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.

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.

Pan-European study on culture-proven Legionnaires' disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups.

This pan-European study included unrelated strains of Legionella pneumophila obtained from 1335 cases of Legionnaires' disease. The isolates were serotyped into the serogroups 1 to 15 by monoclonal antibodies (MAb) and/or rabbit antisera. Additionally, MAb subgrouping was undertaken for isolates belonging to serogroups 1, 4, and 5. Monoclonal types of serogroup 1 were subdivided as having, or not having, the virulence-associated epitope recognized by the MAb 3/1 (Dresden Panel). This epitope is not present on strains belonging to any other serogroups. Taking all Legionella incidents together, MAb 3/1-positive cases were most frequent (66.8%); 11.7% of the isolates belonged to MAb 3/1-negative serogroup 1 subgroups and 21.5% to other serogroups, with serogroups 3 and 6 predominating. Among all serotypes discriminated in this study, monoclonal subtype Philadelphia was the most frequent. If categories of infection were considered, the proportion of MAb 3/1-negative strains differed significantly ( P<0.0005) between community-acquired cases (139/510; 27.3%) and travel-associated (42/295; 14.2%) or hospital-acquired infections (176/329; 53.5%). Moreover, taking distribution in different European areas into account, the proportion of MAb 3/1-negative strains was significantly higher in the Scandinavian region than in the Mediterranean countries or the UK for both community-acquired (48.7% vs. 18.6% or 12.0%; P<0.0005) and nosocomial cases (87.7% vs. 32.6% or 52.6%; P

A point mutation in the active site of Legionella pneumophila O-acetyltransferase results in modified lipopolysaccharide but does not influence virulence.

The majority of clinical isolates of Legionella pneumophila serogroup 1 produce lipopolysaccharide (LPS) that reacts with monoclonal antibody (MAb) 3/1. By using a negative cell sorting method, we isolated a spontaneous LPS mutant from L. pneumophila serogroup 1 strain Corby that lost reactivity with this MAb. The mutant contained a single nucleotide exchange in position 169 of the lag-1 gene that encodes an O-acetyltransferase that is responsible for O-acetylation of the L. pneumophila O-repeat unit (legionaminic acid). This mutation resulted in a single amino acid exchange in a highly conserved motif present in many O-acetyltransferase-like proteins. RT-PCR analysis revealed that the mutant lag-1 gene was transcribed, but the resulting protein lacked O-acetyltransferase activity. Chemical analysis of the mutant LPS revealed that it lacked 8-O-acetyl groups in legionaminic acid. In addition, the mutant failed to produce high-molecular-weight long-chain O-polysaccharide. Complementation of the mutant with the wild-type lag-1 gene restored reactivity with MAb 3/1 and the chemical structure of the wild-type LPS. Strain Corby and its MAb 3/1-negative mutant were indistinguishable in their serum resistance characteristics, and in uptake and intracellular multiplication in Acanthamoeba castellanii and macrophages.

Identification of a homopolymer of 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-talo-nonulosonic acid in the lipopolysaccharides of Legionella pneumophila Non-1 serogroups.

O-Specific polysaccharides (OPS) were isolated by mild acid hydrolysis of the lipopolysaccharides (LPS) of strains of Legionella pneumophila serogroups 2-14, as well as strains Lansing 3 and 16453-92 from newly proposed serogroups. The OPS were studied by (1)H- and (13)C-NMR spectroscopy, GLC/mass spectrometry, and chemical modifications (mild alkaline O-deacetylation and conversion of the N-acetimidoyl group into the N-acetyl group). All OPS were found to be a homopolymer of a 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxynonulosonic acid, which in some strains is 8-O-acetylated. In most strains studied, the monosaccharide has the D-glycero-D-talo configuration and is thus the C4 epimer of legionaminic acid (4-epilegionaminic acid), which has been previously identified as the monomer in the OPS of L. pneumophila serogroup 1. Poly(4-epilegionaminic acid) occurs as a minor polysaccharide in serogroups 5 (strain Dallas 1) and 13 and is absent in serogroups 1 and 7. The chemical basis for serological differentiation of L. pneumophila strains is discussed.

Phase variation among major surface antigens of Mycoplasma penetrans.

The pathogenicity and prevalence of Mycoplasma penetrans, a Mycoplasma species recently isolated from humans, are still debated. A major P35 antigen, which is used as target epitope in serological assays, was shown to be a phase-variable lipid-associated membrane protein (LAMP). In this study, we performed a comparative analysis of the LAMP patterns from five M. penetrans clinical isolates and from the type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and immunoblots with sera serially collected from an M. penetrans-infected patient indicated that these strains expressed different LAMP repertoires. Furthermore, the intraclonal variation in the expression of LAMPs (P34A, P34B, P35, and P38) was monitored by immunoblot analysis with three specific monoclonal antibodies (MAbs) developed in this study and MAb 7 to P35. The phase variation of these LAMPs occurs in an independent manner, with frequencies of variation ranging from 10(-2) to 10(-4) per cell per generation. Consistent with their amphipathic nature, the P34B and P38 antigens were found exposed at the cell surface. The DNA sequence encoding the P38 antigen was defined and found to be related to those of the P35 gene and other putative LAMP-encoding genes, suggesting that these variable antigens are encoded by a family of related genes. Finally, the serum samples from an M. penetrans-infected patient contained antibodies that reacted with a P36 antigen expressed in different M. penetrans strains but not in the isolate recovered from this patient. This result suggested that in vivo phase variation of P36 occurred, which would support a role for these LAMP variations in avoiding the host's immune vigilance.

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.

Pericarditis after allogeneic peripheral blood stem cell transplantation caused by Legionella pneumophila (non-serogroup 1).

A case of Legionella pericarditis caused by a Legionella pneumophila isolate other than serogroup 1 is reported in a 59-year-old man after allogeneic peripheral blood stem cell transplantation. On admission a 5 mm pericardial effusion was detected on echocardiography. Antibodies were detected against L. pneumophila serogroups 7 to 14 using the antigen pool and against serogroup 12 alone. Antibodies were not detected against the serogroup 1 to 6 antigen pool. The patient's clinical condition improved dramatically after treatment with clarithromycin and an echocardiography revealed the total disappearance of the pericardial effusion.

Immunolocalization of the Mip protein of intracellularly and extracellularly grown Legionella pneumophila.

The macrophage infectivity potentiator (Mip) protein is an important factor in the optimal intracellular survival of Legionella pneumophila in protozoa and human cell lines. In this study we have localized the Mip protein in Legionella grown on buffered charcoal yeast extract (BCYE) agar as well as in Legionella which were ingested by Acanthamoeba castellanii. Immunogold techniques have shown that Mip is exposed on the cell surface of extracellularly grown bacteria. In A. castellanii infected with Legionella the Mip protein was also detected on host membranes which exhibited a multilamellar structure. The morphology of these structures is similar to that of respirable vesicles of amoebas by which live legionellas may be transmitted to humans. It can be assumed that the accumulation of Mip protein in the multilamellar host membranes increases the infection potential.

Epitope mapping of immunogenic and adhesive structures in repetitive domains of Mycoplasma bovis variable surface lipoproteins.

The family of variable surface lipoproteins (Vsps) of the bovine pathogen Mycoplasma bovis includes some of the most immunogenic antigens of this microorganism. Vsps were shown to undergo high-frequency phase and size variations and to possess extensive reiterated coding sequences extending from the N-terminal end to the C-terminal end of the Vsp molecule. In the present study, mapping experiments were conducted to detect regions with immunogenicity and/or adhesion sites in repetitive domains of four Vsp antigens of M. bovis, VspA, VspB, VspE, and VspF. In enzyme-linked immunosorbent assay experiments, sera obtained from naturally infected cattle showed antibodies to different repeating peptide units of the Vsps, particularly to units R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1, all of which were found to contain immunodominant epitopes of three to seven amino acids. Competitive adherence trials revealed that a number of oligopeptides derived from various repeating units of VspA, VspB, VspE, and VspF partially inhibited cytoadhesion of M. bovis PG45 to embryonic bovine lung cells. Consequently, putative adherence sites were identified in the same repeating units (R(A)1, R(A)2, R(A)4.1, R(B)2.1, R(E)1, and R(F)1) and in R(F)2. The positions and lengths of the antigenic determinants were mostly identical to those of adhesion-mediating sites in all short repeating units, whereas in the considerably longer R(F)1 unit (84 amino acid residues), there was only one case of identity among four immunogenic epitopes and six adherence sites. The identification of epitopes and adhesive structures in repetitive domains of Vsp molecules is consistent with the highly immunogenic nature observed for several members of the Vsp family and suggests a possible function for these Vsp molecules as complex adherence-mediating regions in pathogenesis.

Diagnostic relevance of the detection of Legionella DNA in urine samples by the polymerase chain reaction.

Urine samples from 317 patients with pneumonia and from 242 patients without pneumonia were tested using a polymerase chain reaction (PCR) system for detection of the Legionella 5S rRNA gene. The results were compared with findings obtained using the established methods for diagnosis of legionellosis. Of the 317 patients with pneumonia, 58 had confirmed legionellosis, 35 had a presumptive Legionella infection, and 224 had no evidence of Legionella infection as determined by conventional methods using published criteria. The PCR was positive for 42 patients with confirmed infections, yielding a sensitivity of 72.4%. Furthermore, 16 (47%) patients with presumptive legionellosis and five (2.2%) patients without other evidence of Legionella infection had positive results. All samples from 242 patients without pneumonia were PCR-negative. When the results for all patients were considered, the specificity of the assay was > or =98.9%. The results demonstrate that the sensitivity and specificity values of urinary PCR are in the same range as those of established methods. The use of PCR in urine complements the repertoire of rapid diagnostic methods, especially for infections caused by legionellae not belonging to Legionella pneumophila serogroup 1, in which tests for detection of urinary antigen often fail.

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.

Molecular cloning and characterization of a locus responsible for O acetylation of the O polysaccharide of Legionella pneumophila serogroup 1 lipopolysaccharide.

Complementation experiments, Tn5 mutagenesis, and DNA sequencing were used to identify a locus (lag-1) that participates in acetylation of Legionella pneumophila serogroup 1 lipopolysaccharide. Nuclear magnetic resonance analyses of lipopolysaccharides from mutant and complemented strains suggest that lag-1 is responsible for O acetylation of serogroup 1 O polysaccharide.

Multiple types of Legionella pneumophila serogroup 6 in a hospital heated-water system associated with sporadic infections.

Five sporadic cases of nosocomial Legionnaires' disease were documented from 1989 to 1997 in a hospital in northern Italy. Two of them, which occurred in a 75-year-old man suffering from ischemic cardiopathy and in an 8-year-old girl suffering from acute leukemia, had fatal outcomes. Legionella pneumophila serogroup 6 was isolated from both patients and from hot-water samples taken at different sites in the hospital. These facts led us to consider the possibility that a single clone of L. pneumophila serogroup 6 had persisted in the hospital environment for 8 years and had caused sporadic infections. Comparison of clinical and environmental strains by monoclonal subtyping, macrorestriction analysis (MRA), and arbitrarily primed PCR (AP-PCR) showed that the strains were clustered into three different epidemiological types, of which only two types caused infection. An excellent correspondence between the MRA and AP-PCR results was observed, with both techniques having high discriminatory powers. However, it was not possible to differentiate the isolates by means of ribotyping and analysis of rrn operon polymorphism. Environmental strains that antigenically and chromosomally matched the infecting organism were present at the time of infection in hot-water samples taken from the ward where the patients had stayed. Interpretation of the temporal sequence of events on the basis of the typing results for clinical and environmental isolates enabled the identification of the ward where the patients became infected and the modes of transmission of Legionella infection. The long-term persistence in the hot-water system of different clones of L. pneumophila serogroup 6 indicates that repeated heat-based control measures were ineffective in eradicating the organism.

Subinhibitory concentrations of antimicrobial agents reduce the uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 cells by altering the expression of virulence-associated antigens.

We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reduced Legionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionella antigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected.

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.

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.