Christian Lück

Community-Acquired Legionella Pneumonia: New Insights from the German Competence Network for Community Acquired Pneumonia

BACKGROUND The Competence Network for Community Acquired Pneumonia (CAPNETZ) offers a unique opportunity to study the epidemiology of legionellosis throughout Germany, applying sophisticated diagnostic tools. METHODS The incidence, clinical characteristics, and outcome of Legionella pneumonia in 2503 adult patients with community-acquired pneumonia, participating in the German Multicenter Study of the CAPNETZ, were studied. RESULTS Legionella pneumonia was diagnosed in 94 patients (3.8%), thus identifying Legionella species as one of the most common pathogens to cause community-acquired pneumonia. It was equally common among ambulatory and hospitalized patients (3.7% and 3.8%, respectively). The predominant species causing community-acquired pneumonia was Legionella pneumophila; however, 10% of cases were caused by other species not detectable by the urinary antigen test. Patients whose disease was diagnosed by urinary antigen testing experienced a more severe clinical course. Compared with hospitalized patients, ambulatory patients with Legionella pneumonia showed an equal sex distribution, were younger, had fewer comorbidities, fewer cases of discordant initial antimicrobial treatment, and a milder clinical course without fatalities. Thirty percent of patients with Legionella pneumonia received discordant initial antimicrobial treatment without increased mortality. CONCLUSIONS Legionella is a leading cause of community-acquired pneumonia in Germany. It needs to be considered equally in hospitalized and ambulatory patients. A positive result of a urine antigen test is associated with a more severe clinical course and leads to a potentially relevant underrecognition of species other than L. pneumophila. Legionella pneumonia in outpatients differs significantly from that in hospitalized patients in terms of clinical presentation and outcome. There was an unacceptably high rate of discordant initial antimicrobial treatment.

Sensitive Detection of Mycoplasma pneumoniae in Human Respiratory Tract Samples by Optimized Real-Time PCR Approach

ABSTRACT To enhance the sensitivity of the available real-time PCR systems for the detection of Mycoplasma pneumoniae, we established a method to amplify copies of the repetitive element repMp1. In a study of respiratory tract samples, we found that, compared to the use of the conserved part of the P1 adhesin gene as a monocopy target, the use of the repMp1-PCR showed an increase in the detected genome equivalents by a factor of 22.

Mycoplasma pneumoniae pneumonia revisited within the German Competence Network for Community-acquired pneumonia (CAPNETZ)

BackgroundCurrently, broad empiric antimicrobial treatment including atypical coverage is recommended for patients with mild to moderate community-acquired pneumonia (CAP). Therefore, the relative impact of each atypical pathogen, particularly Mycoplasma pneumoniae deserves renewed attention.MethodsBased on prospective data from 4532 patients with CAP included in the German CAP-Competence Network (CAPNETZ), we studied the incidence, clinical characteristics, and outcome of patients with Mycoplasma pneumoniae pneumonia (MPP). The diagnosis of MPP was based on a positive PCR from respiratory samples and/or a positive IgM-titer from an acute phase serum sample.Results307 patients (6.8%) had definite MPP (148 with positive PCR, 204 with positive IgM, 46 with positive PCR and IgM). Compared to patients with other definite and unknown etiologies, patients with MPP were significantly younger (41 ± 16 versus 62 ± 17 and 61 ± 18 years), had fewer co-morbidities, presented with a less severe disease, showed a lower inflammatory response in terms of leukocyte counts (median 8850 versus 13200 and 11000 μL) and CRP values (60 versus 173 and 73 mg/L), and had better outcomes, including a shorter length of hospitalization (9 ± 5 versus 14 ± 11 and 12 ± 9 days), fewer patients requiring mechanical ventilation (0.3 versus 4.5 and 2.1%), and a minimal mortality (0.7 versus 8.7 and 6.5%).ConclusionIn this large series of patients with definite MPP according to very strict criteria, MPP appears as a condition with a high incidence, quite specific clinical presentation, and a largely benign course. In view of a widely favorable clinical outcome, recent recommendations including regular coverage of atypical pathogens in patients with mild to moderate CAP might be reconsidered for patients in Germany as well as in other countries with comparable epidemiological settings.

Regrowth of Legionella pneumophila in a heat-disinfected plumbing system

We examined the factors involved in the occurrence of Legionellaceae in a hospital water system and the recontamination by Legionella pneumophila after a thermal disinfection procedure was studied. Three months after the heat treatment (70 degrees C), the regrowth of the two prevalent Legionella strains (L. pneumophila serogroup 1 [Oxford-like] and L. pneumophila serogroup 2) reached the original level of cell numbers. Genomic analysis (pulsed-field gel electrophoresis) revealed the strains to be survivors of the decontamination. Temperature tolerance experiments showed that the serogroup 1 strain exhibited a higher tolerance to 60 degrees C than the serogroup 2 strain, which could account for the order of reappearance of the strains after the heat treatment. Potential host amoebae, including Acanthamoeba spp. and Vahlkampfia spp., which are known to play a critical role in the amplification process of Legionella, were isolated from the plumbing system. In-vitro studies demonstrated both Legionella strains for a similar rate of multiplication in A. castellanii. In competitive coinfections, however, the serogroup 1 strain achieved a higher rate of multiplication if compared with the serogroup 2 strain.

The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells.

Abstract We analysed eight monoclonal antibodies (mAbs) directed against the Mip (macrophage infectivity potentiator) protein, a virulence factor of the intracellular pathogen Legionella pneumophila. Mip belongs to the FK506-binding proteins (FKBPs) and exhibits peptidyl prolyl cis/trans isomerase (PPIase) activity. Five of the mAbs recognised epitopes in the Cterminal, FKBP-homologous domain of Mip, which is highly conserved among all Legionella species. Upon immunological binding to Mip, all but one of these mAbs caused inhibition of the PPIase activity in vitro. mAb binding to the N-terminal domain of Mip did not influence its enzymatic activity. All but one of the PPIase inhibiting mAbs were able to significantly inhibit the early establishment and initiation of an intracellular infection of the bacteria in Acanthamoeba castellanii, the natural host, and in the human phagocytic cell line U937. These data demonstrate for the first time that for the virulence-enhancing property of the L. pneumophila Mip protein, an intact active site of the enzyme is an essential requirement.

The N-Acylneuraminate Cytidyltransferase Gene, neuA, Is Heterogenous in Legionella pneumophila Strains but Can Be Used as a Marker for Epidemiological Typing in the Consensus Sequence-Based Typing Scheme

ABSTRACT Sequence-based typing (SBT) is the internationally recognized standard method for genotyping Legionella pneumophila. To date all strains of serogroup 1 (SG1) and some of SGs 2 to 14 yield a seven-allele profile and can be assigned a sequence type (ST). However, for some strains belonging to SGs 2 to 14, the targeted region of the neuA gene could not be amplified using the published standard primers. We determined the DNA sequence of a neuA gene homolog located in the lipopolysaccharide synthesis locus of strain Dallas-1E. By using newly designed degenerate consensus primers based on the neuA homolog in strains Dallas-1E, Philadelphia-1, Paris, Lens, and Corby, we were able to obtain DNA sequences for all 48 non-SG1 strains which were untypeable by the standard method. Our data show that the neuA gene is present in all L. pneumophila strains but differs significantly in some non-SG1 strains at both the DNA and amino acid levels. The new primers can be used to amplify and sequence the neuA gene in all strains and can substitute for the standard primers. This offers the possibility of assigning an ST to all strains of L. pneumophila.

Severe Legionella pneumophila pneumonia following infliximab therapy in a patient with Crohn's disease

Background: Immunosuppressive therapy with anti‐TNF‐α antibodies is effective in patients with inflammatory bowel disease (IBD). However, there is an increased risk for infections associated with this therapy. Methods: Here, we report the case of a 58‐year‐old patient with Crohns disease (CD) treated with steroids and azathioprine who developed severe Legionella pneumophila pneumonia after 3 infusions of infliximab. The patient presented at our IBD department with severe active CD complicated by inflammatory small bowel stenoses and entero‐enteral fistulas despite long‐term high‐dose steroid therapy. To achieve steroid tapering and control of disease activity, immunosuppressive therapy with azathioprine was initiated. Due to persistent symptoms, infusion therapy with the anti‐TNF‐α antibody infliximab was started, subsequently leading to significant clinical improvement. However, after the third infliximab infusion the patient was hospitalized with fever, severe fatigue, and syncope. Results: Laboratory findings and chest X‐ray revealed left‐sided pneumonia; cultural analysis showed L. pneumophila serogroup 1 leading to respiratory insufficiency, which required mechanical ventilation for 2 weeks in the intensive care unit. After discontinuation of all immunosuppressive agents and immediate antibiotic therapy the patient recovered completely. Conclusions: To our knowledge, this is the third case of L. pneumophila pneumonia in an IBD patient treated with infliximab. Similar to other published cases, concomitant treatment of immunosuppressives and anti‐TNF agents is a major risk factor for the development of L. pneumophila infection, which should be ruled out in all cases of pneumonia in patients with such a therapeutic regimen. Appropriate prevention strategies should be provided in these patients. (Inflamm Bowel Dis 2009)

Typing Methods for Legionella

In this chapter we describe the methods currently used for subgrouping Legionella pneumophila and other non-pneumophila species. In the first part we describe monoclonal antibody (mAb) subgrouping, either by indirect immunofluorescence or indirect ELISA methods. These monoclonal antibodies are not commercially available but can be obtained for noncommercial purposes from one of the authors. Further, we describe pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) and sequence-based typing (SBT) as well standardized and reproducible methods for genotyping. The SBT schema is currently available for L. pneumophila whereas PFGE and AFLP can be used for all Legionella species. For certain applications it might be useful to use spoligotyping to distinguish strains belonging to the same sequence type (ST).

Isolation of ciprofloxacin-resistant Legionella pneumophila in a patient with severe pneumonia

Sir, Legionella species are responsible for 1%– 5% of cases of community-acquired pneumonia. Legionella pneumophila serogroup 1 (SG1) accounts for .90% of Legionnaires’ disease (LD) in North America and Europe and is the cause of significant mortality. The mortality rate among patients with L. pneumophila infections continues to be high, up to 26%. The antimicrobial agents most commonly used for treatment of LD are fluoroquinolones (e.g. ciprofloxacin or levofloxacin) and macrolides. In recent studies, we established wild-type distributions and determined the epidemiological cut-off values (ECOFFs) in clinical L. pneumophila SG1 isolates for 10 antimicrobials commonly used for the treatment of Legionella infections. A patient sought care at his general practitioner after several days of falling and body pains. On examination, the patient appeared ill and was sent to the emergency department of a nearby hospital. The initial chest radiograph demonstrated an infiltrate of the left lower lung field. The patient was admitted to the intensive care unit. Blood cultures were taken and antibiotic treatment was started with cefazolin and gentamicin. Urine was examined for the presence of Legionella antigens and when this test was reported positive, treatment was switched to 400 mg of ciprofloxacin intravenously twice daily. After initial improvement the clinical condition of the patient deteriorated, leading to intubation and mechanical ventilation. A new chest radiograph revealed a diffuse interstitial pneumonia. Bronchoalveolar lavage (BAL) was performed 4 days after treatment with ciprofloxacin was started and the patient slowly recovered. Eventually, culture of the BAL grew L. pneumophila SG1 after 4 days of ciprofloxacin treatment. After 10 days, the patient could be transferred to the ward. Therapy was then switched to 500 mg of clarithromycin orally twice daily. The patient’s further recovery was uncomplicated. The L. pneumophila SG1 strain was sent, as part of a national Legionella outbreak detection programme, to the reference laboratory for Legionella in Haarlem, The Netherlands. Susceptibility testing for ciprofloxacin was performed with Etest and an MIC value of ciprofloxacin of 2 mg/L was found. This value is outside the wild-type distribution range ECOFF1⁄41 mg/L as previously described and therefore potentially resistant. For sequencing of gyrA and gyrB (DNA gyrase) and parC and parE (topoisomerase IV) genes, extraction of L. pneumophila DNA was performed. The DNA extraction was performed by use of Qiagen’s BioRobot EZ1 (Hilden, Germany) according to the manufacturer’s instructions. The sequencing reaction was performed twice by using primer systems previously described for the L. pneumophila SG1 strain Paris. A comparative analysis of the obtained sequences was done using the published L. pneumophila genomes and data from the literature describing mutations in the quinolone resistance-determining region (QRDR) of type II topoisomerase of L. pneumophila by using DNAStar (WI, USA) software and the NCBI database. For control experiments, the wild-type strain MTZ OLDA and a spontaneous quinolone-resistant mutant of this strain were used. MTZ OLDA is an environmental isolate (L. pneumophila SG1), isolated from the water supply of a large building. A point mutation in the QRDR of the gyrA gene was identified and this mutation led to an amino acid exchange at position 83 (Escherichia coli numbering system). The result of this amino acid exchange is a change in ciprofloxacin susceptibility. Mutation at the same position (amino acid 83) has also been reported for other spontaneous quinolone-resistant mutants (Table 1). It is known that, in general, pathogens can become resistant during the course of a patient’s therapy and also induction of resistance upon exposure to antibiotics has been described. The origin of resistance in the clinical isolate is as yet unclear. There are two possibilities. The first is that the patient contracted an L. pneumophila SG1 strain with this point mutation from the environment. Alternatively, the mutation occurred during the Research letters

Hybrid Microgels with Antibacterial Properties

In the present work, we have used aqueous microgels as containers for the deposition of silver nanoparticles (AgNPs). It has been shown that AgNPs can be effectively incorporated in the microgel interior during the in situ reduction of silver ions. Obtained hybrid microgels with variable AgNPs loading (from 1 to 12 wt.-%) have been used as antibacterial agents for two bacteria types. The experimental results indicate that porous microgel structure allows the release of the silver ions from the AgNPs surface into an aqueous phase. This ensures effective reduction in the number of bacterial colonies in test plates and complete bacteria killing. The antibacterial efficiency of the microgel particles increases with AgNPs loading.