In vitro bactericidal activity of piperacillin,gentamicin, and metronidazole in a mixed model containing Escherichia coli,Enterococcus faecalis,and Bacteroides fragilis

An anaerobic, mixed model assay was used to study the bactericidal activities of piperacillin, gentamicin, and metronidazole, alone and in double- and triple-antibiotic combinations against a polymicrobial suspension of E. coli, E. faecalis, and B. fragilis. Only slight differences were noted with the agents when tested against single (10(5) cfu/mL inoculum) versus polymicrobic suspensions (10(6) cfu/mL final inoculum) of susceptible and resistant organisms. Contrary to previous reports in the literature, metronidazole was not active against E. coli in an anaerobic environment (even in the presence of B. fragilis) nor was the activity of metronidazole reduced against B. fragilis in the presence of E. faecalis. Gentamicin demonstrated excellent activity against E. coli when tested in a Bactron anaerobic chamber (5% hydrogen, 5% CO(2,) 90% nitrogen). The pH of the media was only reduced to 6.3-6.7, considerably higher than the pH range of 5-6 needed to significantly reduce the activity of aminoglycosides.     

Comparison of the bactericidal activity of clindamycin and metronidazole against cefoxitin-susceptible and cefoxitin-resistant isolates of the Bacteroides fragilis group.

Time-kill kinetic methodology was used to evaluate the bactericidal activity of cefoxitin, cefotetan, clindamycin, and metronidizole against cefoxitin-susceptible and cefoxitin-resistant isolates of the Bacteroides fragilis group. Overall, metronidazole was the most bactericidal agent, with all isolates being killed with less than or equal to 4 micrograms/ml at 24 hr. Clindamycin was the next most bactericidal agent, with 20 of 26 isolates being killed with less than 16 micrograms/ml. Six isolates with clindamycin MICs greater than or equal to 64 micrograms/ml were not killed at 24 hr, with concentrations as high as 256 micrograms/ml. Cefoxitin and cefotetan were the least bactericidal agents tested. Seven isolates with MICs of greater than or equal to 64 micrograms/ml to each agent demonstrated a lack of killing at 24 hr, with concentrations of the respective agent as high as 256 micrograms/ml. At concentrations with either agent of 32 micrograms/ml, the remaining 19 isolates were killed at 24 hr. Of the six B. fragilis isolates resistant to clindamycin, four were also resistant to both cefoxitin and cefotetan. We conclude that in hospitals with cefoxitin-resistant B. fragilis group isolates, metronidazole would provide appropriate therapy.     

Serum and blister fluid pharmacokinetics and bactericidal activities of ampicillin-sulbactam, cefotetan, cefoxitin, ceftizoxime, and ticarcillin-clavulanate.

Ampicillin-sulbactam, ticarcillin-clavulanate, cefoxitin, cefotetan, and ceftizoxime are promoted for the treatment of mixed aerobic-anaerobic bacterial infections. Their activities have been compared in vitro but not in vivo. In order to assess the in vivo activities of these agents in serum and interstitial fluid, we administered single, intravenous doses of these antimicrobial agents to healthy subjects. Concentrations of the antimicrobial agents in serum and suction-induced blister fluid and bactericidal activity were measured by high-pressure liquid chromatography and the standard methodology of the National Committee for Clinical Laboratory Standards, respectively. The organisms used for bactericidal activity tests were one isolate each of Staphylococcus aureus, Klebsiella pneumoniae, and Bacteroides fragilis. Pharmacokinetic parameters in serum and blister fluid were similar to those derived in other investigations. Of note were the high and prolonged concentrations of ticarcillin and cefotetan in blister fluid, despite high-level serum protein binding. The bactericidal activities in serum and blister fluid reflected the relative in vitro activities and kinetic dispositions of the various antimicrobial agents except for the bactericidal activity of cefotetan, which was substantially lower in blister fluid than serum, despite a blister fluid:serum area under the concentration-time curve ratio of 1.5. Similarly, the activity of ticarcillin-clavulanate in blister fluid was also substantially less than would have been predicted by the blister fluid:serum ratio of the area under the concentration-time curve of 1.1, possibly because of the low concentrations of clavulanate in blister fluid. The rankings of the in vivo bactericidal activities of the five drugs were as follows: for S. aureus, ampicillin-sulbactam > ticarcillin-clavulanate > ceftizoxime > cefoxitin > cefotetan; for K. pneumoniae, ceftizoxime > cefotetan > ampicillin-sulbactam = ticarcillin-clavulanate > cefoxitin; and for B.fragilis, ticarcillin-clavulanate > cefotetan > ceftizoxime > ampicillin-sulbactam = cefoxitin.     

In vitro activities of clindamycin, imipenem, metronidazole, and piperacillin-tazobactam against susceptible and resistant isolates of Bacteroides fragilis evaluated by kill kinetics

The aim of the present study was to investigate the activities of clindamycin, imipenem, metronidazole, and piperacillin-tazobactam against 12 Bacteroides fragilis isolates (resistant and susceptible strains) by kill kinetics over 24 h. In contrast to the other antimicrobial agents, clindamycin did not affect strains with MICs of >8.0 μg/ml. For those strains with MICs of ≤ 8.0 μg/ml, all employed antibiotics except clindamycin showed nearly bactericidal activity. Metronidazole proved to be the most active antimicrobial agent.     

Comparative in vitro activities of piperacillin-tazobactam and ticarcillin-clavulanate.

The in vitro activities of ticarcillin, piperacillin, clavulanic acid, tazobactam, ticarcillin-clavulanate, and piperacillin-tazobactam against 819 bacterial isolates were compared. The two beta-lactamase inhibitors, clavulanic acid and tazobactam, had little useful antibacterial activity but enhanced the activities of the penicillins against beta-lactamase-producing strains of Haemophilus influenzae, Branhamella catarrhalis, and methicillin-susceptible Staphylococcus aureus; all strains were susceptible to both combinations. Both enzyme inhibitors also enhanced the activities of the penicillins against most strains of Escherichia coli, Klebsiella spp., Citrobacter diversus, Proteus spp., Providencia spp., and Bacteroides spp. and against occasional strains of Citrobacter freundii, Enterobacter spp., and Serratia marcescens. Clavulanic acid frequently enhanced the activity of ticarcillin against Xanthomonas maltophilia, and tazobactam frequently enhanced the activity of piperacillin against Morganella morganii. Enhancement was observed primarily with strains relatively resistant to the penicillins. In general, clavulanic acid was more effective than tazobactam in enhancing penicillin activity against Klebsiella spp., C. diversus, X. maltophilia, and Bacteroides spp., whereas tazobactam was more effective against Escherichia coli and Proteeae. There was little or no enhancement of activity against Enterococcus faecalis, Aeromonas hydrophila, Pseudomonas aeruginosa, Pseudomonas cepacia, or Acinetobacter anitratus. Clavulanic acid occasionally antagonized the activity of ticarcillin against ticarcillin-susceptible members of the family Enterobacteriaceae, but those strains were still considered susceptible to the combination. Tazobactam never antagonized the activity of piperacillin. In a direct comparison of the activities of ticarcillin-clavulanate and piperacillin-tazobactam, the two were equally active against H. influenzae, B. catarrhalis, and S. aureus; the latter was more active against E. faecalis. For relatively susceptible strains of members of the family Enterobacteriaceae, neither combination was predictably more active than the other, but relatively resistant strains were generally more susceptible to piperacillin-tazobactam. Piperacillin-tazobactam was more active than ticarcillin-clavulanate against A. hydrophila, P. aeruginosa, and P. cepacia, similar in activity against A. anitratus, and less active against X. maltophilia and Bacteroides spp.     

Activity of newer beta-lactam agents against clinical isolates of Bacteroides fragilis and other Bacteroides species.

The in vitro activities of beta-lactam antibiotics against Bacteroides fragilis and B. fragilis group isolates are presented. Clinical isolates from 1986 were compared with strains from 1979 to 1982. Imipenem, ticarcillin-clavulanic acid, and ceftizoxime were the most active agents. Cefotetan was equivalent to cefoxitin against B. fragilis but less active against B. fragilis group isolates. Enhancement of cefotaxime by its desacetyl metabolite was minimal.     

Bacteriostatic and bactericidal activities of eight fluoroquinolones against MexAB-OprM-overproducing clinical strains of Pseudomonas aeruginosa

OBJECTIVES: To assess the impact of stable overproduction of efflux system MexAB-OprM on the bacteriostatic and bactericidal activities of fluoroquinolones against clinical Pseudomonas aeruginosa strains. METHODS: The minimal inhibitory concentrations (MICs) and the minimal bactericidal concentrations (MBCs) of eight fluoroquinolones (pefloxacin, norfloxacin, ofloxacin, moxifloxacin, levofloxacin, ciprofloxacin, trovafloxacin and grepafloxacin) were determined for nine post-therapy resistant isolates of P. aeruginosa overexpressing MexAB-OprM. Clinical significance of low-level resistance conferred by the efflux mechanism was evaluated with a Monte Carlo simulation. RESULTS: Compared with their pre-therapy susceptible counterparts, seven out of the nine post-therapy efflux mutants exhibited a modest two- to eight-fold increase in resistance to all the fluoroquinolones tested. Interestingly, stronger variations in resistance (up to 64-fold) were observed in two other mutants, one of which had acquired a GyrB target mutation in addition to efflux under chemotherapy. Time-kill experiments showed that MexAB-OprM up-regulation did not confer tolerance to fluoroquinolones as the ratio of MBC to MIC was less than 4 for most of the strains. To gain an insight into the clinical significance of resistance conferred by MexAB-OprM, a Monte Carlo simulation was conducted with various fluoroquinolone regimens. With this model, low levels of resistance to ciprofloxacin (MIC > or = 0.25 mg/L) or levofloxacin (MIC > or = 1 mg/L), such as those due to overproduced MexAB-OprM, were predicted to result in poor clinical outcomes. CONCLUSIONS: Altogether, these data strongly suggest that when derepressed, MexAB-OprM provides P. aeruginosa with a resistance that may be sufficient to impair the efficacy of single therapy with highly potent fluoroquinolones, such as ciprofloxacin and ofloxacin.     

Serum bactericidal activity of piperacillin/tazobactam against Staphylococcus aureus, piperacillin-susceptible and piperacillin-resistant Escherichia coli and Pseudomonas aeruginosa

BACKGROUND: The serum bactericidal test measures the highest level of an antibiotic-containing serum dilution at which 99.9% of bacteria are killed. In this study the serum bactericidal activity of piperacillin/tazobactam was determined for bacteria often involved in severe infections. In earlier studies titres >/=1:8 in the serum bactericidal tests correlated well with clinical success in the treatment of endocarditis and osteomyelitis as well as bacterial eradication. METHODS: Blood samples of 6 healthy volunteers were taken before and 1 and 4 h after piperacillin/tazobactam (4.5 g) administration. Serum concentrations and serum bactericidal activity were determined for 10 strains each of Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, both piperacillin-resistant and piperacillin-susceptible according to NCCLS guidelines. RESULTS: 100% of S. aureus and piperacillin-susceptible E. coli, 90% of piperacillin-resistant E. coli and 80% of P. aeruginosa were killed 1 h after drug administration. 4 h after drug administration serum bactericidal activity decreased to 60% for S. aureus, 90% for piperacillin-susceptible E. coli, 80% for piperacillin-resistant E. coli and 30% for P. aeruginosa. CONCLUSIONS: Excellent serum bactericidal activity of piperacillin/tazobactam was recorded 1 h after drug administration for S. aureus, E. coli and P. aeruginosa. After 4 h limited killing rates for P. aeruginosa could be detected, which supports the idea of a combination therapy.     

Comparison of inhibitory and bactericidal activity of antipseudomonal antibiotics against Pseudomonas aeruginosa isolates from cystic fibrosis patients

Using a broth microtiter dilution method, the minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of so-called antipseudomonal antibiotics were determined against 79 Pseudomonas aeruginosa isolates from cystic fibrosis patients. On the basis of the MIC values and using DIN breakpoints, the percentual susceptibilities led to the following rank order: imipenem (91%), ciprofloxacin (90%), tobramycin (87%), amikacin (87%), ceftazidime (82%), cefsulodin (76%), piperacillin (71%), azlocillin (62%), followed by gentamicin, ceftriaxone, mezlocillin, netilmicin, and cefotaxime (less than 50%). However, evaluating MBC values according to DIN breakpoints, only ciprofloxacin (82%), tobramycin (77%), amikacin (58%), and imipenem (57%) showed a pronounced antipseudomonal effectiveness. The data indicate that MBC determinations are necessary to evaluate the antibiotic susceptibility of P. aeruginosa rather than MIC determinations, at least in patients with impaired defence mechanisms which require bactericidal therapy.     

Comparison of the bactericidal activity of ciprofloxacin alone and in combination with selected antipseudomonal beta-lactam agents against clinical isolates of Pseudomonas aeruginosa

The bactericidal activity of ciprofloxacin against 60 clinical isolates of Pseudomonas aeruginosa was evaluated by exposing replicating microorganisms to concentrations ranging from 0.12 to 2 micrograms/ml for 48 hr. In addition, ciprofloxacin was combined with selected antipseudomonal beta-lactams using subinhibitory concentrations (1/4 x MIC, 1/2 x MIC) of each. We found that a concentration of 2 micrograms/ml of ciprofloxacin resulted in bactericidal activity (greater than or equal to 99.9% killing of the final inoculum) at 8 and 12 hr for the highest percentage of isolates (95 and 96.7%, respectively). At the breakpoint concentration (1 micrograms/ml), there was bactericidal activity against 81.7% of these isolates at 8 hr and 78.7% at 12 hr. By 24 and 48 hr, regrowth was frequently seen for concentrations of 1 and 2 micrograms/m (76.7 and 41.7%, respectively). This regrowth represented limited progression of resistance (4- to 16-fold increase in MICs) to ciprofloxacin without cross-resistance to other classes of antibiotics. The combinations containing ciprofloxacin and beta-lactam agent were synergistic for isolates that were susceptible to both agents. Moreover, the combinations prevented the emergence of resistance to either drug.     

Comparison of the bactericidal activities of ofloxacin and ciprofloxacin alone and in combination with ceftazidime and piperacillin against clinical strains of Pseudomonas aeruginosa.

On the basis of MIC data, ciprofloxacin exhibits superior activity against Pseudomonas aeruginosa than the other currently available fluoroquinolones do. Despite the antipseudomonal advantage noted for ciprofloxacin monotherapy, it is unknown whether this advantage is maintained when the fluoroquinolones are used in combination with antipseudomonal beta-lactams such as ceftazidime and piperacillin. Twelve healthy volunteers were enrolled in this open-label, randomized, steady-state, six-way cross-over, comparative trial. All subjects received the following regimens: (i) 400 mg of ofloxacin given intravenously (i.v.) every 12 h (q12h), (ii) 400 mg of ciprofloxacin given i.v. q12h, (iii) 400 mg of ofloxacin given i.v. q12h plus 1 g of ceftazidime given i.v. every 8 h (q8h), (iv) 400 mg of ciprofloxacin given i.v. q12h plus 1 g of ceftazidime given i.v. q8h, (v) 400 mg of ofloxacin given i.v. q12h plus 4 g of piperacillin given i.v. q8h, and (vi) 400 mg of ciprofloxacin given i.v. q12h plus 4 g of piperacillin given i.v. q8h. Serum bactericidal titers with subsequent calculation of the area under the bactericidal curve were determined against three clinical isolates of P. aeruginosa. As monotherapy, ciprofloxacin demonstrated superior antipseudomonal activity than ofloxacin did; however, combination of these agents with ceftazidime yielded remarkably similar and statistically comparable activity profiles. In contrast, ciprofloxacin-piperacillin retained a bactericidal advantage over ofloxacin-piperacillin. Although ciprofloxacin exhibits superior antipseudomonal activity when used as monotherapy, combination of ofloxacin or ciprofloxacin with ceftazidime yielded equivalent activity profiles against susceptible strains of P. aeruginosa.     

Bactericidal activities of new beta-lactam antibiotics against Bacteroides fragilis.

The bactericidal activities of cefoxitin, cefoperazone, cefotaxime, moxalactam, mezlocillin, and piperacillin were determined in duplicate against low and high inocula of each of 10 Bacteroides fragilis strains. Antibiotic concentrations resulting in 99.9% killing were established by quantitation of the inocula just before anaerobic incubation of tests and by subcultures of volumes sufficient to accurately determine less than or equal to 0.1% survivors. Inoculum effects on inhibitory and bactericidal activities were least for cefoxitin and greatest for cefoperazone and cefotaxime. Bactericidal-to-inhibitory concentration ratios of greater than or equal to 8 occurred most frequently with mezlocillin and piperacillin, regardless of inoculum size; however, 55% of the strains tested at the low inoculum size and 38% of those tested at the high inoculum size against the six antibiotics had bactericidal-to-inhibitory concentration ratios of greater than or equal to 4.     

Comparison of the activities of penicillin G and new beta-lactam antibiotics against clinical isolates of Bacteroides species.

MICs were determined for 218 clinical isolates of Bacteroides by a broth microdilution method. Imipenem was the most active antibiotic tested. Azlocillin, mezlocillin, and cefoxitin had comparable activities, with resistance among members of the B. fragilis group and B. capillosus. Ceftizoxime was the most active cephalosporin tested. Members of the B. fragilis group showed high levels of resistance to cefotetan and ceftazidime. Resistance to penicillin G varied from 0 to 14%.     

Comparison of bactericidal activity of selected beta-lactam antimicrobials against Pseudomonas aeruginosa

The efficacy of ceftazidime, piperacillin, tobramycin and ticarcillin was tested in vitro against 102 clinical isolates of Pseudomonas aeruginosa. The MIC and MBC of each agent alone were determined. In addition, the rate of killing of 27 isolates by each beta-lactam agent was tested both singly and in combination with tobramycin. The concentrations of beta-lactams and of tobramycin were tailored to each organism; these were double the MBC in the case of the beta-lactams and half the MBC for tobramycin. Although 85.2% of isolates were susceptible to piperacillin and only 58.8% were susceptible to ticarcillin, the MBC90 was higher for piperacillin (greater than 256 mg/l) than ticarcillin (199.7 mg/l). The addition of tobramycin in sublethal concentrations increased the rapidity of killing of bacteria.     

Susceptibilities of 200 penicillin-susceptible and -resistant pneumococci to piperacillin, piperacillin-tazobactam, ticarcillin, ticarcillin-clavulanate, ampicillin, ampicillin-sulbactam, ceftazidime, and ceftriaxone.

MICs of eight beta-lactams (piperacillin, piperacillin-tazobactam, ticarcillin, ticarcillin-clavulanate, ampicillin, ampicillin-sulbactam, ceftazidime, and ceftriaxone) were determined by agar dilution against 64 penicillin-susceptible, 70 intermediately penicillin-resistant, and 66 fully penicillin-resistant pneumococci. The MICs of piperacillin with and without tazobactam for 90% of the susceptible, intermediately resistant, and resistant strains tested (MIC90s) were < or = 0.064, 2.0, and 4.0 micrograms/ml, respectively. By comparison, those of ampicillin with and without sulbactam were 0.125, 2.0, and 4.0 micrograms/ml and those of ceftriaxone were < or = 0.064, 1.0, and 2.0 micrograms/ml, respectively. Strains were less susceptible to ticarcillin with and without clavulanate (MIC90s, 2.0, 64.0, and 128.0 micrograms/ml) and ceftazidime (MIC90s, 1.0, 8.0, and 32.0 micrograms/ml).     

Characterization of clinical isolates of beta-lactamase-negative, highly ampicillin-resistant Enterococcus faecalis.

We analyzed the penicillin-binding protein (PBP) profiles of two clinical isolates of Enterococcus faecalis for which ampicillin MICs were 32 and 64 micrograms/ml. Six PBPs were detected in both isolates, demonstrating an apparently increased amount of PBP 5 and decreased penicillin binding of PBPs 1 and 6. These results suggest that ampicillin resistance in the clinical isolates of E. faecalis described could be associated with alterations in different PBPs.     

Efficacy of rifampicin in experimental Bacteroides fragilis and Pseudomonas aeruginosa mixed infections

Experimental intraabdominal abscesses were produced in mice by intraperitoneal injection of Bacteroides fragilis and Pseudomonas aeruginosa. The therapeutic efficacy of rifampicin and cefsulodin alone, and in combination was investigated in this in-vivo experimental mixed intraabdominal abscess model. Treatment with rifampicin at 10, and 25 mg/kg or cefsulodin at 50, and 100 mg/kg singly or in combinations prevented mortality as compared to 68% mortality rate occurring in the untreated mice. Rifampicin, at 25 mg/kg dose, was very effective in preventing abscess formation and produced bacterial eradication. It prevented abscess formation in 80% of the mice and eradicated both Bacteroides and Pseudomonas in 100% and 75% of the abscesses of the mice. Cefsulodin failed to reduce the incidence of abscess formation, and to eradicate Bact. fragilis from the abscesses, although it significantly decreased Ps. aeruginosa in the abscesses. The combination of rifampicin at 10 mg/kg and cefsulodin at 100 mg/kg was more effective than either of the antibiotics alone and was as effective as rifampicin alone at 25 mg/kg levels. This combination was bactericidal against both organisms in the infected mice.     

Conjugative transposition of Tn916-related elements from Enterococcus faecalis to Escherichia coli and Pseudomonas fluorescens.

We studied the ability of transposons Tn916, Tn1545, and Tn916-Km, a Tn916 derivative expressing kanamycin resistance, to be conjugatively transferred from Enterococcus faecalis to various gram-negative bacteria. Our results demonstrate that these types of elements can carry out conjugative transposition from the chromosome of E. faecalis to those of Escherichia coli and Pseudomonas fluorescens and that the accomplishment of this event depends on the donor potential of the E. faecalis transposon delivery strain. Since the tet(M) gene does not confer a selectable level of tetracycline resistance to gram-negative bacteria such as E. coli, the presence of another marker(s) readily expressed in these recipients is required for the detection of this type of transfer. Conjugal transfer of Tn916-Km from E. faecalis to E. coli is not restricted by the EcoK restriction system, nor does it depend on the presence of functional homologous recombination system and integration host factor proteins in the recipient bacteria.     

Comparison of the bactericidal activity of cefotaxime and desacetylcefotaxime alone and in combination against Bacteroides fragilis group organisms

Through the use of time-kill kinetic studies, the bactericidal activity of cefotaxime (CTX) and desacetylcefotaxime (dCTX) alone and in combination against 18 strains of Bacteroides fragilis group was studied. Each isolate was tested at subinhibitory, inhibitory, and suprainhibitory concentrations of each drug as determined from the MIC values. Overall CTX was more bactericidal than dCTX at each of the three concentration levels tested. The combination of CTX and dCTX showed comparable bactericidal activity to CTX at the subinhibitory and inhibitory concentrations, even though each component was present at only one-half the concentration of CTX alone. At suprainhibitory concentrations, the combination of CTX/dCTX appeared synergistic since the combination with each component at a concentration of 1 x MIC was as bactericidal as CTX at a concentration of 4 x MIC. CTX and dCTX alone and in combination exhibited comparable bactericidal activity against test isolates with high (greater than or equal to 32 micrograms/ml) or low (less than or equal to 16 micrograms/ml) MICs. Thus, in vitro the combination of CTX and its naturally occurring metabolite dCTX interacts to produce an additive or synergistic effect against strains of B. fragilis group. Whether the in vitro testing of the combinations is more relevant to clinical outcome than testing CTX alone needs further study.     

Characterization of bactericidal activity of clindamycin against Bacteroides fragilis via kill curve methods.

Kill curves were determined for five isolates of Bacteroides fragilis with clindamycin at concentrations equal to the MIC or to 4, 16, and 64 times the MIC. Examination of plots of log CFU per milliliter versus time revealed no association between the clindamycin concentration and the rate and extent of the bactericidal activity against B. fragilis at or below 64 times the MIC.