Kathrine Dornbusch

Synergism between aminoglycosides and cephalosporins with antipseudomonal activity: interaction index and killing curve method.

Combinations of gentamicin with cefotaxime, moxalactam, and ceftazidime were tested against 43 bacterial strains, most of them blood isolates. With an interaction index of less than or equal to 0.5 as borderline, synergism was demonstrated against 30 to 40% of the strains by the fractional inhibitory concentration index and against 50 to 70% by the fractional bactericidal concentration index. The reproducibility of the index was within +/- 0.2 for two-thirds of 40 repetitive assays and within +/- 0.4 to 0.5 for all of these assays. Similar results were obtained when netilmicin was substituted for gentamicin. The killing curve system for studying antibiotic synergism was standardized to give results comparable to those obtained with the interaction index. This was achieved when one-half of a previously determined minimum bactericidal concentration was used for single drugs and the amount of antibiotic was at least halved again when drugs were used in combination. An initial bacterial concentration of 10(5) to 10(6) colony-forming units per ml is recommended. Given these conditions, synergism could be defined as a 2-log 10 or more decrease in viable count given by both drugs together, as compared with the more active of the pair after 24 h. Prediction of killing curve results could then be obtained with the fractional bactericidal concentration index. When cephalosporins and gentamicin were combined from the start, the beta-lactam antibiotics were less susceptible to inactivation, as demonstrated in time-killing assays. If one of the antibiotics were added after 24 h, synergism was not demonstrable. The results indicate that the new cephalosporins may be advantageously combined with aminoglycosides.

Incidence of Antibiotic Resistance in Blood and Urine Isolates from Hospitalized Patients. Report from a European Collaborative Study

During 1992-93, 2544 isolates from blood cultures, comprising 52% gram-negative bacilli, 24% Staphylococcus aureus, 15% other staphylococci, 7% Enterococcus faecalis and 2% E. faecium, were consecutively collected and identified in 30 laboratories in 21 European countries. In addition 2512 urine isolates, comprising 82% gram-negative bacilli, 3% S. aureus, 4% other staphylococci and 11% enterococci were collected. The bacteria were sent to 3 laboratories for susceptibility testing by the microdilution method in Mueller-Hinton broth. The MICs of penicillins and aztreonam for all susceptible gram-negative bacilli were 0.25-8 mg/l, penems 0.032-2 mg/l, cefotaxime, ceftazidime and cefpirome or cefepime 0.032-0.25 mg/l, gentamicin, tobramycin and netilmicin 0.125-2 mg/l, amikacin 0.5-4 mg/l, ciprofloxacin 0.016-1 mg/l, trimethoprim 0.25-1 mg/l and tetracycline 1-2 mg/l. For susceptible staphylococci the MICs of erythromycin were 0.25-0.5 mg/l, clindamycin 0.125-0.25 mg/l, methicillin 2-8 mg/l, vancomycin and trimethoprim 1-4 mg/l, ciprofloxacin 0.25-1 mg/l, gentamicin and tobramycin 0.25-1 mg/l. For the enterococci the MICs of ampicillin and vancomycin were 2-4 mg/l and of imipenem, teicoplanin and trimethoprim 0.5-1 mg/l. The antibiotic resistance rates varied between laboratories, being lower in northern Europe, except for the penems, cefpirome and cefepime, which showed uniformly lower resistance rates. Compared to the earlier European studies the resistance rates to beta-lactam antibiotics among the gram-negatives have not changed except with an increase to cefotaxime and ceftazidime in central Europe. Resistance to aminoglycosides had also increased in central Europe from 7-8% to 20-21%, but decreased in southern Europe from 22-24% to 13-14% among the blood isolates and from 12-28% to 6-7% among the urine isolates. There was an increase in resistance to ciprofloxacin and gentamicin in staphylococci from southern Europe. The prevalence of MRSA was significant in central and southern Europe. It is of importance that collaborative national and international studies on the incidence of antibiotic resistance are being performed on a repetitive basis.

Factors Contributing to Resistance to Beta-Lactam Antibiotics in Bacteroides fragilis

Fifteen strains of Bacteroides fragilis, five highly resistant, five moderately resistant, and five susceptible to benzylpenicillin and cephaloridine, were tested for β-lactamase production. In the highly resistant and the moderately resistant groups of strains, a correlation between formation of β-lactamase and minimal inhibitory concentrations was demonstrated. In the presence of the β-lactamase inhibitors clavulanic acid and CP-45,899 at concentrations of 1 μg/ml, the susceptibility to cephaloridine increased fourfold or more in the β-lactamase-producing strains. Crypticity measurements (β-lactamase activity broken/intact cells) with cephaloridine as substrate could indicate a diffusion barrier in the cell wall.

Susceptibility to beta-lactam antibiotics and production of beta-lactamase in Bacteroides fragilis.

Using the agar dilution technique, 231 strains ofBacteroides fragilis, isolated during a 2-year period from human infections, were identified at subspecies level and were tested for susceptibility to 13Β-lactam antibiotics. The penicillins were benzylpenicillin, ampicillin, carbenicillin, cloxacillin, and the recently described penicillin derivatives cyclacillin, ticarcillin, and PC-904. The following cephalosporin derivates were tested: cephaloridine, cephalothin, cephalexin, cefamandole and cefuroxime. The cephamycin C derivative cefoxitin was also included in the study. Cefoxitin was the most effective drug tested since more than 80% of the strains were inhibited by 8Μg/ml or less, and no strain had a minimal inhibitory concentration (MIC) of more than 64Μg/ml. There was no marked difference in sensitivity among the subspecies with exception of subspeciesvulgatus, which was slightly more sensitive to all antibiotics tested. The size of the inoculum was an important factor for obtaining reproducible results in the sensitivity tests. Increased inocula resulted in markedly higher MICs for cephaloridine and cefuroxime.Production ofΒ-lactamase was performed on all isolates by a chromogenic cephalosporin substrate and about 90% of the strains were found to beΒ-lactamase producers.

Laboratory- and species-specific interpretive breakpoints for disk diffusion tests of chloramphenicol susceptibility of Haemophilus influenzae.

A total of 601 clinical isolates of Haemophilus influenzae isolated in six different regions of Sweden were tested for chloramphenicol susceptibility by using agar dilution MIC determinations and disk diffusion tests. For seven strains MICs were 4 micrograms/ml or higher, and for one strain the MIC was 2 micrograms/ml. All eight strains produced chloramphenicol acetyltransferase. For the remaining 593 strains, MICs were less than or equal to 1 microgram/ml, and the MICs for 50% and 90% of the strains were both 0.5 microgram/ml. Disk diffusion tests carried out by using revised interpretive criteria introduced in 1984 by the Swedish Reference Group for Antibiotics correctly identified the 593 strains as susceptible and the 8 strains as resistant. Quality assessments were performed in 29 clinical microbiology laboratories. The revised criteria for chloramphenicol disk diffusion testing gave rise to false resistance results in some laboratories. The interpretive accuracy improved when the interlaboratory variation was compensated for by using adjusted breakpoints. Such revision was possible through peak correction, single-strain regression analysis, and standard curve regression analysis. Peak-corrected breakpoints improved the accuracy from an overall incidence of false-resistant isolates of 4.4% to 2.3%. Single-strain regression analysis and standard curve regression analysis provided laboratory- and species-specific breakpoints which reduced false resistance rates of 0.14% and 0%, respectively.

Characterization of three different beta-lactamases from the Bacteroides fragilis group.

beta-Lactamases from five strains of Bacteroides fragilis and two strains of Bacteroides uniformis, all resistant to beta-lactam antibiotics, were compared by means of isoelectric focusing and enzyme kinetic measurements. beta-Lactamases from the five B. fragilis strains were identical, whereas those from the two B. uniformis strains were distinguished from each other and also from the B. fragilis enzymes. The two B. uniformis strains were relatively resistant to cefoxitin (minimal inhibitory concentration, 32 micrograms/ml), but only one of the strains, B. uniformis 2986, was found to rapidly inactivate cefoxitin. This apparently enzymatic inactivation of cefoxitin seemed to be of minor importance, and the main factor for cefoxitin resistance was considered to be a decreased permeability of the drug. Transfer of resistance to beta-lactam antibiotics from these beta-lactamase-producing strains of B. fragilis and B. uniformis to a non-beta-lactamase-producing strain of Bacteroides distasonis was attempted, but with these isolates no transfer of resistance to beta-lactam antibiotics was demonstrated.

In Vitro Aminoglycoside Resistance of Gram-negative Bacilli and Staphylococci Isolated from Blood in Sweden 1980–1984

The in vitro susceptibility to gentamicin, tobramycin, amikacin and netilmicin in septicaemia isolates was followed during 1980-1984 in 6-8 Swedish laboratories. The bacterial distribution was similar over the years and was dominated by Escherichia coli and staphylococci. Resistance to gentamicin was found in 2.3-3.6%, to tobramycin in 1.4-3.4%, to amikacin and netilmicin in 0.5-0.9%. Production of aminoglycoside modifying enzymes was observed among resistant strains.

Antibiotic susceptibility and β‐lactamase production in clinical isolates of Enterobacter spp.

The in vitro susceptibility of 237 clinical isolates of Enterobacter spp. (E. aerogenes, E. agglomerans and E. cloacae; 41, 64 and 132 respectively) to 16 different antibiotics is described. Four quinolones (ciprofloxacin, lomefloxacin, norfloxacin and ofloxacin), two new cephalosporins (cefpirome and cefepime) and imipenem, all showed high activity against the three Enterobacter species tested (MIC50≤ 0.125 mg/l, MIC90≤ 0.5 mg/l). Also the aminoglycosides gentamicin and tobramycin were highly active antibiotics (MIC50≤ 0.5 mg/l, MIC90≤ 1.0 mg/l). The susceptibility of β‐lactam‐antibiotics to β‐lactamase produced by Enterobacter spp. was evaluated, and imipenem and cefepime were found to be most stable. Different methods for detection of inducible β‐lactamases were used, the agar dilution method being more sensitive than the double‐disc diffusion test. Elevated β‐lactamase production was detected, via induction, in 83% of E. aerogenes strains and 70% of E. cloacae strains, with cefamandole used as the substrate and cefoxitin as the inducer. Constitutive, high level enzyme production was detected in 7 and 13% respectively of the E. aerogenes and the E. cloacae strains. In all the strains of E. agglomerans, 10% of E. aerogenes and 13% of E. cloacae, no β‐lactamases could be detected with the methods studied.

Bacteriuria Diagnosis and Antibiotic Susceptibility Testing in a Group Practice by Dipslide Techniques

In group practice, screening for bacteriuria and antibacterial susceptibility testing of bacteria in urine specimens were performed by dipslide methods (Uricult and Sensicult, Orion Diagnostica) and the results were evaluated with respect to conventional cultivation of urine specimens and standardized susceptibility testing by the disc diffusion method at a bacteriological laboratory. Bacteriuria diagnosis by screening by the Uricult method seemed to be satisfactorily performed except for some streptococcal strains. In the case of direct susceptibility testing by the Sensicult dipslide method, however, the results obtained by personnel at the surgeries and by trained bacteriologists displayed unacceptable disparities, despite the fact that a continuously running training programme was established.

In vitro Activity of Cefepime, a New Parenteral Cephalosporin, against Recent European Blood Isolates and in Comparison with Piperacillin/Tazobactam

Cefepime, a new parenteral cephalosporin with broad antibacterial spectrum and stability to the hydrolysis to many bacterial beta-lactamases, was tested against recent blood culture isolates (369 strains of gram-negative bacilli and 131 strains of staphylococci) collected in 29 European laboratories by the microdilution method in Mueller-Hinton broth. Cefepime was very active against the gram-negative bacilli (MIC50 less than or equal to 0.016-0.064 mg/l; MIC90 0.064-4 mg/l) and less active against Pseudomonas (MIC50 4 mg/l; MIC90 greater than 16 mg/l) or Acinetobacter (MIC50 and MIC90 greater than 16 mg/l). The staphylococci were also inhibited (MIC50 8 mg/l; MIC90 16 mg/l). Cefepime was very active against bacteria producing different plasmid-encoded beta-lactamases (MIC 0.016-0.5 mg/l). Piperacillin was not active against the latter strains (MIC from 2 to greater than 64 mg/l), but the presence of the beta-lactamase inhibitor tazobactam restored the activity of piperacillin. The bactericidal activity of cefepime and piperacillin/tazobactam against beta-lactamase-producing strains was confirmed by the killing curve technique.