Mutants of Escherichia coli K-12 exhibiting reduced killing by both quinolone and beta-lactam antimicrobial agents.

Norfloxacin, ofloxacin, and other new quinolones, which are antagonists of the enzyme DNA gyrase, rapidly kill bacteria by largely unknown mechanisms. Earlier, we isolated, after mutagenesis, Escherichia coli DS1, which exhibited reduced killing by quinolones. We evaluated the killing of DS1 and several other strains by quinolones and beta-lactams. In time-killing studies with norfloxacin, DS1 was killed 1 to 2 log10 units compared to 4 to 5 log10 units for the wild-type parent strain KL16, thus revealing that DS1 is a high-persistence (hip) mutant. DS1 exhibited a similar high-persistence pattern for the beta-lactam ampicillin and reduced killing by drugs that differed in their affinities for penicillin-binding proteins, including cefoxitin, cefsulodin, imipenem, mecillinam, and piperacillin. Conjugation and P1 transduction studies identified a novel mutant locus (termed hipQ) in the 2-min region of the DS1 chromosome necessary for reduced killing by norfloxacin and ampicillin. E. coli KL500, which was isolated for reduced killing by norfloxacin without mutagenesis, exhibited reduced killing by ampicillin. E. coli HM23, a hipA (34 min) mutant that was isolated earlier for reduced killing by ampicillin, also exhibited high persistence to norfloxacin. DS1 differed from HM23, however, in the map location of its hip mutation, lack of cold sensitivity, and reduced killing by coumermycin. Results of these studies with strains DS1, KL500, and HM23 demonstrate overlap in the pathways of killing of E. coli by quinolones and beta-lactams and identify hipQ, a new mutant locus that is involved in a high-persistence pattern of reduced killing by norfloxacin and ampicillin.     

In vitro pharmacodynamics of piperacillin, piperacillin-tazobactam, and ciprofloxacin alone and in combination against Staphylococcus aureus, Klebsiella pneumoniae, Enterobacter cloacae, and Pseudomonas aeruginosa.

The time-kill curve methodology was used to determine the pharmacodynamics of piperacillin, ciprofloxacin, piperacillin-tazobactam and the combinations piperacillin-ciprofloxacin and ciprofloxacin-piperacillin-tazobactam. Kill curve studies were performed for piperacillin, ciprofloxacin, and piperacillin-tazobactam at concentrations of 0.25 to 50 times the MICs for 13 strains of bacteria: four Pseudomonas aeruginosa, three Enterobacter cloacae, three Klebsiella pneumoniae, and three Staphylococcus aureus isolates (tazobactam concentrations of 0.5, 4, and 12 micrograms/ml). By using a sigmoid Emax model and nonlinear least squares regression, the 50% lethal concentrations and the maximum lethal rates of each agent were determined for each bacterial strain. For piperacillin-ciprofloxacin and ciprofloxacin-piperacillin-tazobactam, kill curve studies were performed with concentrations obtained by the fractional maximal effect method (R. C. Li, J. J. Schentag, and D. E. Nix, Antimicrob. Agents Chemother. 37:523-531, 1993) and from individual 50% lethal concentrations and maximum lethal rates. Ciprofloxacin-piperacillin-tazobactam was evaluated only against the four P. aeruginosa strains. Interactions between piperacillin and ciprofloxacin were generally additive. At physiologically relevant concentrations of piperacillin and ciprofloxacin, ciprofloxacin had the highest rates of killing against K. pneumoniae. Piperacillin-tazobactam (12 micrograms/ml) had the highest rate of killing against E. cloacae. Piperacillin-ciprofloxacin with relatively higher ciprofloxacin concentrations had the greatest killing rates against S. aureus. This combination had significantly higher killing rates than piperacillin (P < 0.002). For all the bacterial strains tested, killing rates by ciprofloxacin were significantly higher than those by piperacillin-tazobactam (4 and 12 micrograms/ml had significantly higher killing rates than piperacillin alone (P < 0.02 and P < 0.004, respectively). The effect of the combination of piperacillin-ciprofloxacin, in which piperacillin concentrations were relatively higher, was not statistically different from that of piperacillin alone (p > or = 0.71). The combination of ciprofloxacin-piperacillin-tazobactam achieved greater killing than other combinations or monotherapies against P. aeruginosa. The reduction in the initial inoculum was 1 to 4 logs greater with ciprofloxacin-piperacillin-tazobactam at 4 and 12 micrograms/ml than with any other agent or combination of agents. On the basis of the additive effects prevalently demonstrated in the in vitro study, the combinations of piperacillin-ciprofloxacin and piperacillin-tazobactam are rational therapeutic options. Greater killing of P. aeruginosa was demonstrated with ciprofloxacin-piperacillin--tazobactam. Since treatment failure of P. aeruginosa pneumonia is a significant problem, clinical studies are warranted.     

Effect of growth phase and pH on the in vitro activity of a new glycopeptide,oritavancin (LY333328), against Staphylococcus aureus and Enterococcus faecium

Oritavancin (LY333328) is a novel glycopeptide with activity against Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. We compared the effects of pH and growth phase on the activity of oritavancin and vancomycin against methicillin-resistant S. aureus and vancomycin-susceptible and -resistant E. faecium. Killing curve methods were used to evaluate the effect of growth phase (stationary versus exponential) and pH (6.4, 7.4 and 8.0). An inoculum of 10(6) cfu/mL was used for all experiments. Growth phase of S. aureus and vancomycin-susceptible E. faecium did not influence the rate and killing activity of oritavancin. The rate of killing by oritavancin against the vancomycin-resistant E. faecium strain was significantly faster and the reduction in cfu/mL at 24 h was significantly greater when the organism was in exponential compared with stationary growth phase (P < 0.05). In exponential growth phase, time to 99.9% killing was achieved in 0.6 +/- 0.01 h for the vancomycin-resistant strain, whereas in stationary growth phase, oritavancin did not decrease the inoculum by 99.9% within 24 h. Oritavancin's activity against S. aureus and vancomycin-susceptible E. faecium was not influenced by the pH conditions tested. Oritivancin's killing activity against the vancomycin-resistant E. faecium strain was significantly enhanced when tested at pH 7.4 and 8.0 (P < 0.05). Our study has demonstrated that oritavancin's activity does not seem to be influenced by the growth phase of the organisms or the pH of the environment when tested against sensitive strains of S. aureus and E. faecium. However, oritavancin's activity might be reduced against vancomycin-resistant E. faecium strains in stationary growth phase, as seen in infective endocarditis or when organisms are exposed to an acidic environment.     

Bactericidal activity and killing rate of serum against gram-positive cocci in volunteers receiving imipenem, oxacillin, vancomycin or ampicillin plus gentamicin

The serum bactericidal activity and rate of killing of serum was studied in two groups of ten volunteers receiving imipenem 500 mg, oxacillin 3 g and vancomycin 1 g given iv (Group 1) or imipenem 500 mg, vancomycin 1 g, and ampicillin 3 g plus gentamicin 80 mg iv (Group 2). Serum samples collected 30 min, 1 h and 6 h after the start of a 15 min infusion were tested against Staphylococcus aureus susceptible (five strains) or resistant (five strains) to oxacillin, Staphylococcus epidermidis susceptible to (five strains) or resistant (five strains) to oxacillin (Group 1), and against Streptococcus faecalis (four strains), Streptococcus spp. (six strains). All strains of streptococci had been isolated from patients with endocarditis. Imipenem produced the highest bactericidal titres against susceptible staphylococci whereas vancomycin produced the highest titres against oxacillin-resistant staphylococci. Against "non faecalis" streptococci, imipenem was equivalent to ampicillin plus gentamicin, whereas ampicillin plus gentamicin was the most active regimen against Str. faecalis. The study of the rate of killing of serum showed that imipenem was able to kill initially 1.5 log cfu/ml of Str. faecalis but this was followed by regrowth. A similar regrowth was observed with oxacillin-resistant staphylococci. Killing curves in broth were studied with imipenem at two temperatures (30 and 37 degrees C) and two initial inocula (10(5) and 10(6) cfu/ml) for 48 h of incubation against oxacillin-resistant staphylococci; regrowth occurred more rapidly with a high initial inoculum and low temperature of incubation despite a rapid initial killing. Gentamicin was found significantly to increase the rate of killing of imipenem against Str. faecalis in killing curves in broth. In conclusion, imipenem showed excellent activity, as assessed by the measure of the bactericidal titres and rate of killing in serum, against the studied strains with the exception of oxacillin-resistant staphylococci and Str. faecalis.     

Bactericidal activity of ceftizoxime, cefotetan, and clindamycin against cefoxitin-resistant strains of the Bacteroides fragilis group.

The bactericidal activity of ceftizoxime, cefotetan, and clindamycin at 0.5 x MIC, 1 x MIC, and 4 x MIC was determined by killing kinetic studies for 12 cefoxitin-resistant strains of the Bacteroides fragilis group. Ceftizoxime and clindamycin had greater bactericidal activity than cefotetan. The degree of bactericidal activity of each compound increased as the concentration increased from subinhibitory (0.5 x MIC) to suprainhibitory concentrations (4 x MIC). Comparison of the bactericidal activity at the MIC of the test agents with the MIC showed greater killing of susceptible strains than the resistant strains. Interestingly, ceftizoxime was more active than clindamycin, or cefotetan, against strains with high cefoxitin MIC values.     

Antibiotic activity in microbiological media versus that in human urine: comparison of ampicillin, ciprofloxacin, and trimethoprim-sulfamethoxazole.

The activities of three antibiotics in both Mueller-Hinton broth (MHB) and pooled human urine were compared by using an in vitro pharmacodynamic model. Clinical and reference strains of Escherichia coli were exposed to antibiotics at concentrations achievable in human urine. The rate of bacterial killing (time to a reduction of 3 log10 CFU/ml) and the extent of bacterial killing at 24 h were examined. Between MHB and urine, there were no significant differences in the rate or extent of bacterial killing for both ampicillin and ciprofloxacin. For trimethoprim-sulfamethoxazole there was no significant difference in the extent of bacterial killing in urine compared with that in MHB (P > 0.1); however, there was a significant decrease in the rate of bacterial killing in urine compared with that in MHB (P < 0.001). We conclude that with ampicillin and ciprofloxacin, activity against E. coli in MHB is predictive of the effects in human urine. The activity of trimethoprim-sulfamethoxazole in MHB predicts the extent but not the rate of bacterial killing in human urine.     

Comparative killing activity and postantibiotic effect of streptomycin combined with ampicillin, ciprofloxacin, imipenem, piperacillin or vancomycin against strains of Streptococcus faecalis and Streptococcus faecium

Nine strains of Streptococcus faecalis and Streptococcus faecium were studied with respect to ampicillin, ciprofloxacin, imipenem, piperacillin, vancomycin and streptomycin. Two strains were highly resistant (MIC greater than or equal to 2,000 micrograms/ml) to streptomycin. Evaluation and comparison of the killing activity with killing curves, and duration of the postantibiotic effect (PAE) after exposure for 1 h with regrowth curves was done with combinations of antibiotics or alone. The overall killing effect of ciprofloxacin with streptomycin was antagonistic, whereas synergism (greater than or equal to one log10 decrease in viable counts) was observed in streptomycin-susceptible strains with combinations of streptomycin and ampicillin, imipenem, piperacillin or vancomycin. In addition, prolongation of PAE (greater than or equal to 0.5 h) was seen only in streptomycin-susceptible strains. Thus, seven (100%) strains showed a synergistic increase in PAE to combinations with ampicillin and vancomycin, three (43%) to imipenem, four (57%) to piperacillin, but none to the combination of streptomycin and ciprofloxacin. A significant correlation was observed between the magnitude of increased killing and the extent of increase in recovery period with combinations of streptomycin with either ampicillin or vancomycin.     

Protective effect of amdinocillin against emergence of resistance to ceftazidime in Enterobacter cloacae.

Enterobacter cloacae infections have been shown clinically to respond less reliably to monotherapy with broad-spectrum cephalosporins than was initially expected. Selection of populations producing high levels of beta-lactamase has been shown to be the most frequent reason for treatment failure, and the use of these agents with another active antibiotic is recommended. In this study, E. cloacae strains from clinical specimens susceptible to ceftazidime and amdinocillin by broth dilution and disk tests were examined. In the presence of ceftazidime at 10 micrograms/ml, in vitro selection of resistant organisms was demonstrated for 3 of 11 strains. Selection was prevented when amdinocillin was added in combination. A more rapid killing was also demonstrated with this combination. At inocula of 10(8) CFU/ml, ceftazidime-resistant populations were isolated from 6 of 11 strains in vitro, and the emergence of this resistance was prevented by amdinocillin. The enhanced killing effect noted for amdinocillin with ceftazidime may have resulted in part from complementary activity of the antibiotics on penicillin-binding proteins. The ceftazidime-amdinocillin combination offers an interesting prospect for the therapy of infections caused by E. cloacae strains which are initially susceptible to both antibiotics.     

Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype

Escherichia coli is a common cause of meningitis and sepsis in the newborn infant, and the large majority of isolates from these infections produce a polysialic acid (PSA) capsular polysaccharide, the K1 antigen, that protects the bacterial cell from immune attack. We determined whether a capsule-depolymerizing enzyme, by removing this protective barrier, could alter the outcome of systemic infection in an animal model. Bacteriophage-derived endosialidase E (endoE) selectively degrades the PSA capsule on the surface of E. coli K1 strains. Intraperitoneal administration of small quantities of recombinant endoE (20 micro g) to 3-day-old rats, colonized with a virulent strain of K1, prevented bacteremia and death from systemic infection. The enzyme had no effect on the viability of E. coli strains but sensitized strains expressing PSA to killing by the complement system. This study demonstrates the potential therapeutic efficacy of agents that cure infections by modification of the bacterial phenotype rather than by killing or inhibition of growth of the pathogen.     

MIC and time-kill study of antipneumococcal activities of RPR 106972 (a new oral streptogramin), RP 59500 (quinupristin-dalfopristin), pyostacine (RP 7293), penicillin G, cefotaxime, erythromycin, and clarithromycin against 10 penicillin-susceptible and -resistant pneumococci.

Broth MICs and time-kill studies were used to test the activity of RP 59500 (quinupristin-dalfopristin), RPR 106972, pyostacine (RP 7293), erythromycin, clarithromycin, and cefotaxime for four penicillin-susceptible (MICs of 0.008 to 0.03 microgram/ml), two penicillin-intermediate (MIC of 0.25 microgram/ml), and four penicillin-resistant (MIC of 2.0 to 4.0 micrograms/ml) strains of pneumococci: 6 of 10 strains were resistant to macrolides (MICs of > or = 0.5 microgram/ml). MICs of RP 59500 (0.5 to 1.0 microgram/ml), RPR 106972 (0.125 to 0.25 microgram/ml), and pyostacine (0.125 to 0.25 microgram/ml) did not alter with the strain's penicillin or macrolide susceptibility status. Three penicillin-susceptible strains and one penicillin-intermediate strain were susceptible to macrolides (MICs of < or = 0.25 microgram/ml); the macrolide MICs for the remaining strains were > or = 4.0 micrograms/ml. Cefotaxime MICs rose with those of penicillin G, but all strains were inhibited at MICs of < or = 2.0 micrograms/ml. RP 59500 was bactericidal for all strains after 24 h at 2 x MIC and yielded 90% killing of all strains at 6 h at 2 x MIC; at 8 x MIC, RP 59500 showed 90% killing of six strains within 10 min (approximately 0.2 h). In comparison, RPR 106972 was bactericidal for 9 of 10 strains at 2 x MIC after 24 h and yielded 90% killing of all strains at 2 x MIC after 6 h; 90% killing of six strains was found at 8 x MIC at 0.2 h. Results for pyostacine were similar to those of RPR 106972. Erythromycin and clarithromycin were bactericidal for three of four macrolide-susceptible strains after 24 h at 4 x MIC. Clarithromycin yielded 90% killing of three strains at 8 x MIC after 12 h. Cefotaxime was bactericidal for all strains after 24 h at 4 x MIC, yielding 90% killing of all strains after 6 h at 4 x MIC. All three streptogramins yielded rapid killing of penicillin- and erythromycin-susceptible and -resistant pneumococci and were the only compounds which killed significant numbers of strains at 0.2 h.     

Influence of erythromycin resistance, inoculum growth phase, and incubation time on assessment of the bactericidal activity of RP 59500 (quinupristin-dalfopristin) against vancomycin-resistant Enterococcus faecium.

RP 59500, a mixture of two semisynthetic streptogramin antibiotics (quinupristin and dalfopristin), is one of a few investigational agents currently in clinical trials with inhibitory activity against multiple-drug-resistant strains of Enterococcus faecium. We evaluated the bactericidal activity of this antimicrobial against 30 recent clinical isolates of vancomycin-resistant E. faecium, including 23 erythromycin-resistant (MIC, >256 microg/ml) and 7 erythromycin-intermediate (MIC, 2 to 4 microg/ml) strains. All isolates were inhibited by RP 59500 at 0.25 to 1.0 microg/ml. The bactericidal activity of RP 59500 was markedly influenced by the erythromycin susceptibility of the strains and by several technical factors, such as inoculum growth phase and time of incubation of counting plates. As determined by time-kill methods, RP 59500 at a concentration of 2 or 8 microg/ml failed to kill erythromycin-resistant organisms under any conditions. Bactericidal activity was observed against all seven erythromycin-intermediate isolates when log-phase inocula were used and the cells were counted after 48 h of incubation (mean reductions in viable bacteria for RP 59500 at concentrations of 2 and 8 microg/ml, 3.45 and 3.50 log10 CFU/ml, respectively), but killing was diminished when the plates were examined at 72 h (mean killing, 3.06 and 2.95 log10, CFU/ml, respectively). No bactericidal activity was observed when stationary-phase cultures were used. On the basis of these data, we expect that bactericidal activity of RP 59500 against the multiple-drug-resistant E. faecium strains currently encountered would be distinctly uncommon.     

Efficacy of colistin versus beta-lactams,aminoglycosides, and rifampin as monotherapy in a mouse model of pneumonia caused by multiresistant Acinetobacter baumannii

The treatment of life-threatening infections due to carbapenem-resistant Acinetobacter baumannii has become a serious challenge for physicians worldwide. Often, only colistin shows in general good in vitro activity against these carbapenem-resistant strains, but its antibacterial efficacy in comparison with the antibiotics most used in clinical practice is not well known. We studied the efficacy of colistin versus those of imipenem, sulbactam, tobramycin, and rifampin in an experimental pneumonia model with immunocompetent mice. We used three strains of A. baumannii corresponding to the main clones (A, D, and E) involved in the outbreaks of our hospital, with different grades of resistance to imipenem (imipenem MICs of 1, 8, and 512 microg/ml, respectively) and to the other antibiotics. The MIC of colistin was 0.5 microg/ml for the three strains. Reduction of log(10) CFU/g in lung bacterial counts, clearance of bacteremia, and survival versus results with controls were used as parameters of efficacy. Imipenem and sulbactam (Deltalung counts: -5.38 and -4.64 log(10) CFU/ml) showed the highest level of bactericidal efficacy in infections by susceptible and even intermediate strains. Tobramycin and rifampin (-4.16 and -5.15 log(10) CFU/ml) provided good results against intermediate or moderately resistant strains, in agreement with killing curves and pharmacodynamics. On the contrary, colistin showed the weakest antibacterial effect among the antibiotics tested, both in killing curves and in the in vivo model (-2.39 log(10) CFU/ml; P < 0.05). We conclude that colistin did not appear as a good option for treatment of patients with pneumonia due to carbapenem-resistant A. baumannii strains. Other alternatives, including combinations with rifampin, may offer better therapeutic profiles and thus should be studied.     

Effect of pooled human cerebrospinal fluid on the postantibiotic effects of cefotaxime, ciprofloxacin, and gentamicin against Escherichia coli.

The killing and postantibiotic effects (PAE) of cefotaxime, ciprofloxacin, and gentamicin against Escherichia coli were determined in Mueller-Hinton broth (MHB) and pooled human cerebrospinal fluid (CSF). MICs performed in MHB and CSF were within one dilution for all antimicrobial agent-organism combinations. At two times the MIC, CSF significantly (P less than 0.05) increased the duration of the PAE compared with MHB when cefotaxime, ciprofloxacin, and gentamicin were used against all strains tested. This effect occurred despite similar reductions in bacterial growth in both fluids after the 2-h antimicrobial agent exposure. We conclude that pooled human CSF markedly increases the PAE of cefotaxime, ciprofloxacin, and gentamicin against E. coli compared with MHB, without affecting bacterial killing.     

Correlation between growth curves and killing curves of Escherichia coli in the presence of fleroxacin and ampicillin

Fleroxacin, a new long-acting quinolone, induces rapid killing and bacterial filamentation as do other quinolones. Ten strains of Escherichia coli were exposed comparatively to fleroxacin and ampicillin in order to determine the effect of sub- and supra-inhibitory concentrations of each of these two compounds on turbidimetric growth curves and viable counts. By comparing the maximal early increase in optical density (OD, PIOD) as colony-forming units per milliliter (CFU/ml) after 2 and 6 h of exposure to antibiotics, we observed a reduced number of CFU/ml in comparison with the control after the 2-hour exposure at 1/4 the minimum inhibition concentration (MIC) and after 6 h at 1/8 MIC, but a high OD value was also seen among the fleroxacin exposed bacteria. For ampicillin, PIOD rates and killing rates were slower and dose dependent. This discrepancy was due to filament formation, which increased the PIOD value to the same extent as the control curve. After exposure to fleroxacin at 1/2 MIC the PIOD decreased significantly and after 2 and 6 h E. coli killing rates of 99 and 99.9%, respectively, were observed. With exposure to 2 and 4 x MIC, both PIOD values and CFU/ml decreased substantially. Combined analysis of continuous turbidimetric monitoring and viable counts showed that subinhibitory concentrations of fleroxacin and beta-lactam had different effects on E. coli. Fleroxacin's rapid killing rate, despite filament formation, contrasted with the result obtained with ampicillin. The minimum antibiotic concentration of fleroxacin against E. coli was around 1/8 MIC.     

Effect of a single percutaneous abscess drainage puncture and imipenem therapy,alone or in combination,in treatment of mixed-infection abscesses in mice

The importance of supplementary imipenem therapy after a single percutaneous abscess drainage puncture was studied in a mouse model of established mixed-infection abscesses. Animals were treated for 3 days with daily dosing regimens of 384 to 1,536 mg/kg of body weight that took into account the short half-life of this antibiotic in mice. Imipenem therapy in conjunction with abscess drainage was significantly better than drainage alone in reducing the Escherichia coli and Bacteroides fragilis counts in the mixed infections. Furthermore, the killing of B. fragilis by the combination of imipenem therapy and abscess drainage was significantly better than that by imipenem treatment alone. The maximum reductions in E. coli and B. fragilis counts were 1.1 and 2.2 log(10) CFU/abscess, respectively. In contrast, the in vitro activity of imipenem was significantly better (maximum reduction, > or =6.2 log(10) CFU/ml) against mixed cultures of the same strains even when bacterial numbers similar to those found in the abscesses were used. Comparable in vivo activity was achieved only when treatment was started 30 min before inoculation (reduction for both strains, > or =6.1 log(10) CFU/abscess), but this killing was significantly diminished if the start of treatment was delayed until > or =12 h after inoculation. Imipenem concentrations in abscess tissue reached levels above the MIC for E. coli for >60% of the dosing interval. Possible reasons for the reduced activity of imipenem in vivo are discussed, and we conclude that standard susceptibility tests overestimate the efficacy of this antibiotic against the organisms present in these abscesses.     

Photodynamic effects of novel XF porphyrin derivatives on prokaryotic and eukaryotic cells

The worldwide rise in the rates of antibiotic resistance of bacteria underlines the need for alternative antibacterial agents. A promising approach to the killing of gram-positive antibiotic-resistant bacteria of the skin uses light in combination with a photosensitizer to induce a phototoxic reaction. Different concentrations (0 to 100 microM) of porphyrin-based photosensitizers (CTP1, XF70, and XF73) and different incubation times (5 min, 1 h, and 4 h) were used to determine phototoxicity against two methicillin-resistant Staphylococcus aureus strains, one methicillin-sensitive S. aureus strain, one methicillin-resistant Staphylococcus epidermidis strain, one Escherichia coli strain, and human keratinocytes and fibroblasts. Incubation with 0.005 microM XF70 or XF73, followed by illumination, yielded a 3-log10 (> or = 99.9%) decrease in the viable cell numbers of all staphylococcal strains, indicating that the XF drugs have high degrees of potency against gram-positive bacteria and also that the activities of these novel drugs are independent of the antibiotic resistance pattern of the staphylococci examined. CTP1 was less potent against the staphylococci under the same conditions. At 0.005 microM, XF70 and XF73 demonstrated no toxicity toward fibroblasts or keratinocytes. No inactivation of E. coli was detected at this concentration. XF73 was confirmed to act via a reactive oxygen species from the results of studies with sodium azide (a quencher of singlet oxygen), which reduced the killing of both eukaryotic and prokaryotic cells. When a quencher of superoxide anion and the hydroxyl radical was used, cell killing was not inhibited. These results demonstrate that the porphyrin-based photosensitizers had concentration-dependent differences in their efficacies of killing of methicillin-resistant staphylococcal strains via reactive oxygen species without harming eukaryotic cells at the same concentrations.     

Contribution of permeability and sensitivity to inhibition of DNA synthesis in determining susceptibilities of Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis to ciprofloxacin.

To examine the correlation between bacterial cell susceptibility to ciprofloxacin and the magnitude of uptake and cell target sensitivity, the relative contribution of ciprofloxacin accumulation in intact cells and its ability to inhibit DNA synthesis were investigated among strains of Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis. Uptake studies of [14C]ciprofloxacin demonstrated diffusion kinetics for P. aeruginosa and E. coli. Ciprofloxacin was more readily removed from E. coli J53 and A. faecalis ATCC 19018 by washing than from P. aeruginosa PAO503. These results indicate that the process of cell accumulation is different for P. aeruginosa in that the drug is firmly bound at an extracellular site. Whatever the washing conditions, A. faecalis accumulated less drug than either of the other two bacteria. Magnesium chloride (10 mM) caused a substantial decrease of ciprofloxacin accumulated and an increase in the MIC, depending upon the nature of the medium. The addition of carbonyl cyanide m-chlorophenylhydrazone caused a variable increase in drug accumulated, depending on the medium and the bacterial strain. The concentration of ciprofloxacin required to obtain 50% inhibition (ID50) of DNA synthesis for P. aeruginosa PAO503 and A. faecalis ATCC 19018 did not correlate with their corresponding MICs but did for E. coli J53. Treatment with EDTA decreased the ID50 of ciprofloxacin for P. aeruginosa PAO503 and its gyrA derivative by 5- and 2-fold, respectively, and decreased the ID50 for E. coli JB5R, a strain with a known decrease in OmpF, by 1.4-fold but did not decrease the ID50 for the normally susceptible E. coli J53. The ID(50) for P. aeruginosa obtained after EDTA treatment or in ether-permeabilized cells was higher than that obtained for the other two strains. The protonophore carbonyl cyanide m-chlorophenylhydrazone prevented killing by low ciprofloxacin concentrtaions, but sodium azide did not. The latter compound did not enhance killing in association with inhibition of a previously described energy-dependent efflux of ciprofloxacin susceptibility being the susceptibility to inhibition of DNA synthesis in E. coli, poor premeability associated with the small pore size of A. faecalis, and a combination of low permeability and reduced susceptibility of DNA synthesis to inhibition for P. aeruginosa.     

Comparative evaluation of enoxacin, ofloxacin, ampicillin, and chloramphenicol for treatment of experimental Haemophilus influenzae pneumonia.

A murine model of bacteremic Haemophilus influenzae type b pneumonia was used to evaluate the therapeutic efficacies of the quinolone antimicrobial agents enoxacin and ofloxacin compared with those of ampicillin and chloramphenicol. Ampicillin-susceptible (AS) and ampicillin-resistant (AR) challenge strains were employed. Treatment with enoxacin or ofloxacin produced intrapulmonary killing of H. influenzae that was superior to that achieved with ampicillin (P less than 0.01 to P less than 0.001 for both AS and AR strains). Ofloxacin and enoxacin also provided killing greater than that with chloramphenicol for the AS strain (P less than 0.01 to P less than 0.001). For the AR strain, ofloxacin provided killing greater than that obtained with chloramphenicol (P less than 0.001). Survival from AS strain pneumonia was 60% in enoxacin-treated and 78% in ofloxacin-treated animals compared with 41% for chloramphenicol-treated and 23% for ampicillin-treated groups. We conclude that enoxacin and ofloxacin may be effective antimicrobial agents in treating either AS or AR strains causing H. influenzae pneumonia.     

Comparison of ampicillin-sulbactam regimens simulating 1.5- and 3.0-gram doses to humans in treatment of Escherichia coli bacteremia in mice.

A mouse model of bacteremia was used to compare the efficacies of 1.5- and 3.0-g intravenous doses of ampicillin-sulbactam. Seven strains of Escherichia coli producing various levels of TEM-1 beta-lactamase were used as the challenge isolates. These strains included six clinical isolates (MICs from 2/1 micrograms/ml [with 2 and 1 microgram/ml being the respective concentrations of ampicillin and sulbactam] to 32/16 micrograms/ml) with similar degrees of virulence in mice and a laboratory genetic transformant (E. coli AFE) which hyperproduces TEM-1 (MIC = 128/64 micrograms/ml). Human pharmacokinetics were simulated by injecting mice subcutaneously twice (1 h apart) with ampicillin-sulbactam at concentrations of 40 mg/kg of body weight (1.5 g) and 80 mg/kg (3.0 g). Against two clinical isolates for which ampicillin-sulbactam MICs were < or = 8/4 micrograms/ml, no difference was observed in either the rate or level of killing between the two doses, and both doses were 100% protective against lethal infection. Against the four clinical isolates for which ampicillin-sulbactam MICs were between 16/8 and 32/16 micrograms/ml, a slight delay in killing was noted with three of the strains. This delay was followed by a rapid 2- to 3-log drop in the level of bacteremia, and both doses of ampicillin-sulbactam were 100% protective against lethal septicemia. With strain AFE, no killing was observed with the 40-mg/kg dose compared with a 2-log killing with the 80-mg/kg dose. This difference in killing correlated with a decreased protective efficacy of the 40-mg/kg dose. These data suggest that the 1.5-g preparation of ampicillin-sulbactam is as effective as the 3.0-g dose in the treatment of experimentally induced E. coli bacteremia, as long as ampicillin-sulbactam MICs are 32/16 micrograms/ml or less.     

Antimicrobial activity of quinupristin-dalfopristin combined with other antibiotics against vancomycin-resistant enterococci

Interactions between quinupristin-dalfopristin and six other antimicrobials were examined by checkerboard arrays against 50 clinical isolates of vancomycin-resistant Enterococcus faecium selected to represent a range of susceptibilities to individual agents. Unequivocal synergistic or antagonistic interactions at clinically relevant concentrations were infrequently encountered when the streptogramin was combined with chloramphenicol, ampicillin, imipenem, vancomycin, or teicoplanin. Combinations with doxycycline resulted in synergistic inhibition in 36% of checkerboards. Against 10 strains of Enterococcus faecalis, synergistic interactions were found when quinupristin-dalfopristin was combined with doxycycline (four strains), either glycopeptide (three strains), or ampicillin (two strains). Combination with quinupristin-dalfopristin increased the ampicillin MIC from 1 to 4 microg/ml for one strain. For 10 strains of E. faecium, interactions were also assessed by time-kill methods using concentrations of the agents attainable in human serum. Most of these antimicrobials augmented killing by quinupristin-dalfopristin to a minor degree. Against 2 of the 12 strains in this collection that were not highly resistant to gentamicin, the combination of quinupristin-dalfopristin (2 microg/ml) plus gentamicin (5 microg/ml) resulted in killing approaching 3 log(10) CFU/ml. With the exception of doxycycline, inhibitory interactions between quinupristin-dalfopristin and other agents tested against vancomycin-resistant strains of E. faecium were uncommon at clinically relevant concentrations.