Jette Marie Bangsborg

Excretion of ciprofloxacin in sweat and multiresistant Staphylococcus epidermidis

BACKGROUND Staphylococcus epidermidis develops resistance to ciprofloxacin rapidly. That this antibiotic is excreted in apocrine and eccrine sweat of healthy individuals might be the reason for the development of such resistance. We assessed whether S epidermidis isolated from the axilla and nasal flora of healthy people could develop resistance to ciprofloxacin after a 1-week course of this antibiotic. METHODS The concentration of ciprofloxacin in sweat was measured in seven volunteers after oral administration of 750 mg ciprofloxacin twice daily for 7 days, and the development of resistance in S epidermidis from axilla and nostrils was monitored during and 2 months after the treatment. Genotyping of S epidermidis was done by restriction fragment length polymorphism. FINDINGS The mean concentration of ciprofloxacin in sweat increased during the 7 days of treatment-from 2.2 micrograms/mL 2.5 h after the first tablet to 2.5 micrograms/mL after the fifth tablet, and 5.5 micrograms/mL after the 13th tablet. All persons harboured susceptible S epidermidis (minimal inhibitory concentration [MIC] 0.25 microgram/mL) in axilla and nostrils before treatment. Four resistant strains were detected, two intermediate-level (MIC 4-12 micrograms/mL) and two high-level (MIC > 32 micrograms/mL). Three of these strains were found in all the participants, and a ciprofloxacin-sensitive variant of one of the high-level resistant strains was also found before the start of the treatment. The high-level resistant strains were also resistant to methicillin, erythromycin, gentamicin, sulphonamide, and trimethoprim. A mean of 2.7 days after the start of the treatment, development of ciprofloxacin resistance was detected in S epidermidis from the axilla of all persons, compared with 11 days for the appearance of resistant S epidermidis in nostrils. The resistant strains persisted for an average of 37 and 39 days in axilla and nostrils, respectively, after the end of the treatment. INTERPRETATION The rapid development of resistance to ciprofloxacin due to excretion of this drug into the sweat might be involved in the development of multiresistant S epidermidis and possibly other skin bacteria in hospitals and in communities with high use of ciprofloxacin or related drugs.

Designation of the European Working Group on Legionella infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing using a standard protocol

Abstract.The utility of amplified fragment length polymorphism (AFLP) analysis as a genotyping method for the epidemiological typing of Legionella pneumophila serogroup 1 has been previously demonstrated. This study (i) reports recommendations for the designation of the European Working Group on Legionella Infections (EWGLI) AFLP types, (ii) describes the EWGLI AFLP types identified for the 130 strains in the EWGLI culture collection, and (iii) reports the results of a newly introduced international programme of proficiency testing. Following preliminary analysis of 20 epidemiologically unrelated isolates, 16 major AFLP types were identified. A coded proficiency panel, comprising 12 additional isolates representing 9 of these 16 AFLP types, was sent to 17 centres in 14 European countries where it was analysed following a previously determined standard protocol. The identity of each coded strain (recorded as AFLP type 001–016 or untypeable) was determined by participants with reference to these 16 AFLP types, either visually or using gel analysis software where available, and reported to the coordinating centre. Nine of the 12 strains, including an epidemiologically related pair and two pairs of unrelated isolates of the same type, were correctly identified to the correct AFLP type by all or all but one of the participants. Seven laboratories correctly identified all 12 isolates, and a further seven laboratories correctly identified 11. Type identification scores ranged from 75% (1 centre), 83% (2 centres), and 92% (7 centres) to 100% (7 centres). The AFLP method as described is robust and rapid and allows the genotypic comparison of isolates of Legionella pneumophila between different testing centres without the need for exchange of the strains studied.

Assessment of intercentre reproducibility and epidemiological concordance of Legionella pneumophila serogroup 1 genotyping by amplified fragment length polymorphism analysis

Abstract The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a “reproducibility” panel (n=20) and an “epidemiologically related” panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each centre following a previously determined standard protocol. Results were analysed by the participants, using gel analysis software where available, and submitted to the coordinating centre. The coordinating centre reanalysed all results visually and selected data-sets with gel analysis software. Data analysis by participants yielded reproducibility (R) values of 0.20–1.00 and epidemiological concordance (E) values of 0.11–1.00, with 6 to 34 types. Following visual analysis by the coordinating centre, R=0.78–1.00, and E=0.67–1.00, with 10–20 types. Analysis of three data-sets by the coordinating centre using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1.00) and epidemiologically concordant (E=1.00), with good discrimination. The electropherograms generated are amenable to computer-aided analysis, but strict adherence to a previously defined laboratory protocol is required. Following designation of representative type strains and patterns, this method will be adopted by the European Working Group on Legionella Infections as the first internationally standardised typing method for use in the investigation of travel-associated Legionella infections.

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.

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.

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

ABSTRACT Rifampin in combination with erythromycin is a recommended treatment for severe cases of legionellosis. Mutations in therpoB gene are known to cause rifampin resistance inEscherichia 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 therpoC 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.

Legionellosis in patients with HIV infection

SummaryDuring the five-year period 1984–1988 we received 192 specimens from 180 patients infected with the human immunodeficiency virus (HIV) for investigation ofLegionella infection. The majority of specimens were bronchoalveolar lavage (BAL) fluids (84%), but tracheal suctions and lung tissue from autopsies were also examined. The diagnostic methods used were a direct immunofluorescence assay (DFA) for the detection ofLegionella antigen, and culture on buffered charcoal yeast extract (BCYE-α) media. All specimens were also examined for the presence of other bacterial lung pathogens, and all BAL specimens additionally forPneumocystis carinii and mycobacteria. Legionellosis was not found to be common among HIV-infected patients, as only six specimens (3%) from six patients were found positive by DFA, and no specimens were culture-positive forLegionella species. Dual infection withLegionella andP. carinii occurred in two patients. Clinical data of the six patients are presented, and currently used methods for diagnosing legionellosis are discussed.ZusammenfassungIn einem Zeitraum von fünf Jahren (1984–1988) erhielten wir 192 Proben von 180 mit dem menschlichen Immunschwächevirus (HIV) infizierten Patienten zur Untersuchung aufLegionella-Infektion. Die meisten Proben (84%) waren Bronchoalveolarlavageflüssigkeit, außerdem wurde durch Trachealabsaugung gewonnenes Material und autoptisch entnommenes Gewebe untersucht. Zum Nachweis von Legionella-Antigen wurde ein direkter Immunfluoreszenztest (DFA) verwendet; die Kultivierung erfolgte auf gepufferten Kohle-Hefeextrakt-(BCYE-α)-Medien. Alle Proben wurden auch auf andere bakterielle Erreger von Atemwegsinfektionen untersucht; Bronchoalveolarlavage-Proben wurden in allen Fällen aufPneumocystis carinii und Mykobakterien getestet. Es zeigte sich, daß die Legionellose bei HIV-infizierten Patienten nicht häufig vorkommt. Nur sechs der Proben (3%), die von sechs Patienten stammten, waren im DFA positiv; in keinem Fall konntenLegionella-Spezies kultiviert werden. Eine Doppelinfektion mitLegionella undP. carinii wurde bei zwei Patienten festgestellt. Die klinischen Daten der sechs Patienten werden mitgeteilt und derzeit gebräuchliche Methoden zur Legionellose-Diagnostik diskutiert.During the five-year period 1984–1988 we received 192 specimens from 180 patients infected with the human immunodeficiency virus (HIV) for investigation ofLegionella infection. The majority of specimens were bronchoalveolar lavage (BAL) fluids (84%), but tracheal suctions and lung tissue from autopsies were also examined. The diagnostic methods used were a direct immunofluorescence assay (DFA) for the detection ofLegionella antigen, and culture on buffered charcoal yeast extract (BCYE-α) media. All specimens were also examined for the presence of other bacterial lung pathogens, and all BAL specimens additionally forPneumocystis carinii and mycobacteria. Legionellosis was not found to be common among HIV-infected patients, as only six specimens (3%) from six patients were found positive by DFA, and no specimens were culture-positive forLegionella species. Dual infection withLegionella andP. carinii occurred in two patients. Clinical data of the six patients are presented, and currently used methods for diagnosing legionellosis are discussed. In einem Zeitraum von fünf Jahren (1984–1988) erhielten wir 192 Proben von 180 mit dem menschlichen Immunschwächevirus (HIV) infizierten Patienten zur Untersuchung aufLegionella-Infektion. Die meisten Proben (84%) waren Bronchoalveolarlavageflüssigkeit, außerdem wurde durch Trachealabsaugung gewonnenes Material und autoptisch entnommenes Gewebe untersucht. Zum Nachweis von Legionella-Antigen wurde ein direkter Immunfluoreszenztest (DFA) verwendet; die Kultivierung erfolgte auf gepufferten Kohle-Hefeextrakt-(BCYE-α)-Medien. Alle Proben wurden auch auf andere bakterielle Erreger von Atemwegsinfektionen untersucht; Bronchoalveolarlavage-Proben wurden in allen Fällen aufPneumocystis carinii und Mykobakterien getestet. Es zeigte sich, daß die Legionellose bei HIV-infizierten Patienten nicht häufig vorkommt. Nur sechs der Proben (3%), die von sechs Patienten stammten, waren im DFA positiv; in keinem Fall konntenLegionella-Spezies kultiviert werden. Eine Doppelinfektion mitLegionella undP. carinii wurde bei zwei Patienten festgestellt. Die klinischen Daten der sechs Patienten werden mitgeteilt und derzeit gebräuchliche Methoden zur Legionellose-Diagnostik diskutiert.

Serotype Distribution in Non-Bacteremic Pneumococcal Pneumonia: Association with Disease Severity and Implications for Pneumococcal Conjugate Vaccines

Background There is limited knowledge of serotypes that cause non-bacteremic pneumococcal pneumonia (NBP). Here we report serotypes, their associated disease potential and coverage of pneumococcal conjugate vaccines (PCV) in adults with NBP and compare these to bacteremic pneumonia (BP). Methods Adults with pneumonia and Streptococcus pneumoniae isolated from the lower respiratory tract or blood were included 1 year in a population-based design in Denmark. Pneumonia was defined as a new infiltrate on chest radiograph in combination with clinical symptoms or elevated white blood count or plasma C-reactive protein. All isolates were serotyped using type-specific pneumococcal rabbit antisera. All values are medians with interquartile ranges. Results There were 272 cases of NBP and 192 cases of BP. Ninety-nine percent were hospitalized. NBP and BP cases were of comparable age and sex but NBP cases had more respiratory symptoms and less severe disease compared to BP cases. In total, 46 different serotypes were identified. Among NBP cases, 5 serotypes accounted for nearly a third of isolates. PCV10 and -13 types covered 17% (95% confidence interval (CI): 11-23%) and 34% (95% CI: 25-43%) of NBP isolates, respectively. In contrast, the five most frequent serotypes accounted for two-thirds of BP isolates. PCV10 and -13 types covered 39% (95% CI: 30-48%) and 64% (95% CI: 48-79) of BP isolates, respectively. More severe NBP disease was associated with infection with invasive serotypes while there was an inverse relationship for BP. Conclusions Only a third of cases of adult non-bacteremic pneumococcal pneumonia would potentially be preventable with the use of PCV13 and just one sixth of cases with the use of PCV10 indicating that PCVs with increased valency are needed to increase vaccine coverage for NBP in adults. PCV13 could potentially prevent two-thirds of adult bacteremic pneumococcal pneumonia.

Utilization of serology for the diagnosis of suspected Lyme borreliosis in Denmark: Survey of patients seen in general practice

BackgroundSerological testing for Lyme borreliosis (LB) is frequently requested by general practitioners for patients with a wide variety of symptoms.MethodsA survey was performed in order to characterize test utilization and clinical features of patients investigated for serum antibodies to Borrelia burgdorferi sensu lato. During one calendar year a questionnaire was sent to the general practitioners who had ordered LB serology from patients in three Danish counties (population 1.5 million inhabitants). Testing was done with a commercial ELISA assay with purified flagella antigen from a Danish strain of B. afzelii.ResultsA total of 4,664 patients were tested. The IgM and IgG seropositivity rates were 9.2% and 3.3%, respectively. Questionnaires from 2,643 (57%) patients were available for analysis. Erythema migrans (EM) was suspected in 38% of patients, Lyme arthritis/disseminated disease in 23% and early neuroborreliosis in 13%. Age 0-15 years and suspected EM were significant predictors of IgM seropositivity, whereas suspected acrodermatitis was a predictor of IgG seropositivity. LB was suspected in 646 patients with arthritis, but only 2.3% were IgG seropositive. This is comparable to the level of seropositivity in the background population indicating that Lyme arthritis is a rare entity in Denmark, and the low pretest probability should alert general practitioners to the possibility of false positive LB serology. Significant predictors for treating the patient were a reported tick bite and suspected EM.ConclusionsA detailed description of the utilization of serology for Lyme borreliosis with rates of seropositivity according to clinical symptoms is presented. Low rates of seropositivity in certain patient groups indicate a low pretest probability and there is a notable risk of false positive results. 38% of all patients tested were suspected of EM, although this is not a recommended indication due to a low sensitivity of serological testing.

Bacteremia and Candidemia in Hematological Malignancies: Microbiological Findings and Antibiotic Susceptibilities

The microorganisms isolated in 1981-1985 from 171 cases of septicemia in patients with hematological malignancies were on the whole the same as those found in 1970-1972. The distribution between species was also quite similar for the two periods except within staphylococci, where the isolation rate of coagulase-negative staphylococci was higher in the latter period while that of Staphylococcus aureus was lower. Of 67 strains of Enterobacteriaceae tested for an aminoglycoside, 6% were found to be resistant, whereas 8% of 48 Enterobacteriaceae strains were found to be cefotaxime resistant. Methicillin- or aminoglycoside resistant S. aureus did not occur.