Epidemic of Mycoplasma pneumoniae infection in Denmark, 2010 and 2011.

Denmark experienced two waves of Mycoplasma pneumoniae infection during autumn and early winter in 2010 and 2011, respectively. Both affected the whole country. The proportion of positive results was almost the same for both, indicating that the two waves were probably of equal size. High macrolide consumption during the epidemics did not seem to affect levels of macrolide resistance in M. pneumoniae, which remain low in Demark (1% to 3%).

Effect of Legionella pneumophila sonicate on killing of Listeria monocytogenes by human polymorphonuclear neutrophils and monocytes.

Legionella pneumophila shares with other intracellular pathogens the ability to resist intracellular killing within phagocytes. An increasing number of cellular components of L. pneumophila are proposed as pathogenic factors of the organism. At the site of infection, the phagocytic cells will be exposed to bacterial components, either expressed on the surface of the organisms or released in the environment upon cell lysis. In this study, we have investigated the effect of water-soluble bacterial components present in L. pneumophila sonicate on the phagocytosis and bactericidal activity of human polymorphonuclear neutrophils and monocytes. Preincubation of neutrophils with L. pneumophila sonicate did not affect phagocytosis of L. monocytogenes, whereas Listeria killing was significantly inhibited at sonicate concentrations of 1 and 2 mg/ml. The phenol phase of a phenol-water extraction, containing most of the lipopolysaccharide (LPS), had no inhibitory effect on the listericidal activity of neutrophils. Killing of Listeria by monocytes was inhibited in a similar manner. The inhibitory activity was mainly recovered in the sonicate fraction above 100 kDa, suggesting that components organized in larger molecular complexes are most likely to represent the inhibitory factors. The inhibitory activity of L. pneumophila sonic extract appears to be related to inhibition of killing mechanisms since uptake of Listeria was not affected by the sonicate. Our observations indicate that as Legionella infection progresses, bacterial components liberated by cell lysis could exert a detrimental effect on the antimicrobial function of phagocytes, stressing the importance of early treatment of Legionnaires' disease to reduce bacterial numbers in the infected tissues.

Cloning and expression of the Legionella micdadei "common antigen" in Escherichia coli.

To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly with a monospecific rabbit antiserum raised against L. micdadei "common antigen" (CA), and an additional 13 K L. micdadei protein. The region encoding these two proteins from the 17 kb recombinant plasmid (pBA 2) was subcloned in pBGS18+. The DNA sequence of the CA encoding region in the 2.7 kb subcloned fragment will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function.

Performance of four different indirect enzyme-linked immunosorbent assays (ELISAs) to detect specific IgG, IgA, and IgM in Legionnaires' disease.

Currently recommended methods in Legionnaires' disease serology are based upon crude whole-cell antigenic preparations. To investigate whether purified antigens would perform better in a given diagnostic test for antibodies against Legionella pneumophila, we compared the performance of three antigenic preparations of L. pneumophila serogroup 1 consisting of outer membrane protein (OMP), flagellin (FLA), and lipopolysaccharide (LPS) to a sonic extract (SON) in indirect immunosorbent assay (ELISA) measuring both IgG, IgA, and IgM. The reactivity of sera from 20 patients with culture-verified Legionnaires' disease and sera from 12 patients with pneumonia and a diagnostic rise in titre by a microagglutination test (MA) was studied. Our results indicated that the SON IgA assay was the most sensitive test in both groups of patients. The LPS IgG and IgM assays, however, were the most specific tests, closely followed by the corresponding SON tests. By combining two individual assays, a maximum nosographic sensitivity of 85% could be obtained. Whereas no benefit of using purified outer membrane protein or flagella instead of a sonic extract in the indirect ELISAs was found, the LPS antigen provided a sensitive and specific alternative to the sonic extract.

Cross-reactive Legionella antigens and the antibody response during infection.

In order to define cross-reactive Legionella antigens suitable for diagnostic purposes, we investigated sonicate antigens from two Legionella species, including two serogroups of L. pneumophila. The antigens were reacted with heterologous and homologous rabbit antisera in Western blot. Sera from seven patients with culture-verified L. pneumophila infection and nine patients with serologically confirmed L. micdadei infection were also investigated for reactivity with the corresponding antigens. Among the cross-reactive Legionella antigens defined, non-specific reactivity in patients' sera with the 58-kDa common antigen (CA) was noted. Specific reactions were observed with the Legionella flagellum antigen and with the macrophage infectivity potentiator (Mip) protein; with both antigens, however, the reactive sera were too few to suggest the use of a single antigen in a diagnostic test.

The Legionella micdadei flagellin: expression in Escherichia coli K 12 and DNA sequence of the gene.

To study the structure and function of the Legionella flagellum, we screened a genomic L. micdadei library in Escherichia coli for expression of the flagellin (Fla) subunit. One recombinant clone, JM105 (pHI5588), producing a truncated Fla protein of 40.5 kDa was identified. The plasmid pHI5588 carried a L. micdadei DNA insert of 5 kb, containing ca 95% of the fla gene. The complete DNA sequence of the L. micdadei fla gene was obtained by combining sequence data from pHI5588 with results using a polymerase chain reaction-based system for genome walking (vectorette PCR). The L. micdadei fla gene shared a high degree of homology with other flagellin genes in the amino- and carboxy termini, whereas the central region was found to be nonconserved. The fla sequence will facilitate the cloning of Fla proteins from other Legionella species and the study of flagella in the pathogenesis of Legionnaires' disease.

The E. coli immunosorbent as used in serodiagnosis of Legionella infections studied by crossed immunoelectrophoresis.

In this study we investigated an immunosorbent, E. coli blocking fluid (BF), proposed for use in the Legionella Indirect Fluorescent Antibody Test (IFA). With crossed immunoelectrophoresis (CIE) of clinically relevant Legionella species, only one heat-stable antigen (no. 1) cross-reacted with the BF preparation. Patients' sera with elevated Legionella IFA titres did not react with this antigen in CIE. Out of 23 IFA positive patients' sera, six had titres lowered significantly to negative, when BF was applied as serum diluent for the titration (IFA BF negative sera). All six sera were negative in the micro agglutination test (MA). None of the IFA BF negative sera contained any Legionella precipitins in CIE, whereas nine out of the remaining 17 IFA BF positive sera unchanged by BF contained one or more precipitins. CIE results could not explain the effect of BF in Legionella IFA, and further studies are needed to sufficiently define the use of immunosorbents in diagnostic Legionella serology.

Susceptibility of Legionella species to five antibiotics and development of resistance by exposure to erythromycin, ciprofloxacin, and rifampicin.

The minimal inhibitory concentration (MIC) values of erythromycin, ciprofloxacin, ofloxacin, rifampicin, and clindamycin were determined for 56 strains of Legionella pneumophila (38 patient, 3 environmental, and 15 reference strains) and 37 strains of other Legionella species (7 patient, 2 environmental, and 28 reference strains) using the epsilon-test system on BCYEalpha agar plates. High-level resistance (MIC > or = 4 microg/mL) was found only for clindamycin (57%), with MIC values ranging from 0.25-32 microg/mL. Low-level resistance was found for erythromycin (18%) (0.5 < MIC < 8), ciprofloxacin (1%) (1 < MIC < 4), and clindamycin (40%) (0.5 < MIC < 4), but not for ofloxacin and rifampicin. MIC50 for the 45 Danish clinical Legionella strains were 0.25 microg/mL (erythromycin), 0.25 microg/mL (ciprofloxacin), 0.19 microg/mL (ofloxacin), below 0.016 microg/mL (rifampicin), and 4 microg/mL (clindamycin). Of the clinical isolates, 64% were resistant to clindamycin. There were no significant differences between the MIC50 values obtained for clinical and nonclinical Legionella strains. Selected susceptible strains were exposed to increasing concentrations of either erythromycin, ciprofloxacin, or rifampicin to select for resistance. Isolates resistant to erythromycin (MIC 0.75-32 microg/mL) or ciprofloxacin (MIC 2-3 microg/mL) could be selected by a two-step procedure. One single strain recovered from media containing 50 microg/mL of erythromycin had an MIC value higher than 256 microg/mL to erythromycin. In contrast, high-level resistance toward rifampicin with MIC > or = 256 microg/mL developed as a one-step phenomenon in several strains.

Adult bacterial meningitis: aetiology, penicillin susceptibility, risk factors, prognostic factors and guidelines for empirical antibiotic treatment.

Episodes of adult bacterial meningitis (ABM) at a Danish hospital in 1991-2000 were identified from the databases of the Department of Clinical Microbiology, and compared with data from the Danish National Patient Register and the Danish National Notification System. Reduced penicillin susceptibility occurred in 21 (23%) of 92 cases of known aetiology, compared to an estimated 6% in nationally notified cases (p < 0.001). Ceftriaxone plus penicillin as empirical treatment was appropriate in 97% of ABM cases in the study population, and in 99.6% of nationally notified cases. The notification rate was 75% for penicillin-susceptible episodes, and 24% for penicillin-non-susceptible episodes (p < 0.001). Cases involving staphylococci, Pseudomonas spp. and Enterobacteriaceae were under-reported. Among 51 ABM cases with no identified risk factors, nine of 11 cases with penicillin-non-susceptible bacteria were community-acquired. Severe sequelae correlated independently with age, penicillin non-susceptibility, mechanical ventilation and non-transferral to a tertiary hospital (p < 0.05; logistic regression). Other factors that correlated with severe sequelae by univariate analysis only were inappropriate clinical handling, abnormal consciousness, convulsions and nosocomial infection. Overall, the data indicated that neither age alone, community-acquired infection nor absence of identified risk factors can predict susceptibility to penicillin accurately. Recommendations for empirical antibiotic treatment for ABM should not be based exclusively on clinical notification systems with possible unbalanced under-reporting.

Epidemiology of Pseudomonas aeruginosa in cystic fibrosis and the possible role of contamination by dental equipment.

Cystic fibrosis (CF) patients often suffer from Pseudomonas aeruginosa lung infection yet the source of this organism is not known. In order to determine whether CF patients might be contaminated with P. aeruginosa from dental equipment, a total of 103 water samples from 25 dental sessions in Frederiksberg Municipal Oral Health Care Service were examined. Three samples (2.9%) were positive for P. aeruginosa. Three hundred and twenty-seven water samples from 82 dental sessions from various other Municipal Oral Health Services in Denmark, attended by CF patients, were also examined. Eighteen of 327 samples (5.5%) from nine sessions (11%) were positive for P. aeruginosa. In one case, genotypically identical (RFLP, pulsed-field gel electrophoresis) P. aeruginosa strains were found both in water from the dental equipment and in the CF patients sputum. This indicates a small risk for acquiring P. aeruginosa from dental sessions, which is however equal to the yearly 'natural background' incidence (1-2%) of acquisition of P. aeruginosa in our CF centre.

Haemophilus segnis endocarditis.

Haemophilus segnis is a rarely recognised commensal in the oropharynx. We wish to report the first published case of endocarditis caused by H. segnis. The patient, a 76-year-old female did not recover until after 2 courses of ampicillin given for a total of 57 days. In the second course of treatment, ampicillin was combined with 10 days of netilmicin therapy.

Dysgonic fermenter 3-associated abscess in a diabetic patient.

We report a case in which a strain of the U.S.A. Centers for Disease Control (CDC) dysgonic fermenter (DF) 3, together with Citrobacter freundii, was isolated from an abscess in a diabetic patient. DF 3 may be easily overlooked due to its fastidious nature, a characteristic shared with two former DF groups now placed in the genus Capnocytophaga. To our knowledge, this is the first European case report of DF 3-associated infection.

[Bacterial infections as complications of dog bites]. / Bakterielle infektioner som komplikation til hundebid.

Dog bites may result in serious bacterial infections with e.g. the gram-negative rods Capnocytophaga canimorsus and Pasteurella multocida. Human disease caused by these microorganisms can be complicated by acute development of septicaemia and/or meningitis followed by disseminated intravascular coagulation syndrome, peripheral gangrene and renal failure. The mortality of C. canimorsus septicaemia is about 23-31%. These severe infections are most often reported in immunocompromised patients and occur a few days after the bite. By reviewing the literature it is concluded that the broadest prophylactic coverage is obtained by amoxicillin/clavulanic acid and that antibiotic prophylaxis should be given to all immunocompromised patients experiencing a dog bite. Moreover, prophylactic treatment should be initiated for all patients with greater penetrating wounds and those involving the hands.

Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein.

After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L. pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern. Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L. micdadei has extensive homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).

Sequencing of the rpoB gene in Legionella pneumophila and characterization of mutations associated with rifampin resistance in the Legionellaceae.

Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in the rpoB gene are known to cause rifampin resistance in Escherichia coli and Mycobacterium tuberculosis, and the purpose of the present study was to investigate a possible similar resistance mechanism within the members of the family Legionellaceae. Since the RNA polymerase genes of this genus have never been characterized, the DNA sequence of the Legionella pneumophila rpoB gene was determined by the Vectorette technique for genome walking. A 4,647-bp DNA sequence that contained the open reading frame (ORF) of the rpoB gene (4,104 bp) and an ORF of 384 bp representing part of the rpoC gene was obtained. A 316-bp DNA fragment in the center of the L. pneumophila rpoB gene, corresponding to a previously described site for mutations leading to rifampin resistance in M. tuberculosis, was sequenced from 18 rifampin-resistant Legionella isolates representing four species (L. bozemanii, L. longbeachae, L. micdadei, and L. pneumophila), and the sequences were compared to the sequences of the fragments from the parent (rifampin-sensitive) strains. Six single-base mutations which led to amino acid substitutions at five different positions were identified. A single strain did not contain any mutations in the 316-bp fragment. This study represents the characterization of a hitherto undescribed resistance mechanism within the family Legionellaceae.

Ribotyping on small-sized spirochetes isolated from subgingival plaque.

In the present study DNA restriction patterns and corresponding ribotypes of 17 subgingival small-sized spirochetes (1:2:1 and 2:4:2 isolates), 2 Treponema socranskii strains and two Treponema denticola strains were examined. Purified chromosomal DNA was digested by BamHI, HindIII, PstI and ClaI. The DNA fragments were separated in a horizontal slab of 0.7% agarose containing ethidium bromide and transferred by nylon membranes. Hybridization was carried out with digoxigenin-labelled copy DNA of 16S and 23S ribosomal RNA from Escherichia coli. Depending on the restriction endonuclease used, up to 4 distinct bands were observed for the 2:4:2 isolates and the T. denticola strains. For each of the endonucleases used, identical band patterns were always observed for this group of isolates, and these patterns differed persistently from the T. denticola strains. For the 1:2:1 strains, up to 11 distinct bands were observed after digestion with HindIII, whereas a maximum of 6 bands were observed when PstI or ClaI was used. By using ClaI, the examined 1:2:1 isolates were separated into 8 groups, whereas PstI and HindIII separated these isolates into 5 groups. The ribotyping showed that the tested 1:2:1 spirochetes were more heterogeneous than the 2:4:2 spirochetes examined.

Immunochemical characterization of and isolation of the gene for a Borrelia burgdorferi immunodominant 60-kilodalton antigen common to a wide range of bacteria.

By crossed immunoelectrophoresis and Western blotting (immunoblotting), it was shown that Borrelia burgdorferi expresses the 60-kilodalton Common Antigen (CA) that is cross-reactive with an equivalent antigen in a wide range of remotely related bacteria. B. burgdorferi CA is strongly immunogenic. A B. burgdorferi genomic library was constructed by using a plasmid cloning system. Escherichia coli recombinants were screened for expression of immunodominant B. burgdorferi antigens. One of the recombinant clones expressed the 60-kilodalton CA of B. burgdorferi. The DNA region encoding B. burgdorferi CA was localized on a 2.3-kilobase fragment of the plasmid pKH1. CA may have pathogenetic implications in Lyme borreliosis, since the CA of mycobacteria recently has been shown to play a role in the etiology of experimental autoimmune arthritis. The extensive cross-reactivity of this antigen may account for the low diagnostic specificity of the currently used serological tests in Lyme borreliosis.

Identification of mip-like genes in the genus Legionella.

The mip gene of Legionella pneumophila serogroup 1 strain AA100 encodes a 24-kilodalton surface protein (Mip) and enhances the abilities of L. pneumophila to parasitize human macrophages and to cause pneumonia in experimental animals. To determine whether this virulence factor is conserved in the genus Legionella, a large panel of Legionella strains was examined by Southern hybridization and immunoblot analyses for the presence and expression of mip-related sequences. Strains representing all 14 serogroups of L. pneumophila contained a mip gene and expressed a 24-kilodalton Mip protein. Although the isolates of the 29 other Legionella species did not hybridize with mip DNA probes under high-stringency conditions, they did so at reduced stringency. In support of the notion that these strains possess mip-like genes, these species each expressed a protein (24 to 31 kilodaltons in size) that reacted with specific Mip antisera. Moreover, the cloned mip analog from Legionella micdadei encoded the cross-reactive protein. Thus, mip is conserved and specific to L. pneumophila, but mip-like genes are present throughout the genus, perhaps potentiating the intracellular infectivity of all Legionella species.