protection against strand breakage was also seen in Ficoll-separated mononuclear cells but evidence was not obtained for protection of separated, mitogen stimulated T-lymphocytes either against ionising radiation cell killing in a clonal assay, or against clastogenicity assessed by micronucleus formation following one cell division. Exposure of separated lymphocytes in vitro to vitamin C, at doses greater than 200 microM, did not offer protection but induced strand breakage. Our results raise the possibility that variation in normal diet may not only affect susceptibility to endogenous oxidative damage, but may affect some responses of the individual to radiation.

Increased activity of a new chlorofluoroquinolone, BAY y 3118, compared with activities of ciprofloxacin, sparfloxacin, and other antimicrobial agents against anaerobic bacteria.

A total of 435 clinical isolates of anaerobes were tested with a broth microdilution method to determine the activity of BAY y 3118 compared with those of other agents against anaerobic bacteria. All strains of Bacteroides capillosus, Prevotella spp., Porphyromonas spp., Fusobacterium spp., Clostridium spp., Eubacterium spp., Peptostreptococcus spp., and Veillonella parvula were susceptible (MICs of < or = 2 micrograms/ml) to BAY y 3118. Against the 315 strains of the Bacteroides fragilis group, five strains required elevated MICs (> or = 4 micrograms/ml) of BAY y 3118. Only imipenem and metronidazole were active against all anaerobes. Overall, BAY y 3118 was more active than ciprofloxacin, sparfloxacin, piperacillin, cefotaxime, and clindamycin against the test isolates.

In vitro activity of biapenem (L-627), a new carbapenem, against anaerobes.

We tested 441 clinical strains of anaerobes by using a broth microdilution method to determine the in vitro activity of biapenem for comparison with those of other agents. Biapenem had activity comparable to those of imipenem and meropenem against all groups of anaerobes with MICs for 90% of the strains tested of 0.06 to 2 micrograms/ml. Against the Bacteroides fragilis group, biapenem was more active than ampicillin-sulbactam, ticarcillin-clavulanate, piperacillin, cefoxitin, cefotaxime, and ceftriaxone. Biapenem was also active against all of the B. capillosus, Prevotella, Clostridium, and Eubacterium strains and anaerobic cocci tested. Against all of the anaerobes tested, biapenem was 32- and 4-fold more active than clindamycin and metronidazole, respectively. These data indicate broad-spectrum activity by biapenem against anaerobes.

A five-year multicenter study of the susceptibility of the Bacteroides fragilis group isolates to cephalosporins, cephamins, penicillins, clindamycin, and metronidazole in the United States.

Over 2800 clinical strains of the Bacteroides fragilis group were collected during a 5-year period from ten geographically separate sites and tested for their susceptibility to various antimicrobial agents using a broth microdilution method. Among the cephalosporins, ceftizoxime was the most active (13% resistance) and importantly exhibited relatively equal activity against both B. fragilis species and non-B. fragilis species. Cefotaxime exhibited similar activity with an overall resistance rate of 18%. Both ceftriaxone and cefoperazone were appreciably less active cephalosporins especially against non-B. fragilis species. With regard to cephamycins, cefoxitin (MIC90, 32 micrograms/ml) was more active than cefotetan (MIC90, > or = 256 micrograms/ml) and cefmetazole (MIC90, 64 micrograms/ml). Non-B. fragilis species were highly resistant to cefotetan and cefmetazole. Imipenem was highly active against all strains with the exception of four strains of B. fragilis. Ampicillin-sulbactam, amoxicillin-clavulanate, piperacillin-tazobactam, and cefoperazone-sulbactam were all highly active with resistance rates < 2%. No resistance was detected to metronidazole, whereas 14% of isolates were resistant to clindamycin. When compared with other studies, these findings underscore the wide variability in susceptibility patterns reported nationwide and the need to continue monitoring these patterns to aid in choosing the most active compounds for therapy.

Anaerobic susceptibility testing. Slight differences in inoculum size can make a difference in minimum inhibitory concentrations.

In the present study, we compared the actual inoculum density from inoculated broth microdilution wells to the targeted inoculum size (10(5) CFU/well) when the inoculum was prepared using a McFarland nephelometer to achieve the standard density. Three target inoculum sizes (10(5), 5 x 10(5), and 10(6) CFU/well) were used to compare the effect of slight inoculum size increases of both ATCC and clinical strains of anaerobes on MICs of various antimicrobials. Actual colony counts of Bacteroides fragilis, Bacteroides thetaiotaomicron, Eubacterium lentum, and Veillonella parvula ranged from 0.7 x 10(5) to 1.4 x 10(5) CFU/well. As the inoculum size rose above the desired 10(5) CFU/well level, the MICs of certain antimicrobials became elevated. Ceftizoxime, cefotaxime, and ceftriaxone MICs rose 4- to 16-fold with as little as 0.5 log10 increase in inoculum size. Other increases were also noted with E. lentum and Clostridium perfringens, but were primarily between the low and high inoculum sizes. Results with cefoxitin, cefotetan, mezlocillin, and imipenem did not show an appreciable increased inoculum effect. This study demonstrates that the variation in organism size among anaerobes (both between species and within species) does make a difference in actual inoculum size and certain anaerobes may require special adjustment to ensure proper MIC results from susceptibility testing.

Anaerobes in polymicrobial surgical infections: incidence, pathogenicity, and antimicrobial resistance.

Many types of anaerobic bacteria have been isolated from clinical infections. Although most of these infections are polymicrobial and involve facultative Gram-negative bacilli, some are strictly anaerobic. For most of them, surgical intervention such as drainage of an abscess and debridement of devitalised tissue is the primary treatment and re-establishes good blood flow to the affected area. Appropriate antimicrobial treatment is also important to kill both residual organisms and those that may have spread from the site of primary infection. Several groups of anaerobes (for example, Bacteroides fragilis group, Prevotella, Porphyromonas, and Fusobacterium) have developed mechanisms of resistance to beta-lactam agents, the most common of which is production of beta-lactamases. A recent approach to neutralising these enzymes has been to combine the beta-lactam agent with an irreversible beta-lactamase inhibitor. Because of their potency against both aerobes and anaerobes, these combinations may replace traditional combination treatment (gentamicin/clindamycin) for polymicrobial infections. Piperacillin/tazobactam was the beta-lactam/beta-lactamase combination that was most active against the B fragilis group in the present study.