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%).

Pseudomonas aeruginosa and risk of death and exacerbations in patients with chronic obstructive pulmonary disease: an observational cohort study of 22 053 patients.

OBJECTIVES: The role of Pseudomonas aeruginosa in the long-term prognosis of chronic obstructive pulmonary disease (COPD) is unknown. The purpose of this study was to determine whether P. aeruginosa is associated with increased risk of exacerbations or death in patients with COPD. METHODS: This is a multiregional epidemiological study based on complete data on COPD outpatients between 1 January 2010 and 31 October 2017 and corresponding microbiology and national register data. Time-dependent Cox proportional hazards models and propensity matching was used to estimate hospitalization-demanding exacerbations and death after 2 years, separately and in combination. RESULTS: A total of 22 053 COPD outpatients were followed for a median of 1082 days (interquartile-range: 427-1862). P. aeruginosa was present in 905 (4.1%) patients. During 730 days of follow-up, P. aeruginosa strongly and independently predicted an increased risk of hospitalization for exacerbation or all-cause death (HR 2.8, 95%CI 2.2-3.6; p <0.0001) and all-cause death (HR 2.7, 95%CI 2.3-3.4; p <0.0001) in analyses adjusted for known and suspected confounders. The signal remained unchanged in unadjusted analyses as well as propensity-matched subgroup analyses. Among patients 'ever colonized' with P. aeruginosa, the incidence of hospital-demanding exacerbations doubled after the time of the first colonization. CONCLUSIONS: COPD patients in whom P. aeruginosa can be cultured from the airways had a markedly increased risk of exacerbations and death. It is still not clear whether this risk can be reduced by offering patients targeted antipseudomonal antibiotics. A randomized trial is currently recruiting patients to clarify this (ClinicalTrials.gov: NCT03262142).

The impact of HIV-1 co-infection on long-term mortality in patients with hepatitis C: a population-based cohort study.

OBJECTIVE: To investigate the impact of HIV co-infection on mortality in patients infected with hepatitis C virus (HCV). METHODS: From a nationwide Danish database of HCV-infected patients, we identified individuals diagnosed with HCV subsequent to an HIV diagnosis. For each co-infected patient, four control HCV patients without HIV were matched on age, gender and year of HCV diagnosis. Data on comorbidity, drug abuse, alcoholism and date of death were extracted from two healthcare databases. We constructed Kaplan-Meier curves and used Cox regression analyses to estimate mortality rate ratios (MRRs), controlling for comorbidity. RESULTS: We identified 483 HCV-HIV co-infected and 1932 HCV mono-infected patients, yielding 2192 and 9894 person-years of observation with 129 and 271 deaths, respectively. The 5-year probability of survival was 0.74 [95% confidence interval (CI) 0.69-0.80] for HCV-HIV co-infected patients and 0.87 (95% CI 0.85-0.89) for HCV mono-infected patients. Co-infection was associated with substantially increased mortality (MRR 2.1, 95% CI 1.7-2.6). However, prior to the first observed decrease in CD4 counts to below 300 cells/muL, HIV infection did not increase mortality in HCV-infected patients (MRR 0.9, 95% CI 0.5-1.50). CONCLUSIONS: HIV infection has a substantial impact on mortality among HCV-infected individuals, mainly because of HIV-induced immunodeficiency.

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.

Killing curve activity of ciprofloxacin is comparable to synergistic effect of beta-lactam-tobramycin combinations against Haemophilus species endocarditis strains.

Nine Haemophilus species strains, all beta-lactamase negative, isolated from patients with endocarditis were tested in killing curve experiments. Antibiotics used were penicillin, amoxicillin, aztreonam alone and in combination with tobramycin, as well as ciprofloxacin alone. Synergism between beta-lactams and tobramycin with reduction of colony counts to zero was seen after 24 h for H. influenzae, H. parainfluenzae and H. segnis strains. Ciprofloxacin was as effective as beta-lactam-tobramycin combinations. The H. aphrophilus strain was not killed as effectively as other strains by any of the antibiotics.

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.

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.

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.

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.

Sequence analysis of the Legionella micdadei groELS operon.

A 2.7 kb DNA fragment encoding the 60 kDa common antigen (CA) and a 13 kDa protein of Legionella micdadei was sequenced. Two open reading frames of 57,677 and 10,456 Da were identified, corresponding to the heat shock proteins GroEL and GroES, respectively. Typical -35, -10, and Shine-Dalgarno heat shock expression signals were identified upstream of the L. micdadei groEL gene. Further upstream, a poly-T region, also a feature of the sigma 32-regulated Escherichia coli groELS heat shock operon, was found. Despite the high degree of homology of the expression signals in E. coli and L. micdadei, Western blot analysis with an L. micdadei specific anti-groEL antibody did not reveal a significant increase in the amount of the GroEL protein during heat shock in L. micdadei or in the recombinant E. coli expressing L. micdadei GroEL.

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.

Outbreak of listeriosis caused by infected beef meat from a meals-on-wheels delivery in Denmark 2009.

An outbreak of listeriosis in Denmark occurred in May 2009. Multilocus variable number of tandem repeats analysis typing, later confirmed by pulsed-field gel electrophoresis typing, showed that isolates from eight patients had identical patterns and were distinguishable from Listeria monocytogenes isolates from other Danish patients. Seven out of eight patients had received a meal with beef from the same meals-on-wheels delivery catering company 3 weeks prior to onset of disease. Two patients died of their infection. Large-scale delivery of precooked meals to a vulnerable population represents a threat if proper measures against listeriosis are not taken.