Subtypes and variants of Mycoplasma pneumoniae: local and temporal changes in Germany 2003-2006 and absence of a correlation between the genotype in the respiratory tract and the occurrence of genotype-specific antibodies in the sera of infected patients.

Mycoplasma pneumoniae is a frequent cause of community-acquired pneumonia. Three subtypes and three variants of M. pneumoniae have been described showing sequence differences in the main P1 adhesin. Between 2003 and 2006 we collected respiratory tract samples of adult outpatients with symptoms of pneumonia in a German nationwide network and detected M. pneumoniae by real-time PCR in 140 specimens. The strains were typed by sequencing and demonstrated the circulation of subtypes 1 and 2 and variants 2a and 2b. The overall number of isolates belonging to the two variant genotypes increased during the investigation period but the relationship of subtypes and variants within the participating local centres varied strongly. ELISA experiments using sera of acute-phase patients with a known M. pneumoniae type in the respiratory tract resulted in no correlation of IgA and IgG antibodies to subtype- and variant-specific regions of the P1 gene with the genotype of the M. pneumoniae strain causing the actual infection.

Distribution of legionellae in a hospital water system: prevalence of immunologically and genetically related Legionella pneumophila serogroup 6 isolates.

A hospital warm water system was monitored for the presence and distribution of legionellae. Subtyping of ten selected Legionella pneumophila isolates, originating from four different sites in the system by using serogroup specific antisera in an indirect immunofluorescence test, revealed that nine of the ten isolates belong to serogroup 6, while the remaining one was serogroup 10. Two monoclonal antibodies (mAbs) specific for a subgroup of serogroup 6 strains were further used for characterization. None of the strains reacted with these mAbs. Genome analysis by elaborating NotI profiles using the pulsed field gel electrophoresis (PFGE) technique revealed that nearly all serogroup 6 isolates derived from different sites, including a new building connected by a ring pipe, were identical according to restriction fragment patterns. The patterns were distinguishable from those of the two L. pneumophila serogroup 6 reference strains, and from that of the L. pneumophila serogroup 10 isolate. These data argue for a relatively homogeneous L. pneumophila serogroup 6 population in the entire water system.

The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila.

The lly locus confers fluorescence, haemolysis, brown pigmentation and an increased resistance to light in Legionella pneumophila. In this study, we correlated the pigment production of two lly-positive L. pneumophila isolates and a recombinant lly-positive Escherichia coli strain with the presence of homogentisic acid (HGA) in the culture supernatant. The detection of HGA by high performance liquid chromatography and the data analysis of the deduced amino acid sequence of the lly gene indicate that the lly locus codes for a p-hydroxyphenylpyruvate dioxygenase (HPPD). This enzyme catalyses the transformation of p-hydroxyphenylpyruvate into HGA, which subsequently oxidises and polymerises into a melanin-like pigment. One open reading frame (ORF 1) in the lly region exhibited homologies with genes of Synechocystis sp., Petroselium crispum and Streptomyces mycarofaciens that code for methyltransferases. By screening a genomic library of L. pneumophila (serogroup 1) strain Corby with a monoclonal antibody against the legiolysin (lly), we identified two recombinant E. coli clones that did not produce the brown pigment and showed no haemolysis and fluorescence. DNA sequencing revealed that both clones contained 874 nucleotides of the N-terminal part of the lly gene. The recombinant strains expressed truncated legiolysin proteins of 39.5 and 35.7 kDa and did not produce HGA. Considering the highly conserved structure of legiolysin-like HPPD genes from other organisms, we suggest that the C-terminus of the legiolysin may be essential for the enzymatic activity that conferred pigmentation via HGA polymerisation, haemolysis and fluorescence.

An oxaloacetate decarboxylase homologue protein influences the intracellular survival of Legionella pneumophila.

Legionella pneumophila is a facultative intracellular parasite which is able to survive in various eukaryotic cells. We characterised a Tn5-mutant of the L. pneumophila Corby strain and were able to identify the insertion site of the transposon. It is localised within an open reading frame which shows high homology to the alpha-subunit of the oxaloacetate decarboxylase (OadA) of Klebsiella pneumoniae. The OadA homologous protein of L. pneumophila was detected in the wild-type strain by Western blotting. Since the intracellular multiplication of the oadA- mutant strain is reduced in guinea pig alveolar macrophages and human monocytes, it is concluded that the oadA gene product has an effect on the intracellular survival of L. pneumophila.

Genomic heterogenicity amongst phenotypically similar Legionella micdadei strains.

Nine unrelated Legionella micdadei strains isolated from clinical and environmental samples have been characterized biochemically, serologically using polyclonal and monoclonal antibodies and by macrorestriction analyses using pulsed-field gel electrophoresis. All strains were positive in the Bromocresol purple spot test and grew as blue colonies on dye-containing media. They were positive for catalase, weakly positive for oxidase, and negative for sodium-hippurate hydrolysis, beta-lactamase and gelatinase. None of the strains showed autofluorescence under long-wave ultraviolet light. A panel of six monoclonal antibodies raised against the ATCC strain TATLOCK revealed no significant differences in the surface antigen composition of the L. micdadei strains. None of these monoclonal antibodies reacted with L. maceachernii and L. longbeachae serogroup 2, the only species that cross-react with polyclonal antisera. Each of the nine L. micdadei strains showed individual restriction patterns of the genomic DNA when using both SfiI and NotI restriction enzymes in the pulsed-field gel electrophoresis. Macrorestriction analysis is a valuable tool for studies on the molecular epidemiology of L. micdadei.

Correlation of MAb subgroups with genotype in closely related Legionella pneumophila serogroup 1 strains from a cooling tower.

Legionella pneumophila serogroup 1 strains isolated from a cooling tower during the investigation of an outbreak of Legionnaires' disease were shown previously to be related closely or indistinguishable by hybridisation-based restriction fragment length polymorphism analysis. However, these strains could be differentiated into five different MAb subgroups by comparison of their reactivity patterns with a recognised panel of monoclonal antibodies (MAbs). Pulsed-field gel electrophoresis (PFGE) of genomic fragments obtained after cleavage with rare-cutting restriction endonucleases also differentiated these strains. Four different restriction patterns were obtained with SfiI, EagI and SmaI, three restriction patterns with NotI, ApaI and SacII, and two patterns with NaeI. Generally, the restriction patterns were related closely, differing in only one or two bands. The combined results of the restriction endonuclease digestions allowed the strains to be differentiated into groups that correlated to the MAb subgroups. Both PFGE patterns and MAb subgroups were found to be stable markers. The findings demonstrated that the MAb variability seen amongst the L. pneumophilia serogroup 1 strains from this cooling tower was not solely phenotypic.

Detecting legionellosis by unselected culture of respiratory tract secretions and developing links to hospital water strains.

For a 13-month period, all respiratory tract secretions submitted for routine bacteriology from a large hospital complex were cultured for legionella, irrespective of clinical diagnosis and laboratory requests. Ten cases of legionellosis were detected in this manner, three of which met a strict epidemiological definition of hospital-acquired. Therefore, the 16 warm-water systems of the hospitals, spread out over two locations, were examined for the presence of legionella. Legionella pneumophila was found in 15 warm water systems, with a distinct pattern of serogroups between the two locations. Legionella of the same serogroups as those isolated from patients were present in each hospital water supply. The isolates were further typed by monoclonal antibodies and by genomic macrorestriction analysis. Similarity between clinical and environmental isolates was found in seven cases. In these cases, acquisition from the hospital water supply appears very likely. The strains of the remaining three patients did not match those in hospital water, suggesting that community-acquired legionellosis was occurring as well. This study suggests that routinely culturing respiratory tract secretions of pneumonia patients for legionella can help diagnose unsuspected cases of legionellosis. Typing legionella strains beyond the serogroup level with tools such as macrorestriction analysis is useful to define sources of infection, which can then be targeted for control measures.

Immunofluorescent staining of influenza virus antigen in fixed and paraffin-embedded tissue of experimentally infected hamsters.

Sections of formalin-fixed, paraffin-embedded tissue of experimentally influenza virus-infected hamsters were treated with 0.25% trypsin and tested for virus antigen by indirect immunofluorescent staining. The results were comparable to those obtained with aceton-fixed cryo-microtome sections. As far as we know, this is the first description of influenza virus demonstration in formalin-fixed, paraffin-embedded tissue after reactivation by trypsin-treatment. This technique may be useful for influenza virus detection in human autopsy cases. It allows an etiological diagnosis even when fresh tissue for cryocut sections or virus cultivation is not available.

Occurrence of macrolide-resistant Mycoplasma pneumoniae strains in Germany.

In a total of 167 respiratory tract specimens from adult outpatients with confirmed Mycoplasma pneumoniae pneumonia, sampled between 2003 and 2008, and a further 99 isolates obtained from patients between 1991 and 2009 in Germany, M. pneumoniae was tested for macrolide resistance. Using PCR, real-time PCR and sequencing of the 23S rRNA gene, 1.2% of M. pneumoniae in the respiratory tract samples and 3.0% of the isolates were found to be resistant. The results indicate a limited but not negligible importance of macrolide-resistant M. pneumoniae in the population investigated, which requires the monitoring of macrolide susceptibility of isolates or the testing of respiratory samples by molecular methods.

Occurrence and distribution of sequence types among Legionella pneumophila strains isolated from patients in Germany: common features and differences to other regions of the world.

A total of 105 unrelated clinical isolates of Legionella pneumophila were randomly selected from the German National Legionella strain collection and typed by monoclonal antibody (MAb) subgrouping and a seven-gene locus sequence-based typing (SBT) scheme. According to the case definitions of the European Working Group for Legionella Infections, 19 of the isolates tested were travel-associated, 38 were community-acquired and 48 were of nosocomial origin. Eighty-four of these strains belonged to serogroup 1, 20 belonged to other serogroups, and one isolate could not be serogrouped. The majority of strains among the travel-associated and community-acquired cases were MAb3-1-positive. The most common sequence type (1, 4, 3, 1, 1, 1, 1) was found in 20 isolates in 11 cities; other allelic profiles also found in Europe (2, 3, 9, 10, 2, 1, 6), (1, 3, 9, 10, 2, 1, 6), (2, 6, 17, 14, 13, 11, 11) and (3, 4, 1, 1, 1, 9, 1) were detected among the German isolates but at a low frequency. In contrast, some SBT are unique to Germany, including (3, 4, 1, 3, 35, 9, 11), which was found among five isolates from patients in Berlin. In concordance with European data, a significant portion of the L. pneumophila strains isolated from patients in Germany belong to clones that occur throughout the world and which are responsible for the majority of clinical cases.

Random mutagenesis of Legionella pneumophila reveals genes associated with lipopolysaccharide synthesis and recognition by typing monoclonal antibodies.

AIMS: To use random mutagenesis for the characterization of Legionella pneumophila lipopolysaccharide (LPS) components and serotypes. METHODS AND RESULTS: Five strains belonging to different serogroups and/or monoclonal subgroups were mutagenized using a mini-Tn10 transposon. Exactly 11 819 mutants were checked for alterations in LPS using at least 11 monoclonal antibodies (mAbs) that define L. pneumophila serotypes. Among the mutants, five different mini-Tn10 insertions were identified. Four mutants originating from serogroup-1 did not lose their serogroup-specific epitope, but did sustain subtler changes that resulted in switches to different mAb subgroups. In contrast, a mutant from serogroup-6 lost its serogroup-specific epitope, while retaining a serogroup-cross-reacting epitope. CONCLUSIONS: Random mutagenesis is a valuable tool for LPS epitope mapping. While some characteristics of L. pneumophila LPS can be altered, others appear resistant to mutagenesis. This underscores both the flexibility and rigidity of LPS architecture in L. pneumophila. SIGNIFICANCE AND IMPACT OF THE STUDY: Losses of L. pneumophila LPS epitopes can result in new serotypes, changes that might escape detection by current DNA-based typing schemes. But, as the frequency of these changes is rare, based upon our observations, serotyping should remain an important tool for identifying L. pneumophila in water systems that are implicated in human infection.

Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources.

AIMS: To validate identification methods for Legionella pneumophila strains that cannot be serotyped into the known serogroups and to characterize their antigenic diversity. METHODS AND RESULTS: Fifty L. pneumophila strains that could not be serogrouped, but which had been confirmed as L. pneumophila by mip gene sequencing, were further identified phenotypically. We used (i) MONOFLUO anti-Legionella Staining Reagent (Bio-Rad) (50/50), (ii) an in-house prepared immunoblot assay for the detection of L. pneumophila- specific Mip protein epitope (50/50), (iii) fatty acid analysis using the Microbial Identifications System (MIDI) (47/50) and (iv) Oxoid agglutination tests (44/50). The serological diversity was further characterized by testing with five serogroup-cross-reactive monoclonal antibodies, resulting in nine phenons. CONCLUSIONS: The division of L. pneumophila into 15 serogroups does not reflect the serogroup heterogeneity. Results of these tests indicate that there are more serogroups. SIGNIFICANCE AND IMPACT OF THE STUDY: MONOFLUO anti-Legionella Staining Reagent is the only commercially available tool for identifying atypical strains of L. pneumophila. If necessary for epidemiological purposes, the antigenic heterogeneity of these strains can be analysed by monoclonal antibodies.

[Pathogenesis, diagnosis and therapy of Legionella infections]. / Pathogenese, Diagnostik und Therapie der Legionella-Infektion.

Legionella species are ubiquitous in aquatic environments. About 50 years ago they entered the engineered (technical) environment, i.e. warm water systems with zones of stagnation. Since that time they represent a hygienic problem. After transmission to humans via aerosols legionellae might cause Legionella pneumonia (legionnaires' disease) or influenza-like respiratory infections (Pontiac fever). Epidemiological data suggest that Legionella strains might differ substantially in their virulence properties. Although the molecular basis is not understood L. pneumophila serogroup 1 especially MAb 3/1-positive strains cause the majority of infections. The main virulence feature is the ability to multiply intracellularly. After uptake into macrophages legionellae multiply in a specialized vacuole and finally lyse their host cells. Several bacterial factors like surface components, secretion systems and iron uptake systems are involved in this process. Since the clinical picture of Legionella pneumonia does not allow differentiation from pneumoniae caused by other pathogens, microbiological diagnostic methods are needed to establish the diagnosis. Cultivation of legionellae from clinical specimens, detection of antigens and DNA in patients' samples and detection of antibodies in serum samples are suitable methods. However, none of the diagnostic tests presently available offers the desired quality with respect to sensitivity and specificity. Therefore, the standard technique is to use several diagnostic tests in parallel. Advantages and disadvantages of the diagnostic procedures are discussed. Therapeutic options for Legionella infections are newer macrolides like azithromycin and chinolones (ciprofloxacin, levofloxacin and moxifloxacin).

[Molecular typing of Legionella for determining the source of infection]. / Molekulare Typisierung von Legionellen zum Nachweis von Infektketten.

The ubiquitous occurrence of Legionellae requires an exact typing of isolated strains in order to demonstrate the source of infection. Monoclonal antibodies, analysis of genomic and plasmid DNAs, and the typing of alloenzymes are suitable for this purpose. Typing of Legionella pneumophila serogroup 1 strains by using monoclonal antibodies was found to be a rapid and adequate method. Other serogroups of L. pneumophila and non-pneumophila species are of considerably less antigenic diversity, so that the use of monoclonal antibodies is not particular profitable. In such cases, genotypic methods are needed to discriminate between unrelated strains. There are no changes in the genome structure, defined as restriction patterns, during passages on artificial media and cultured Acanthamoeba. The possibility that different species, serogroups and monoclonal or genomic subtypes can be isolated in a given water supply points to necessity to test a sufficiently large number of colonies grown from the water samples. A clonal distribution of some Legionella strains has been observed.

Serogroup-specific and serogroup-cross-reactive epitopes of Legionella pneumophila.

A panel of monoclonal antibodies was used to serotype 343 Legionella pneumophila isolates, using the indirect immunofluorescence test and ELISA. In addition, the isolates were typed by means of absorbed rabbit antisera to provide a reference procedure. As shown by a comparison of reaction patterns, serogroup-specific monoclonal antibodies were found for serogroups 1, 2, 3, 4, 6, 7, 8, and 10. Monoclonal subtypes were found to exist within serogroups 1, 2, 5, and 6. Using the monoclonal antibody panel introduced for serogroups 1 to 8 and 10, it was possible to serotype or subtype 92% of the isolates tested. The remaining isolates belonged to serogroups 9, 11 to 14 and to a monoclonal subtype of serogroup 5. 8 monoclonal antibodies recognized serogroup-cross-reactive epitopes. Except for serogroups 1, 7, and 11, all others shared a common antigenic determinant. Another common epitope was shared by strains of serogroups 2 and 3. Additional cross-reactivity was associated in particular with strains of serogroups 5, 8, and 10.

[Diagnosis of Legionella pneumonia by detection of antigenuria using an enzyme immunoassay with 6 antibody specificities]. / Diagnostik von Legionella-Pneumonien durch Nachweis der Antigenurie mittels Enzymimmunoassay unter Verwendung von 6 unterschiedlichen Antikörperspezifitäten.

In patients with microbiologically and clinically suspected Legionella caused pneumonia antigenuria was investigated by means of a direct two-site binding assay (ELISA) with polyclonal antibodies against Legionella (L.) pneumonia serogroup 1, 2, 3, 5 and 6 and L. micdadei. By application of antibodies only against L. pneumonia serogroup 1 antigenuria was found in 27 of 66 patients (= 41%). The expanding of the used specificities of antibodies in 47 out of this cases resulted in an increase of positive urinary antigen findings from 38% to 55%. Possibilities and limits of the detection of antigenuria with regard to efficient and rapid diagnostics of legionellosis are discussed.

[Determination of Candida albicans mannan antigen in serum]. / Bestimmung von Candida albicans-Mannan-Antigen im Serum.

An enzyme-linked immunosorbent assay (ELISA) for detection of Candida albicans mannan antigen in sera pretreated with pronase was used for investigation of antigenemia in 3 groups of patients: Group A: No antigen was detected in patients (n = 270), which were under control by a mycological surveillance programme. They were clinically not suspicious of candidosis and had no remarkable mycological findings. Group B: Candida antigen was detected in 13 cases of 158 patients (= 8.2%), which suffered from unclear clinical symptoms. Therefore a mycological laboratory diagnosis was performed yielding no remarkable findings neglecting the positive antigen detection. Group C: Candida antigen was also detected in 9 cases of 64 patients (= 14.0%), which were suspicious of candidosis and/or had remarkable mycological laboratory findings. The difference between the frequency of antigenemia in group B and C was not significant. According to our preliminary experiences the detection of Candida mannan antigen may support the early diagnosis of invasive candidosis. Yet antigen findings should not be separately interpreted but included in all available clinical and mycological results.