Pseudomonas aeruginosa extracellular toxicity in infant mice and the protective effect of antitoxin A.

The toxin A-content of crude extracellular preparations from Pseudomonas aeruginosa has been measured by ELISA. Using infant mice as test animal, the toxicity of these preparations was evaluated. The LD 50 in infant mice was determined at 80 ng of purified toxin A in saline. With ten microliters of rabbit antitoxin A serum given together with purified toxin, the LD 50 increased to 2,500 ng. Generally there was no correlation between the LD 50 of the extracellular preparations, their quantity of toxin A as measured by ELISA, and the protective effect of antitoxin A.

Rapid identification of Legionella pneumophila zinc metalloprotease using chromogenic detection.

Strains of Legionella spp. produce extracellular proteases than can be detected using synthetic chromogenic peptides. Chromogenic tri- and tetrapeptides show a high degree of sensitivity, specificity and reagent stability when linked to para-nitroaniline (pNA). For example, SucOMe-Arg-Pro-Tyr.pNa (S-2586) is specifically hydrolysed by proteases of Legionella pneumophila and some other Legionella species. A paper disc method to sample protease directly from agar plates has been used to evaluate chromogenic peptides as reagents for diagnostic purposes. Strains of Legionella spp., Pseudomonas spp. and Enterobacteriaceae were examined, together with a recombinant Escherichia coli strain containing the cloned 38 kDa zinc metalloprotease from L. pneumophila, S-2586 was hydrolysed by 282 out of 283 L. pneumophila strains, and by the recombinant E. coli. Two of the six strains representing other Legionella species, and 22 of the 50 strains from the Pseudomonas group were also positive. No reaction was seen with any of the Enterobacteriaceae strains. Although there was functional homology between proteases from several bacterial groups, the high prevalence of S 2586-hydrolysing proteases within L. pneumophila indicates a potential usefulness for phenotypic identification.

Susceptibility pattern of Scandinavian Francisella tularensis isolates with regard to oral and parenteral antimicrobial agents.

Some recently introduced antimicrobial agents have only been incompletely evaluated for use in Francisella tularensis infections. The present study evaluated the susceptibility pattern of Scandinavian human, rodent, and hare F. tularensis isolates with respect to a selection of traditional as well as recently introduced antimicrobial agents. All strains were resistant to the following beta-lactams: penicillin, cephalexin, cefuroxime, ceftazidime, aztreonam, imipenem, and meropenem with minimal inhibitory concentrations > 32 mg/l. Against macrolides, a mixed susceptibility/resistance pattern appeared. All strains were susceptible to gentamicin, chloramphenicol, doxycycline, and four quinolones. Since the quinolones showed the lowest MIC values, and in addition give a good intracellular penetration, we conclude that future drugs to consider against tularemia should definitely include this group of antibiotics. The outpatient mode of antibiotic treatment is especially relevant as the Scandinavian variant of F. tularensis infection is nonlethal, usually pustuloglandular, and not septicemic. Therefore, oral drugs must be sought, and the quinolone group also satisfies this requirement.

Vesicular stomatitis virus infection enhances invasiveness of Salmonella typhimurium.

When mouse fibroblast L-929 cells were pre-infected with vesicular stomatitis virus, an enhancement of invasiveness by Salmonella typhimurium was observed. The effect was more pronounced when higher virus doses were used. Short-time (5 h) pre-incubation with virus caused a moderate enhancement of invasiveness. When virus pre-incubation time was increased to 8 h or 13 h, a further enhancement was observed. Results obtained after pre-incubation with UV inactivated virus were similar to that achieved by the short-time pre-incubation with the corresponding viable virus preparation. This indicates (i) an early phase of virus infection, when virus causes enhancement of invasiveness that is not dependent on viral nucleic acid induced metabolism, and (ii) a later phase, when virus-induced metabolism is necessary for the enhancement. When virus and bacteria were given concomitantly to infant mice, lethality was increased compared to groups that only received virus or bacteria. The data indicate that vesicular stomatitis virus aggravates infection with a facultatively intracellular bacterium, partly by enhancing the invasiveness of the bacteria.

Legionella antibodies in human and animal populations in Kenya.

Using a microagglutination method, domestic and wild animal sera, together with human patient and healthy blood donor sera, have been analysed for titres against various Legionella species, comprising fourteen different serogroups. Generally, the level of moderately elevated titres, i.e. greater than or equal to 64, was low for all the aforementioned serum groups. Within the domesticated animals, cattle, pigs and dogs presented a much lower prevalence in Kenya than found elsewhere, whereas it was the other way round for goats. Human sera, either from patients or from healthy donors, did not react against L. pneumophila serogroups 1, 6, or 3, which in that sequence are the most common L. pneumophila serogroups in Europe, and in other geographic areas where legionellosis is common. High titres of greater than or equal to 256 against L. pneumophila serogroup 6 (two cattle) or against L. bozemanii strain Mi-15 (two cattle, one dog) indicate that although the epidemiological picture may be different from other parts of the world, Legionella infections exist in Kenya as well.

Field investigations of tularemia in Norway.

In Norway, tularemia is a common disease in small rodent and hare populations, where large outbreaks can be observed. In humans, the yearly number of cases is low, usually less than ten, with peaks up to 44 recorded in recent years. Serological investigations on hunters and healthy school children nevertheless indicate, with up to 4.7% positivity in the latter group, that Francisella tularensis low-grade infection is widespread. F. tularensis in co-culture with amoebae, e.g. Achantamoeba castellanii, may grow after internalization and kill the amoeba. As with Legionella, Francisella virulence may be enhanced after protozoan ingestion. This suggests a mechanism that can explain the pattern of dissemination and infection in our region.

Antibiotic resistance of pneumococci in Norway.

A collection of 178 pneumococcal isolates found in Norway during the period 1987-1994 were tested for their susceptibility to benzylpenicillin, macrolides (azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin), fluoroquinolones (ciprofloxacin, sparfloxacin), imipenem, chloramphenicol, and vancomycin by a standard agar dilution procedure. To benzylpenicillin, two strains (1%) showed resistance and 14 strains (8%) intermediate susceptibility. Towards erythromycin, eight strains (4%) showed resistance and four strains (2%) intermediate susceptibility. Cross-resistance was demonstrated among the macrolides. Among the fluoroquinolones, intermediate susceptibility occurred with 42% of the isolates for sparfioxacin and 90% for ciprofloxacin; to the latter 5.1% proved resistant. The sum of intermediate and highly resistant isolates was 53% for chloramphenicol. Both penicillin-resistant strains were isolated during the last 2 years of collection and came from patients of non-Norwegian ethnic background. Imported strains appeared over represented among the strains resistant to penicillin and macrolides. Only imipenem and vancomycin showed full susceptibility for all pneumococci tested. An over representation of serogroup 6 strains was apparent among the strains with intermediate susceptibility and high resistance to benzylpenicillin. It is apparent that high-level resistance has, not so far, become a difficult problem in Norway. Nevertheless, the situation requires monitoring of the resistance level, particularly in meningitis and septic patients, and certainly in patients who cntail a higher than usual possibility of acquiring pneumococci from pools of resistant strains outside Norway (visitors, immigrants and recent returness from abroad).

Ground penetrating radar surveys to locate 1918 Spanish flu victims in permafrost.

The "Spanish Flu" killed over 40 million people worldwide in 1918. Archival records helped us identify seven men who died of influenza in 1918 and were interred in Longyearbyen, Svalbard, Norway, 1,300 km from the North Pole. Ground Penetrating Radar (GPR) was used successfully, in a high-resolution field survey mode, to locate a large excavation with seven coffins, near the existing seven grave markers. The GPR indicated that the ground was disturbed to 2 m depth and was frozen below 1 m. Subsequent excavation showed that: a) the GPR located the position of the graves accurately, b) the coffins were buried less than 1 m deep, and c) that the frozen ground was 1.2 m deep where the coffins were located. The GPR assisted in planning the exhumation, safely and economically, under the high degree of containment required. Virologic and bacteriologic investigations on recovered tissues may give us an opportunity to isolate and identify the micro-organisms involved in the 1918 influenza and expand our knowledge on the pathogenesis of influenza.

[Bacteremia in the county of Ribe]. / Bakteriaemi i Ribe Amt.

The Department of Microbiology at the Central Hospital of Esbjerg, established in 1987, serves the five hospitals in Ribe county, Denmark. From early on, the department has endeavoured to guide the hospital's antimicrobial policy. In order to investigate whether this involvement had any measurable impact on the antimicrobial resistance pattern in our region, we compared the resistance patterns of 212 strains isolated from the blood of bacteraemic patients in 1988 to those of 317 strains isolated in 1992. No increase in antibiotic resistance was revealed. This is noteworthy since new specialties have been established at the Central Hospital during this period, with an increased number of patients requiring antimicrobial therapy. It is important to survey the antibiotic resistance pattern closely, and that this is done locally.

Chlamydophila pneumoniae diagnostics: importance of methodology in relation to timing of sampling.

The diagnostic impact of PCR-based detection was compared to single-serum IgM antibody measurement and IgG antibody seroconversion during an outbreak of Chlamydophila pneumoniae in a military community. Nasopharyngeal swabs for PCR-based detection, and serum, were obtained from 127 conscripts during the outbreak. Serum, drawn many months before the outbreak, provided the baseline antibody status. C. pneumoniae IgM and IgG antibodies were assayed using microimmunofluorescence (MIF), enzyme immunoassay (EIA) and recombinant ELISA (rELISA). Two reference standard tests were applied: (i) C. pneumoniae PCR; and (ii) assay of C. pneumoniae IgM antibodies, defined as positive if >or=2 IgM antibody assays (i.e. rELISA with MIF and/or EIA) were positive. In 33 subjects, of whom two tested negative according to IgM antibody assays and IgG seroconversion, C. pneumoniae DNA was detected by PCR. The sensitivities were 79%, 85%, 88% and 68%, respectively, and the specificities were 86%, 84%, 78% and 93%, respectively, for MIF IgM, EIA IgM, rELISA IgM and PCR. In two subjects, acute infection was diagnosed on the basis of IgG antibody seroconversion alone. The sensitivity of PCR detection was lower than that of any IgM antibody assay. This may be explained by the late sampling, or clearance of the organism following antibiotic treatment. The results of assay evaluation studies are affected not only by the choice of reference standard tests, but also by the timing of sampling for the different test principles used. On the basis of these findings, a combination of nasopharyngeal swabbing for PCR detection and specific single-serum IgM measurement is recommended in cases of acute respiratory C. pneumoniae infection.

Extracellular proteases from Legionella.

Bacterial endopeptidase (proteinase) activity can be specifically registered using synthetic chromogenic peptides. This method does not replace, but adds to the previously existing methods for measuring proteolytic activity. The use of concentrated broth filtrates from Legionella bacteria on a number of different peptides has permitted the establishment of peptide hydrolysis patterns. To some extent these patterns appear species specific. Thus, a close similarity is demonstrated between strains belonging to Legionella pneumophila, as well as a difference between L. pneumophila and other species within the genus Legionella. L. micdadei strain Tatlock does not present extracellular endopeptidase activity. Recently, a number of cell- or cell wall-bound endopeptidases has also been registered. They are found in all Legionella strains hitherto assayed, including the Tatlock strain.

[Acute bronchitis in adults. Clinical findings, microorganisms and use of antibiotics]. / Akutt bronkitt hos voksne. Kliniske funn, mikroorganismer og bruk av antibiotika.

Among 72 adult patients with a diagnosis of acute bronchitis, serological investigation established the presence of an aetiologic agent in 29 (40%). Influenza virus was the most common pathogen. Seven patients had bacterial infection, caused by pneumococci in four patients and Mycoplasma pneumoniae in three. Five of the patients had pneumonia as diagnosed by radiography, and mycoplasmal aetiology was established in one of these. Altogether, 11 patients either had bacterial infection or radiographic pneumonia. Although the doctors' recording of wheezes was strongly associated with prescription of antibiotics (p < 0.0001), wheezes were heard only in two of the 11 patients with pneumonia or bacterial infection, compared with 30 of the 61 patients with viral or unspecified bronchitis. The median value of C-reactive protein (CRP) was 52 mg/l in the 11 patients, significantly higher than < 11 mg/l in the 61 other patients (p < 0.0001). The corresponding values for erythrocyte sedimentation rate were 45 and 14 mm/h (p < 0.0005). The results indicate that certain patients with acute bronchitis should be treated with antibiotics, and that the erythrocyte sedimentation rate and the CRP-test may be useful in detecting which patients this applies to.

[Chlamydia pneumoniae--pathogenesis and perspectives]. / Chlamydia pneumoniae--patogenese og perspektiver.

Chlamydia pneumoniae, a Gram-negative bacterium, formerly named TWAR but identified as a distinct species since 1988, is now considered to be the most common agent of chlamydial infection in Scandinavia. C pneumoniae has a different tissue trophism from that of Chlamydia trachomatis, since C pneumoniae may infect bronchi and lungs, macrophages, monocytes, and endothelial cells. C pneumoniae, like other chlamydiae, has a slow, intracellular life cycle. An absence of reaction from the host cells, combined with scant tissual reaction owing to the low endotoxic activity of chlamydial lipopolysaccharide, may help to explain the usually discreet clinical picture. Atherosclerosis and coronary heart disease may follow chronic lung infection, and acute pneumonic episodes can trigger myocardial infarct. Asymptomatic infection with C pneumoniae is widespread. Intriguing diagnostic questions are the possible existence of a non-pathogenic carrier state, and the conceivable sensitization of the host with respect to a heterotypic, secondary chlamydial infection by, for example, C trachomatis, giving rise to an aggravated clinical picture. Early antibiotics are indicated to avoid the development of chronic disease.

Legionella extracellular protease activity on chromogenic peptides: a simplified procedure for biochemical enzyme identification.

Legionella strains produce extracellular proteases. One method to demonstrate these is through the activity of broth culture filtrates on para-nitroanilide (pNA)-derivatized peptides. Previously, this method has comprised a 100-times concentration of the filtrates, in order to bring the protease activity, as measured by the liberation of free pNA from hydrolysed peptides, up to easily recordable values. By introducing a diazotation step, the sensitivity of the test has been increased sufficiently to omit the time-consuming concentration procedure. Accordingly, the analysis of extracellular Legionella proteases by pNA-derivatized peptides has become rapid and straight-forward.

Demonstration of Escherichia coli heat-labile enterotoxin using bacterial cell sonicates.

Agar-plate colonies of Escherichia coli were suspended in 0.5 ml distilled water and sonicated for 15 s. The sonicate was tested for E. coli heat-labile enterotoxin by an enzyme-linked immunosorbent assay (ELISA). Sonication of enterotoxin-producing E. coli cells gave detectable toxin concentrations, which correlated well with the results obtained using culture broth supernatants for toxin detection in the ELISA. By using sonicates of isolated bacterial colonies from primary isolation agar plates, the subcultivation on agar plates or in culture broth tubes can be omitted. This may reduce the possibility of losing the plasmids coding for enterotoxin production. The time needed for analysis of suspected toxin producing E. coli strains will be reduced accordingly. Suspensions with bacterial counts of 10(6) per ml or more of E. coli, gave detectable levels of heat-labile enterotoxin in the sonicates.

Bacterial antigen detection in body fluids: methods for rapid antigen concentration and reduction of nonspecific reactions.

We sought procedures which would allow a rapid concentration in high yield of bacterial antigens from tissue fluids of patients and which could be applied also to protein-rich fluids like serum. Ethanol precipitation at a subzero temperature with albumin added as an antigen coprecipitant made it possible to achieve a more than 20-fold concentration of antigen in 15 min and a 200-fold concentration in 45 min. Heat-stable antigens could be concentrated from protein-rich fluids (like serum) after the sample had been deproteinized by boiling. Such heating (100 degrees C, 3 min) also liberated bacterial polysaccharides from antibody complexes and elminated the nonspecific interference of serum in enzyme-linked immunosorbent assay.

Coreactivity of Legionella pneumophila immune sera in the Mycoplasma pneumoniae complement fixation test.

Sheep and guinea pigs were immunized with cellular and extracellular antigen from Legionella pneumophila bacteria. After immunization, the animals developed immunoglobulin G titers against the immunizing agent. The same sera were also tested in a Mycoplasma pneumoniae complement fixation test. All the preimmunization sera from sheep showed positive M. pneumoniae complement fixation tests of varying titers, with significant antibody rises in two of five sheep as a result of the Legionella immunizations. In contrast to the sheep, all the guinea pigs were negative in the M. pneumoniae complement fixation test, both in their preimmunization sera and after completion of the Legionella immunizations. The results obtained with the sheep sera may be explained as a nonspecific booster effect of Legionella bacteria upon previously elicited immune responses.