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In order to see whether the susceptibility of Danish Listeria monocytogenes strains has changed over the years we examined a collection of human isolates from the period 1958-2001. We, furthermore, wanted to compare L. monocytogenes susceptibility testing using a disc diffusion assay with MIC measurements performed by the E-test. 106 strains isolated predominantly from blood cultures and cerebrospinal fluids were examined together with three reference strains. Susceptibility to the following antibiotics was tested by the E-test and by Oxoid discs using Iso-sensitest agar: penicillin G, ampicillin, meropenem, gentamicin, sulphamethoxazole, trimethoprim, ciprofloxacin, erythromycin, vancomycin, linezolid, chloramphenicol and tetracycline. The strains were in the main sensitive to all antibiotics examined using both methods, except for ciprofloxacin, where the strains were intermediate sensitive. However, for penicillin, ampicillin and sulphamethoxazole, while the disc diffusion assay found the strains to be sensitive, MIC measurements generally placed the strains one dilution above the breakpoint for sensitivity in the intermediate sensitive group. Based on the MIC measurements, the antibiotic susceptibility of L. monocytogenes has not changed in Denmark from 1958 to 2001, and the multiresistant strains found in human infections elsewhere have not been found in Denmark.

An epidemiological survey of the increase in Acinetobacter species isolates occurring in the intensive care unit of a Spanish teaching hospital during 1993 and 1994 was carried out. Different laboratory methods were used to find out, whether there was a genetic linkage. The isolates were divided into three main groups according to the resistance patterns to 11 drugs. Using API 20NE biotyping, eight different types were found. The two most common contained 20 and 11 isolates, respectively. Five different plasmid profile types were observed, although plasmids were only demonstrated in 40% of the isolates. Ribotyping with EcoRI, SalI and ClaI enzymes revealed 10, 9, and 8 different patterns, respectively. In total, 15 different ribotypes were identified using these three enzymes. Twenty-one isolates belonged to exactly the same ribotype, and 13 were associated with two highly related ribotypes. In the first ribotype, only five isolates harboured plasmids. The ribotyping method produced 100% typability and ribotypes were easy to compare; it also had taxonomic value. Ribotyping allowed us to determine the genetic linkage between Acinetobacter isolates recovered from ICU patients.

One hundred and twelve clinical strains of Acinetobacter were collected during a 7-month period in 1990-1991 from Danish clinical microbiology departments. According to the old notation, 37 strains were biovar anitratus and 75 b.lwoffii. Using the newest terminology, 35 strains were unambiguously identified as the species A. haemolyticus, A. johnsonii, A. radioresistens, and as the unnamed phenons 6, 10, and 14, by a numerical approach using a simplified phenotypical identification scheme. Most of the remaining strains were identified to the genotypically heterogeneous A. calcoaceticus-A. baumannii complex and the A. lwoffii phenotype. Sixteen strains (14%) were left unidentified. Eight A. lwoffii strains were glucose-oxidizing and were thus classified as b. anitratus using the old terminology. The strains were tested for susceptibility to ampicillin, piperacillin, ticarcillin, cefotaxime, ceftazidime, imipenem, gentamicin, tetracycline, sulphonamide, and nalidixic acid. All strains were susceptible to imipenem. The susceptibility to the remaining beta-lactams was variable, the A. lwoffii isolates being the most and the A. calcoaceticus-A. baumannii complex strains the least susceptible. All non-A. calcoaceticus-A. baumannii complex strains were susceptible to all other antibiotics tested, except for one A. lwoffii strain that was sulphonamide resistant. Twenty-two percent of the strains in the A. calcoaceticus-A. baumannii complex showed reduced susceptibility or resistance to gentamicin, but only sporadic resistance to sulphonamides, tetracyclines, and nalidixic acid. Eight infections were recorded: three cases of septicaemia, three cases of peritonitis related to continuous ambulatory peritoneal dialysis, and two cases of recurrent urinary tract infection. No epidemics were detected.

The Acinetobacter calcoaceticus-Acinetobacter baumannii complex consists of four closely related "genospecies" or DNA groups: DNA group 1 (A. calcoaceticus), DNA group 2 (A. baumannii), DNA 3, and Tjernberg & Ursing's DNA group 13. Strains in this complex are so similar phenotypically that it is often impossible to identify them to the DNA group level by the use of biochemical tests. Twenty-three Danish clinical strains from 23 patients phenotypically identified to the A. calcoaceticus-A. baumannii complex were studied by ribotyping, plasmid profiling, and DNA/DNA hybridization. Multiple isolates were recovered from four patients. These were identical in each patient as judged by phenotype, ribotype and plasmic profile. Seventeen different ribotypes were observed among the 23 strains, and by using this method 19 out of the 23 strains could be identified to the DNA group level. Five strains were allocated to DNA group 2 (A. baumannii), eight to DNA group 3, and six to DNA group 13. These findings were confirmed by DNA/DNA hybridization. Two of the four unidentified strains were genotypically most closely related to but different from DNA groups 1 and 3. The last two strains were most closely related to DNA group 13. These four strains represent two new DNA groups within the A. calcoaceticus-A. baumannii-complex. One to four plasmids in the size range 2.1 kb- > kb were detected in 13 of the strains. Nine plasmid profiles were seen, indicating the usefulness of this typing method if the strains contain plasmids. The study also indicates that ribotyping is useful both for typing and for identification purposes, and that the genetic relationship in this area are more diverse than hitherto perceived. Taxonomic reconsiderations are warranted.

A phage typing system was developed for Listeria monocytogenes. Phages were released from clinical and from food strains of L. monocytogenes by mitomycin C induction. The system consists of two subsystems, one of which is used to type L. monocytogenes serotype 1 strains, containing 12 phages, and a second which is used to type serotype 4 strains, containing 14 other phages. For the serotype 1 subsystem, the reproducibility was > or = 90%, the typability 92%, and the discriminatory power, as judged by the discriminatory index, 80%. The corresponding figures for the serotype 4 subsystem were: reproducibility > or = 94%, typability 87%, and discriminatory index 87%. The performance of the whole typing system is sufficient for it to be used for screening purposes.

328 clinical isolates of Acinetobacter calcoaceticus from hospitalized patients and persons attending general practitioners were characterized according to biotype and resistance-pattern. 117 strains of biotype (b.) anitratus, 200 of b.lwoffi, 11 of b. haemolyticus and no strains of b. alcaligenes were found. B. anitratus was more frequently isolated from patients in hospitals than b.lwoffi; b.lwoffi was more often found in general practice. Strains of b. anitratus had more resistance traits than b.lwoffi. Strains of b. anitratus obtained in hospitals were more resistant than strains from general practice. No such difference was found with b.lwoffi.

Population analyses of the susceptibility to ciprofloxacin of eight strains of Pseudomonas aeruginosa, including mucoid strains and strains resistant to aminoglycosides or anti-Pseudomonas beta-lactams, were carried out. All strains were sensitive as judged by the broth-dilution technique, but four strains were found to yield resistant mutants with a frequency of less than 10(-6). Two strains yielded mutants homogeneously sensitive to ciprofloxacin at a level 8-16 times the MIC of the parent strains. Two other strains yielded mutants resistant to different higher concentrations of ciprofloxacin. One of these mutants was examined for production of ciprofloxacin-inactivating enzyme; no enzyme production could be detected. Cross-resistance was found with another quinolone antibiotic, ofloxacin, but not with aminoglycosides or beta-lactam antibiotics.

Time-kill studies were made with thirteen strains of Pseudomonas aeruginosa, comparing the activity of ciprofloxacin plus piperacillin with the combination of tobramycin and piperacillin. Synergism was demonstrated in an equal number of strains with both combinations. No antagonism was demonstrated. The reproducibility of the killing-curve method suggests that at least two different concentrations should be used and that a decrease in viable counts below 2 log10 after 24 hours does not exclude a synergistic action.