Legionella pneumophila urinary antigen subtyping using monoclonal antibodies as a tool for epidemiological investigations.

Legionnaires' disease is diagnosed predominantly by urinary antigen detection, and patient isolates are rarely available. The lipopolysaccharide (LPS) epitope pattern of isolates detected by monoclonal antibodies is an accepted marker for the phenotyping of L. pneumophila serogroup 1 strains into monoclonal subgroups. L. pneumophila LPS is the dominant antigen in patients' urinary specimens. By using commercially available microtiter wells coated with rabbit anti-Legionella serogroup 1 IgG as the catching antibody, LPS components in urine specimens were bound and detected separately by corresponding monoclonal antibodies of the Dresden Panel. The subtyping of LPS on urinary antigen molecules by using enzyme-linked immunosorbent assay (ELISA) allows deducing of first evidences for the identity/non-identity of environmental isolates and the legionellosis pathogen. Most importantly in our study, urinary antigen typing possesses high probability to distinguish (or does not distinguish) if the pathogen belongs to the MAb 3/1-negative L. pneumophila strains, which are widespread contaminants of water systems, but represent the minority of patient isolates.

Evaluation of an immunomagnetic separation assay in combination with cultivation to improve Legionella pneumophila serogroup 1 recovery from environmental samples.

AIMS: Legionella isolation from environmental samples is often difficult because of the presence of heterotrophic-associated bacteria that frequently overgrow when using standard culture (ISO 11731, 1998; NF T90-431, 2003) methods. To improve Legionella pneumophila recovery from complex water samples (water from cooling towers, biofilms), we evaluated an immunomagnetic separation (IMS) assay using a monoclonal antibody raised against the lipopolysaccharide of Leg. pneumophila sg1 in combination with culture. METHODS AND RESULTS: This study was conducted on 51 environmental specimens. The comparison between IMS-culture and standard culture (ISO 11731, 1998; NF T90-431, 2003) methods was made using ISO 17994, 2004 criteria for establishing equivalence between microbiological methods based on the upper and lower (XH and XL) values of the relative difference (95% confidence limit) and D as maximum acceptable deviation (value of the confidence limit). CONCLUSIONS: We found that the average performance of IMS culture was higher than the reference method.

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.

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.

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.

Structure of a decasaccharide isolated by mild acid degradation and dephosphorylation of the lipopolysaccharide of Pseudomonas fluorescens strain ATCC 49271.

Mild acid degradation of the Pseudomonas fluorescens strain ATCC 49271 lipopolysaccharide resulted in a core oligosaccharide containing D-glucose, 2-acetamido-2-deoxy-D- glucose, 2-(L- alanylamino)-2-deoxy-D-galactose, 2-acetamido-2,6-dideoxy-D-glucose (QuiNAc), 2-acetamido- 2,6-dideoxy-L-galactose (FucNAc), L-glycero-D-manno-heptose (Hep), 3-deoxy-D- manno-octulosonic acid (Kdo, present in multiple forms), and 5-acetamidino-7-acetamido-3,5,7,9- tetradeoxy- L-glycero-D-galacto-nonulosonic acid (a di-N-acyl derivative of legionaminic acid, Non) as well as O-acetyl, O-carbamoyl, and phosphate groups, including triphosphate groups. The dephosphorylated (HF) decasaccharide and products of its partial and full O-deacylation were studied by methylation analysis, GLC-MS, and 1H NMR spectroscopy, including 1D NOE and 2D shift-correlated spectroscopy (COSY). The core oligosaccharide of P. fluorescens strain ATCC 49271 was found to be a decasaccharide (with partially degraded Kdo region) and one O-antigen repeating unit (di-N-acyllegionaminic acid, Non) attached. The following structure of the dephosphorylated core oligosaccharide was established: [sequence: see text]

Structures of decasaccharide and tridecasaccharide tetraphosphates isolated by strong alkaline degradation of O-deacylated lipopolysaccharide of Pseudomonas fluorescens strain ATCC 49271.

Mild hydrazinolysis of Pseudomonas fluorescens strain ATCC 49271 lipopolysaccharide (LPS) followed by strong alkaline degradation and purification by anion-exchange HPLC resulted in two phosphorylated oligosaccharides (1 and 2). On the basis of compositional analysis and 1H, 13C, and 31P NMR spectroscopy, including 2D correlation spectroscopy (COSY), 2D rotating frame NOE spectroscopy (ROESY), and 2D inverse mode H-detected heteronuclear 1H-13C and 1H-31P correlation spectroscopy, the following two structures (1 and 2) could be identified [formula: see text] where Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-octulosonic acid, Non is 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonulosonic acid, and P is phosphate. Decasaccharide 1 and tridecasaccharide 2 represent an incomplete core and the complete core carrying one O-antigen repeating unit, respectively. Both are attached to the lipid A backbone but, due to their degradation protocol, they lack N- and O-acyl substituents, including N- and O-acetyl groups, the 5-N-acetimidoyl group of Non, the 2-N-alanyl group of GalN, and the 7-O-carbamoyl group of Hep as well as diphosphate, triphosphate, and, probably, some of the monophosphate groups that are present in the intact core oligosaccharide.

Chemical characterization of a new 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acid released by mild acid hydrolysis of the Legionella pneumophila serogroup 1 lipopolysaccharide.

A derivative of a new 5,7-diamino-3,5,7,9-tetradeoxynonulosonic acid was released from the lipopolysaccharide of Legionella pneumophila serogroup 1 (strain Philadelphia 1) by mild acid hydrolysis, and identified, using NMR spectroscopy and GLC-MS, as 5,7-diacetamido-8-O-acetyl-3,5,7,9-tetradeoxy-L-glycero-D-talo- nonulosonic acid or its enantiomer.

Identification of an alpha-D-Manp-(1-->8)-Kdo disaccharide in the inner core region and the structure of the complete core region of the Legionella pneumophila serogroup 1 lipopolysaccharide.

A disaccharide alpha-D-mannopyranosyl-(1-->8)-3-deoxy-D-manno-octulosonic acid [alpha-D-Manp-(1-->8)-Kdo] was released by mild acid degradation of Legionella pneumophila serogroup 1 (strain Philadelphia 1) lipopolysaccharide (LPS) and identified using NMR spectroscopy and GLC-MS of derived products. These data, together with methylation analysis of the native LPS and previously reported data [Y.A. Knirel, H. Moll, and U. Zähringer, Carbohydr. Res., 293 (1996) 223-234], allowed elucidation of the complete core region of the LPS as having the following nonasaccharide structure: [Sequence: see text]

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 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.

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.

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.

[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.

[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.

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.

Nosocomial pneumonia caused by three genetically different strains of Legionella pneumophila and detection of these strains in the hospital water supply.

A 44-year-old woman developed Legionella pneumophila pneumonia after cerebral surgery. Initially, one colony from a clinical specimen and two colonies from water samples, all belonging to serogroup 12, did not match when their DNA restriction patterns were compared. When additional colonies from the water specimens were analyzed, a serogroup 12 strain complementary to that found in the clinical specimen was identified. Other colonies from the clinical specimen were identified as serogroup 12 strains complementary to those identified from the water. In addition, the same serogroup 1 strain was isolated from the patient and the water system.

[Legionella in water of medical facilities--a hazard for patients and personnel?]. / Legionellen im Wasser medizinischer Einrichtungen--eine Gefahr für Patienten und Personal?

Since 1987 warm water samples of public health facilities in Dresden were cultured for Legionellae. In 59 of 220 (26.8%) samples and in 15 of 20 facilities Legionella pneumophila was detected. Most frequently serogroups (SG) 1 (5 times), 6 (4 times), 8 (times) and 5 (twice) were found. By reason of the ubiquity of the aerogenic transmissible Legionellae, nosocomial legionellosis can occur at any time. Most legionellosis patients suffered from severe underlying diseases. Therefore the risk for immunocompetent staff should be assessed lower. In dentists who used Legionella-contaminated dental units we could demonstrate more frequently high antibody titers against Legionellae. Measures to eradicate Legionellae from air conditioning systems and hot water supplies are discussed.