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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.

The in vitro susceptibility of 124 Xanthomonas maltophilia isolates was tested by four methods: Agar dilution (reference method), E-test, a disk diffusion and a tablet diffusion method. Trimethoprim-sulfamethoxazole had the highest activity against X. maltophilia, followed by a combination of aztreonam-clavulanic acid at different ratios, the ratio 1:1 being the most active with a susceptibility rate of 85% as compared to 2% for aztreonam alone. Addition of the beta-lactamase inhibitor tazobactam to piperacillin enhanced the rate of susceptible isolates from 31% to 53%, Relatively few isolates were susceptible to ciprofloxacin (27%) and gentamicin (9%). Generally, the disk diffusion method had a considerably higher frequency of "very major" discrepancies when compared with the agar dilution method than with the other methods. The susceptibility of X. maltophilia to trimethoprim-sulfamethoxazole and ciprofloxacin could reliably be determined by all the diffusion methods tested, but otherwise the agar dilution method is to be preferred. A standardized and reliable diffusion method for susceptibility testing of X. maltophilia remains to be found. Trimethoprim-sulfamethoxazole must be considered the drug of choice in the treatment of severe X. maltophilia infections. The combination aztreonam-clavulanic acid is promising, but must be proved in a clinical setting.

DNA-DNA hybridization studies on 42 stains presumptively identified as members of Flavobacterium group IIb and Flavobacterium breve indicated pronounced genotypic heterogeneity within these taxa. Three large groups highly related to the type strains of F. gleum, F. indologenes and F. breve respectively, and eight small groups were found. The group containing the type strain of F. breve was phenotypically indistinguishable from another genomic group, and these two groups were significantly separated from the other flavobacteria studied. The other nine genomic groups, representing Flavobacterium group IIb, could not with certainty be differentiated from each other by phenotypic characteristics, and there is no evidence indicating that these genomic groups differ from each other with respect to pathogenicity or ecology. Thus, it is suggested that for the time being the name "Flavobacterium group IIb" rather than specific epithets continue to be used for these bacteria.

Branhamella catarrhalis isolates from lower respiratory tract specimens collected in 1983/84 (n = 50) and 1988 (n = 30) were examined for beta-lactamase production. The percent of beta-lactamase-producing strains increased from 25% to 63% from 1983/84 to 1988. beta-lactamases from 30 strains could be typed, and of these 28 were of the BRO-1 type and two the BRO-2 type. The two beta-lactamase inhibitors, clavulanic acid and brobactam were very active against beta-lactamase extracts whether of the BRO-1 or the BRO-2 type. Susceptibility to a number of antimicrobial agents or combinations of agents was determined by a plate dilution method (MICs) and by a tablet diffusion method. The penicillin tablet (Rosco Neo-Sensitabs) was found to be useful in discriminating between beta-lactamase-producing and non-beta-lactamase- producing strains of B. catarrhalis.

Lactate concentrations in the pleural fluid of 50 patients were determined by the Monotest Lactate Kit. Lactate values were found higher in bacterial pleural infections than in cases of hydrothorax. Elevated levels were also found in most cases of histologically confirmed cases of pleural neoplasms and in some cases of non-bacterial pleuritis exsudativa . The highest levels were found in cases of empyema, but there was considerable overlapping between the groups. Pleural lactate thus appears to have little diagnostic value in the differential diagnosis between empyema and non-bacterial pleural effusions.

An attempt has been made to further characterize the Flavobacterium groups found in an earlier study by determining utilizable carbon-energy sources. As a preliminary, it was initially tried to define minimal growth factor requirements. It was found likely that most of the Flavobacterium groups have amino acid requirements, but a requirement for vitamins was also established for some strains. Testing Flavobacteria for carbon-energy sources by the replica plating method was found sufficiently technically difficult as to result in dubious readings at times. However, the overall results of the carbon-energy tests are in good agreement with the subdivision resulting from the previously performed biochemical tests, and the validity of the groups delimited in the earlier study is confirmed.

The results of blood cultures from a large number of Danish hospitals examined at Statens Seruminstitut in 1977 and 1978 have been analysed. Out of a total of 17, 518 cultures in 1977 and 17,820 in 1978, 1,626 from 830 patients and 1,544 from 832 patients yielded growth of pathogenic microorganisms. Cases of bacteremia with Enterobacteriaceae constituted approximately 50% of cases both in 1977 and 1978. Polymicrobial bacteremia occurred in 8.1% of bacteremic cases in 1977 and 7.5% of those in 1978. The microorganisms isolated and their frequencies were surprisingly similar to those found in 1965 and 1971 in an earlier investigation from this department. No change in antibiotic susceptibility was observed between 1977 and 1978. Retesting of 50 E. coli strains from 1965, 1971 and 1978 for susceptibility towards ampicillin, cephalotin, streptomycin and tetracycline indicated that no significant changes had occurred, contrary to the results of the earlier investigation. This fact stresses the importance of exploring the possibility of unnoticed methodological changes having occurred in the sensitivity testing systems before any conclusions can be drawn concerning apparent differences in antibiotic susceptibilities.

Two cases of CNS infections caused by Actinomyces israelii one a fatal case of brain abscess and the other a case of subdural empyema with complete recovery, are reported. One of the patients suffered from a congenital ventricular septal defect, and in both patients a pulmonary focus was found. When slow-growing bacteria such as Actinomyces may be suspected, it is important to cultivate anaerobically for at least five days.

The genus Flavobacterium has undergone a number of changes during the last years resulting in a revised description of the genus in which restriction of Flavobacterium to certain low G + C content species is proposed. The purpose of the present study has been to see how a collection of 184 clinical and environmental strains of Flavobacterium-like organisms isolated in Denmark would fit into this latest revision. It was found that most of the different biochemical patterns represented gradually merged into each other, with certain patterns, however, occurring more frequently than others and corresponding to the already described taxa: F. meningosepticum, Flavobacterium group IIb, F. breve, F. odoratum, F. multivorum and F. spiritivorum. The lately proposed subdivision of the genus will need either revision or extension or both to cope satisfactorily with the situation revealed in this study.

By means of crossed immunoelectrophoresis and crossed-line immunoelectrophoresis, antigens were compared from four strains and five strains, respectively, of Flavobacterium meningosepticum DNA hybridization groups I and II. Two reference antisera raised against the pooled antigens of each of the two DNA groups were used. The two reference antigen-antibody systems produced 81 and 51 immunoprecipitates, respectively, and within each group nearly all antigens were common to all strains, giving matching coefficients of 0.98-1.00. Group specific antigens were not detected. When antigens from individual strains from each group were compared to individual strains of the other group, however, 5-15 of the antigens were not cross-reactive, giving matching coefficients of 0.83-0.94. The antigenic relatedness between strains belonging to the two different groups was, therefore, just below the level commonly found for strains belonging to the same species. The results thus confirm the previously published differences detected by a DNA hybridization technique.

Antimicrobial susceptibilities of 52 strains of Flavobacterium meningosepticum were determined by an agar plate dilution technique and by a tablet diffusion method. No useful susceptibility differences between the two main DNA relatedness groups within F. meningosepticum were found. All strains produced beta-lactamase and were resistant to most beta-lactam antibiotics. Ciprofloxacin, rifampicin and clindamycin appear to be the most promising for treating infections caused by F. meningosepticum.

A case of Branhamella catarrhalis bacteremia without known infectious focus in a granulocytopenic patient is reported. There were no unusual clinical features of bacteremia, such as purpura, in this case which distinguished it from bacteremia due to other bacteria. The isolated strain was beta-lactamase producing. The patient recovered uneventfully on antibiotics; at the same time, however, the bone marrow was also normalized.

47 staphylococcal reference strains representing 13 species were tested for polymyxin sensitivity using tablet and disc diffusion methods. Corresponding MIC and IC50 values were determined with a plate dilution assay. Coagulase-positive strains were found to be more resistant towards polymyxin, and could thereby be separated from coagulase-negative strains. Furthermore, 100 recently isolated staphylococci from clinical specimens were tested, and again the coagulase-positive strains could be identified by their smaller tablet inhibition zones. We conclude that the polymyxin test is an aid in the fast differentiation of staphylococci in laboratories performing primary sensitivity tests.

DNA-DNA reassociation studies on 52 strains of Flavobacterium meningosepticum showed two main hybridization groups about 40-55% interrelated and comprising 4 and 48 strains respectively. The larger group could be further divided into four subgroups if differences in thermal stability of the reassociated duplexes were taken into consideration. Of a total of 20 strains isolated from cerebrospinal fluid, 18 were found in one subgroup of 28 strains, which also contained seven blood isolates. The results indicate that genetically defined subgroups within the species might differ with regard to pathogenic significance.

A case of pasteurella multocida meningitis in a 75-year old woman with chronic otitis media is reported. There were no unusual clinical features of meningitis in this case which distinguished it from meningitis due to other pyogenic bacteria. P. multocida is characterized by a distinctive biochemical pattern, and correct identification should not be difficult provided that the possibility of its occurrence in meningitis is kept in mind.

A total of 55 different antigens were demonstrated in pooled lysate of six Flavobacterium meningosepticum strains by crossed immunoelectrophoresis against homologous rabbit antibody. The six strains used represented the two DNA relatedness groups within this species. All 52 strains of F. meningosepticum investigated were found to cross-react with 53 to 55 of these F. meningosepticum reference antigens. Using this F. meningosepticum reference system, cross-reactions between the F. meningosepticum reference antigen and antigens from representatives of other Flavobacterium taxa were also investigated. A positive correlation was demonstrated between the number of cross-reacting antigens and the taxonomic relatedness of the investigated taxa as determined by other classification methods, including DNA-DNA hybridization. The crossed immunoelectrophoretic techniques were not useful in differentiating the two main DNA groups of F. meningosepticum.

Two cases of endocarditis caused by Cardiobacterium hominis are reported. There were no unusual clinical features of endocarditis in the patients except for the extensive valvular damage present at time of hospitalization. Properties with distinguish C. hominis from other fastidious, gram negative rods are oxidase and catalase reactions, indole production and carbohydrate fermentation pattern.

A case of Haemophilus parainfluenzae bacteremia without known infectious focus is reported. Phenotypically, the isolated strain is a typical H. parainfluenzae except for its ability to produce indole and beta-lactamase. Beta-lactamase producing H. parainfluenzae organisms are encountered occasionally, but to the best of our knowledge this is the first reported blood culture isolate with this ability. We propose a new biotype (IV) of H. parainfluenzae to accommodate strains that are indole, urease and ornithine decarboxylase positive.

Lactate concentrations in the cerebrospinal fluid of 104 patients were determined by the Monotest Lactate Kit. Lactate values were found higher in cases of bacterial meningitis than in patients not suffering from acute CNS disorders. Elevated lactate levels were also found in patients suffering from aseptic meningitis, septicemia, CNS trauma and cerebrovascular accidents, seizures and diabetes mellitus. The highest levels were found in cases of bacterial meningitis, but there was considerable overlapping between the groups. CSF lactate thus appears to have limited diagnostic value in the differential diagnosis between bacterial meningitis and other diseases with meningeal involvement.

A two-year survey at our hospital revealed 19 patients suspected of pulmonary infection from whom Neisseria meningitidis was isolated from lower respiratory tract specimens, in 17 cases in pure culture. Of the isolates, 16, two and one belonged to serogroup B, C and 29E, respectively. All except three patients suffered from chronic pulmonary disease, notably chronic bronchitis. It was found likely or possible that N. meningitidis was the causative agent in 12 patients with symptoms of acute bronchitis. Results of tests for complement-fixing antibodies against N. meningitidis (MAT) were generally not found to be useful when assessing the possible causative role of N. meningitidis in non-pneumonic pulmonary infections.

A three-month survey revealed 29 patients at our hospital with symptoms of acute pulmonary infection, from whom Branhamella catarrhalis was isolated from lower respiratory tract specimens, in 18 cases in pure culture. Approximately 2% of all respiratory tract specimens examined during the period yielded growth of B. catarrhalis. All except one patient suffered from chronic pulmonary disease, notably chronic bronchitis. A phenotypic comparison was made between 55 strains of B. catarrhalis, of which 50 were recent isolates from lower respiratory tract specimens, and 23 Neisseria strains representing Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria cinerea, Neisseria flavescens, Neisseria mucosa, Neisseria pharyngis, and Neisseria lactamica. The morphology of B. catarrhalis colonies is very characteristic, and when the diagnosis is suspected, testing for the ability to hydrolyze tributyrin may confirm it within hours. Ability to produce deoxyribonuclease is another property which differentiates B. catarrhalis from the Neisseria species. Otherwise, the combination of nitrate reduction and failure to produce acid from glucose, maltose, and sucrose establishes the diagnosis.

Fifty-two strains found to belong to Flavobacterium meningosepticum on the basis of DNA-DNA hybridization analyses were characterized and found to be phenotypically homogeneous. All strains were oxidase, catalase, indole, gelatinase and beta-galactosidase positive, and produced acid from glucose, mannose, fructose, maltose and mannitol; nitrate was not reduced. F. meningosepticum could be differentiated from Flavobacterium group IIb by its ability to produce beta-galactosidase, and by the latter taxon's ability to produce a bright yellow pigment in contrast to the weak or non-existing pigmentation of F. meningosepticum. Phenotypic characteristics that could differentiate between the two main DNA relatedness groups of F. meningosepticum were not discovered, wherefore a subdivision of the present species into two species cannot be recommended. Strains from the DNA relatedness groups comprising 19 of 20 CSF isolates were found to be able to grow at 40 degrees C and to produce a weak yellow pigment; in contrast, strains from the three other DNA relatedness groups were unable to grow at 40 degrees C and produced no pigment.

A prospective study evaluating the usefulness of polymyxin susceptibility testing in unveiling coagulase-negative S. aureus strains was undertaken. During a six months study period, 14 staphylococcal isolates from four patients were initially found to lack coagulase activity and to be polymyxin resistant; in comparison, approximately 1500 ordinary, coagulase-positive, polymyxin resistant S. aureus isolates were found during the same period. One isolate from each of the four patients together with a previously isolated coagulase-negative S. aureus strain from our own collection were further characterized. Two of the strains turned out to show delayed coagulase activity on retesting, while the other three strains failed to produce clotting in any coagulase assay. Judged by thermostable nuclease activity, phage typing and biochemical profiles these three coagulase-negative strains are bona fide S. aureus strains. The polymyxin test thus appears to be useful in disclosing S. aureus variants not identified as S. aureus by routinely performed coagulase assays.