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The effects of imipenem on the growth of Escherichia coli ATCC 25922 were studied using a bioluminescence assay of bacterial ATP, microscopy and viable counting in iso-osmotic Mueller-Hinton broth (MHB) and hypo-osmotic nutrient broth (NB). Imipenem showed a post-antibiotic effect (PAE) of > 2 h for E. coli in both MHB and NB after 2 h exposure to 1 and 8 mg/L of imipenem when determined by bioluminescence and microscopy. The intracellular ATP level increased after 2 h exposure of E. coli to 1 mg/L of imipenem in MHB. In this culture there was a predominance of spheroplasts. These spheroplasts were large and osmotically fragile and a 10 min treatment in water-diluted MHB (hypo-osmotic) prior to the assays lysed the large spheroplasts. This reduced the intracellular ATP level and shortened the PAE when determined by bioluminescence, and caused more rapid initial killing and a negative PAE when determined by viable counting. At 8 mg/L imipenem in MHB and at all concentrations in NB there was a predominance of rods and only a small number of spheroplasts which all disappeared when the cultures resumed logarithmic growth. In these cultures there was a significant initial decrease in intracellular ATP. This study showed reasonable agreement between microscopy and bioluminescence, which are direct methods, for determining the initial killing and PAE of imipenem on E. coli. More rapid initial killing and shorter or no PAEs, were in general, obtained in both MHB and NB when determined by viable counting. However, the effective regrowth time, defined as the time for the bacterial density to increase 1 log10 from the pre-exposure inoculum, was independent of the method used for measuring regrowth in both MHB and NB.

Three hundred and sixteen patients with serious infections verified or suspected to be of Gram-negative aetiology were treated in an open, randomized, comparative multicentre study with amikacin 15 mg/kg/day given either as a single dose or in two divided doses at 12 h intervals. Two hundred patients were evaluated for efficacy and all 316 for safety. The efficacy of both dosage regimens was very good with a satisfactory clinical response in 90% of the patients. There were no significant differences between the two regimens regarding efficacy and safety. This was also confirmed in an analysis according to the principle of 'intention-to-treat' including all randomized patients. In 218 patients additional therapy, most commonly with piperacillin or ampicillin, was considered necessary. The mean peak serum concentration of amikacin was 40.9 mg/L in the once-daily group, which is 10 x MIC for most Gram-negative bacteria, compared to 24.4 mg/L in the twice-daily group, which is 6 x MIC. Mean trough serum concentrations after 24 h were 1.8 mg/L in the once-daily group and 3.1 mg/L after 12 h in the twice-daily group. These serum concentrations were often close to or just below the MICs of the isolated pathogens. Drug related adverse reactions were seen in 40 (13%) of the patients. Among the adverse reactions with possible or probable relation to amikacin were 20 nephrotoxic events, nine in the once-daily group and 11 in the twice-daily group. A multivariate analysis of selective causative factors and nephrotoxic events gave a low correlation for once- vs twice-daily amikacin therapy. Five ototoxic events were observed, three in the once-daily group and two in the twice-daily group. One patient in the once-daily group experienced nausea in connection with amikacin infusions.

Two different isoxazolylpenicillins (cloxacillin and dicloxacillin) were compared regarding impairment of renal function after total hip arthroplasty. 85 patients received dicloxacillin and 93 patients received cloxacillin as antibiotic prophylaxis. A total dose of 6 grams was given during a 36-hour period in doses of 1 gram pre-, per- and postoperatively. Creatinine in serum and beta 2-microglobulin in serum and urine were determined preoperatively and 2, 4, and 10 days after the operation. The dicloxacillin-treated patients had an increase in creatinine and beta 2-microglobulin in serum that was not seen in the cloxacillin group. The increase indicates a transient injury in the process of glomerular filtration. Although the increase was temporary and subclinical, a dose reduction is nevertheless recommended for older patients.

In a prospective pilot study 70 patients (age greater than 65 years) who underwent hip arthroplasty were treated with dicloxacillin, a total of 6 g given pre-, per- and postoperatively as antibiotic prophylaxis. Creatinine in serum and beta 2-microglobulin in serum and urine were determined as estimates of renal function. Values were obtained preoperatively and on days 2, 4 and 10 after operation. A slight but significant increase of serum creatinine was seen on day 2 with a gradual decrease almost down to the preoperative baseline value on day 10. Serum beta 2-microglobulin increased more gradually; the increase was significant on day 10. Raised levels of beta 2-microglobulin in urine were most pronounced: a 20-fold increase on day 2, then a slow decrease, still significant increase on day 10. This may indicate a reversible damage of proximal tubules with blocked tubular reabsorption of beta 2-microglobulin. The slightly increased levels of serum creatinine and beta 2-microglobulin would also indicate a minor reversible decrease in glomerular filtration rate. Whether these effects are caused by the operation trauma per se or by the dicloxacillin prophylaxis cannot be determined from this pilot study. It seems quite clear that hip arthroplasty with short term prophylaxis with dicloxacillin does not result in clinically important changes in renal function.

Selection and regrowth of resistant variants, which are present in low frequencies in the initial inoculum, were seen when large inocula of five strains of Staphylococcus aureus and four strains of Staphylococcus epidermidis were incubated in broth with amikacin, gentamicin, netilmicin and tobramycin. Statistical analysis showed no significant difference between the aminoglycosides in the selective growth of resistant variants (P0.5). Vancomycin differed significantly from the aminoglycosides in both the frequency of, and selection of resistant variants (P0.001). No bacteria resistant to1 x MIC was seen in the vancomycin-exposed cultures of S. aureus and S. epidermidis, while in most aminoglycoside-exposed cultures, bacteria resistant to 4-16 x MIC were seen.

The in vitro post-antibiotic effect (PAE) of cefepime, cefotaxime, ceftazidime and imipenem on reference strains of Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens were evaluated by bioluminescence assay of bacterial ATP. In parallel with the PAE determination, initial killing and morphology studies were performed. Imipenem produced greater than 1 h PAE on all strains tested, cefepime and cefotaxime on four strains and ceftazidime only on one of the strains tested. The length of the PAE on different strains did not correlate in the same way to MIC. Imipenem induced greater than 1 h PAE at 1/4-2 MIC while the cephalosporins caused greater than 1 h PAE at 4-256 x MIC. A PAE exceeding 1.2h was seen concomitantly with spheroplasts but there was not necessarily strong (greater than or equal to 99%) initial killing at the same time. The PAE duration at greater than or equal to 99% initial killing varied between 2.0 h and 5.0 h. When the cephalosporins produced less than 1 h PAEs, this was seen concomitantly with production on filaments and weak initial killing. The bioluminescence method was not jeopardized by filament formation and no negative PAE was found in contrast to the viable count method. The study showed that neither a certain multiple of MIC, the presence of spheroplasts nor strong initial killing can predict the length of PAE for beta-lactam antibiotics on gram-negative bacteria.

The pharmacodynamics of daptomycin and vancomycin on Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were investigated by studying the postantibiotic effect (PAE) and initial killing. The influence of Ca2+ and albumin on these drugs was also evaluated. The PAE was studied by use of bioluminescence assay of bacterial ATP. Daptomycin at clinically achievable concentrations produced a dose-dependent PAE on E. faecalis (0.6 to 6.7 h) and S. aureus (1.0 to 6.3 h). The long PAE of daptomycin was seen simultaneously with a potent dose-dependent initial killing assayed by viable count determination. The initial change in bacterial ATP was not as extensive as the decrease in viability. Vancomycin at corresponding concentrations produced shorter PAEs on E. faecalis (0.5 to 1.0 h) and S. aureus (1.3 to 1.8 h). This coincides with a weak non-dose-dependent initial change in viability and intracellular ATP. The MICs of vancomycin were not influenced by different Ca2+ concentrations or by the addition of albumin to the broth. The MICs of daptomycin for both strains were lowered, and the PAEs were prolonged with increasing concentrations of Ca2+ in the broth. The PAE of daptomycin was Ca2+ dependent to the same extent as the MIC was. In the presence of physiological concentrations of albumin and free Ca2+, the PAEs of daptomycin on both strains were reduced and the MICs were increased in comparison with the results obtained in pure Mueller-Hinton broth with approximately the same free Ca2+ concentration. This decrease in daptomycin activity was considered to be due to the albumin binding of daptomycin. Despite the albumin binding of daptomycin, the PAE produced on E. faecalis and S. aureus in the presence of a physiological free Ca2+ concentration was still over 6 h at clinically achievable concentrations.

The in-vitro post-antibiotic effect (PAE) of amikacin alone and in combination with ceftazidime, ceftriaxone and piperacillin was studied for two strains of Enterococcus faecalis using a bioluminescent assay of bacterial ATP. The two strains of E. faecalis were resistant to amikacin, ceftazidime and ceftriaxone but sensitive to piperacillin. The bacterial cultures were incubated with the beta-lactam antibiotics for 1 h and concentrations of amikacin between 2-64 mg/l were then added. Thereafter, incubation continued with the combinations for one more hour. After dilution, regrowth was monitored by measuring bacterial ATP every hour. Increasing concentrations of amikacin (2-64 mg/l), ceftazidime (8-32 mg/l) and ceftriaxone (32-128 mg/l) resulted in little or no PAE (0-0.3 h) on these strains. PAEs of 0.5 to 1.6 h resulted from exposure to piperacillin (4-32 mg/l). In combination amikacin and piperacillin increased the PAE to 5.5 h. A synergistic PAE was also seen when the enterococci were exposed to amikacin combined with ceftazidime or ceftriaxone in concentrations close to the MICs of the latter antibiotics.

The synergic in-vitro post-antibiotic effect (PAE) of mecillinam, in combination with either ampicillin, aztreonam, ceftazidime or piperacillin, on a reference strain of Escherichia coli was evaluated by bioluminescence assay of bacterial ATP. Ampicillin, ceftazidime and mecillinam alone induced a concentration dependent PAE (greater than 3 h) on E. coli, whereas aztreonam and piperacillin alone induced a short (less than 1 h) non-dose dependent PAE. At most concentrations, the combination of mecillinam and ampicillin, aztreonam, ceftazidime or piperacillin induced longer PAEs on E. coli than the sum of the individual antibiotics' PAEs. Long PAEs were seen concomitantly with the presence of spheroplasts. In addition to the synergistic PAE, the decrease in colony counts and changes in ATP values after a 2 h exposure to mecillinam, in combination with the other beta-lactam antibiotics, were more prominent than the respective values after exposure to the individual antibiotics. The change in ATP was generally less pronounced than the decrease in colony counts. This could be due to lysis of spheroplasts on agar plates, leading to an over-estimation of the initial killing when assayed by viable counting. Mecillinam, which induced long PAEs on E. coli at almost all concentrations in this study, has a high affinity for penicillin binding protein 2 (PBP 2) and induced spheroplast formation at all concentrations. However, mechanisms other than the affinity for PBP 2 and spheroplast formation are involved in the PAE of beta-lactam antibiotics on Gram-negative bacteria; since the PAE was prolonged when mecillinam was combined with ampicillin, aztreonam, ceftazidime or piperacillin, which bind preferentially to PBP 1 and 3.

Two hundred and twenty patients with serious infections verified or suspected to be of Gram-negative aetiology were treated in an open randomized comparative multicentre trial with amikacin 15 mg/kg/day given either as a single dose or in two divided doses at 12-h intervals. Amikacin was administered as a short-term iv infusion. When additional therapy was considered necessary piperacillin or ampicillin was recommended. The trial continues and an interim report on data from the 12 participating Scandinavian hospitals is presented. One hundred and forty-four patients have been evaluated for efficacy and 213 patients for safety. There were no significant differences between the two dosage regimens regarding efficacy and safety. A satisfactory clinical response was recorded in 129 (90%) of the evaluable patients. One serious adverse reaction was seen in a patient in the once-daily group. This was ototoxicity which was superimposed on a long standing hearing defect possibly caused by previous streptomycin therapy.

Once-daily dosing of amikacin is a novel therapy regimen which seems pharmacokinetically appropriate for the primary group of patients considered for aminoglycoside therapy. In this study of 29 elderly patients with serious infections, amikacin 11 mg/kg or 15 mg/kg bw was administered as a short-term (30 min) intravenous infusion. The amikacin serum concentration-time profile was best described by a bi-exponential equation with a half-life of about 4.8 h. A triexponential equation was not applicable because the slow terminal elimination phase was not detected during the 24 h dosing interval. In practice, a uni-exponential equation is often used, and this may lead to incorrect conclusions about the elimination rate of amikacin. Amikacin clearance provides more direct information about elimination of amikacin than does serum half-life. Thus, there was a better correlation between the individual amikacin clearances and creatinine clearances (r = 0.89), than between the serum half-lives of amikacin and the creatinine clearances (r = 0.71). For elderly patients a smaller dose of amikacin than the regular daily dose of 15 mg/kg bw, i.e. about 11 mg/kg bw, seems recommendable, when it is given once daily. From the data obtained it is also obvious that once-daily dosing of amikacin does not eliminate the need for checking serum concentrations of the drug.

The in vitro postantibiotic effects (PAE) of aztreonam, ceftazidime, cefuroxime, imipenem, and piperacillin on Escherichia coli ATCC 25922 were studied by a bioluminescence assay of bacterial ATP. In parallel with the PAE investigation, viability and morphology studies were performed. The strain was exposed for 2 h to different concentrations of beta-lactam antibiotics. The antibiotic activity was eliminated by 10(-4) dilutions, and regrowth of bacteria was monitored hourly by the bioluminescence assay of bacterial ATP. The length of PAE was dose dependent for ceftazidime (0.5 to 2.6 h), cefuroxime (0.4 to 2.6 h), and imipenem (0.3 to 4.5 h). The long PAE for these antibiotics at higher concentrations was associated with a potent initial killing and the presence of spheroplasts. Aztreonam and piperacillin produced a short, non-dose-dependent PAE (0.4 to 0.95 h). Short PAEs (below 1 h) were seen concomitantly with production of filaments, except in the case of imipenem, which only produced spheroplasts. The bioluminescence method was not jeopardized by filament formation, in contrast to the viable count assay which is normally used for PAE investigations. This makes it possible to study PAE for beta-lactam antibiotics on gram-negative bacteria with bioluminescence.

The post-antibiotic effect (PAE) of amikacin alone and in combination with ceftazidime, ceftriaxone and piperacillin was studied for two strains each of Pseudomonas aeruginosa and Serratia marcescens using a bioluminescent assay of bacterial ATP. Two models were used for combining beta-lactam antibiotics and amikacin: in one model the cultures were incubated with 32 mg/L of ceftazidime, 128 mg/L of ceftriaxone or 32 mg/L of piperacillin for 1 h. Different concentrations of amikacin (0.5-64 mg/L) were then added. Incubation of the combinations continued for one more hour. The antibiotics were eliminated by dilution. In the second model tested, one strain of S. marcescens was simultaneously exposed to amikacin and a beta-lactam antibiotic for 2 h. The PAEs produced by the drugs in combination were longer than the sum of the individual effects of the drugs when they were used alone. Results were equally good with both models. A synergic PAE was also found with amikacin concentrations close to the MIC in combination with low concentrations of ceftazidime, ceftriaxone and piperacillin.

Forty-five mostly elderly patients with serious infections were treated in a prospective, comparative and randomized pharmacokinetic study with amikacin 11.0 or 15.0 mg/kg administered in a single daily dose as an intravenous, short-term infusion or with amikacin 7.5 mg/kg administered twice daily in the same way. The results indicate that administration of amikacin 15 mg/kg in a single daily dose should be a practical and safe principle of administration. However elderly patients often have reduced creatinine clearance and should preferably be given a lower dose of 11 mg/kg bw. The risk of nephrotoxicity did not increase, but conclusions on ototoxicity and clinical efficacy cannot be drawn from this limited study. This should be considered as an initial part of a future multicentre trial.

Measurements of aminoglycosides by an agar disk diffusion assay are inhibited by heparin in a dose-dependent way. When assayed by a homogeneous immunoassay, this was only evident for tobramycin. This indicates that specimens for aminoglycoside measurement should not be obtained in heparinized tubes. When heparin is used clinically as an anticoagulant, the amount in blood does not reach levels that affect the aminoglycoside activity.

The in-vitro postantibiotic effect (PAE) of amikacin, gentamicin, netilmicin and tobramycin was investigated by a bioluminescent assay of bacterial ATP. Two strains each of Escherichia coli and Pseudomonas aeruginosa were exposed for 1 h to different concentrations of the aminoglycosides. The aminoglycoside was removed by a 10(-3) dilution, and regrowth of bacteria was followed at hourly intervals by monitoring bacterial ATP. This method simplified the PAE studies and made such studies possible at high aminoglycoside concentrations. The length of the PAE was dose-dependent for all the aminoglycosides studied. The PAEs ranged between three and seven hours for all four strains at the aminoglycoside concentrations normally reached in serum during standard dosing. The long PAE of aminoglycosides, especially after exposure to high drug concentrations, constitutes an argument in favour of administering aminoglycosides in higher-than-usual doses with longer intervals between doses. This proposal is also supported by recent pharmacokinetic, bacteriological and toxicity data.