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).

Characterization of legionella lipopolysaccharide.

The lipopolysaccharide(LPS) of Legionella spp. is an immuno-dominant antigen and the basis for Legionella pneumophila serogroup classification. The LPS shows a peculiar structure composed of a very hydrophobic lipid A acylated by long chain fatty acids and an O-antigen-specific chain consisting of homopolymeric legionaminic acid. In this chapter we describe a method for the isolation of LPS from L. pneumophila. In the first part we describe the chemical purification, in the second part we outline the application of monoclonal antibody (mAb) in Western blot and immuno-localization by indirect immunofluorescence. This report does not describe physico-chemical methods that analyze the structure of lipopolysaccharide entities.

Distribution of monoclonal antibody subgroups and sequence-based types among Legionella pneumophila serogroup 1 isolates derived from cooling tower water, bathwater, and soil in Japan.

Legionella pneumophila serogroup (SG) 1 is the most frequent cause of legionellosis. This study analyzed environmental isolates of L. pneumophila SG 1 in Japan using monoclonal antibody (MAb) typing and sequence-based typing (SBT). Samples were analyzed from bathwater (BW; n = 50), cooling tower water (CT; n = 50), and soil (SO; n = 35). The distribution of MAb types varied by source, with the most prevalent types being Bellingham (42%), Oxford (72%), and OLDA (51%) in BW, CT, and SO, respectively. The ratios of MAb 3/1 positive isolates were 26, 2, and 14% from BW, CT, and SO, respectively. The environmental isolates from BW, CT, and SO were divided into 34 sequence types (STs; index of discrimination [IOD] = 0.973), 8 STs (IOD = 0.448), and 11 STs (IOD = 0.879), respectively. Genetic variation among CT isolates was smaller than seen in BW and SO. ST1 accounted for 74% of the CT isolates. The only common STs between (i) BW and CT, (ii) BW and SO, and (iii) CT and SO were ST1, ST129, and ST48, respectively, suggesting that each environment constitutes an independent habitat.

Legionella pneumophila carrying the virulence-associated lipopolysaccharide epitope possesses two functionally different LPS components.

Phase-variable expression of Legionella pneumophila lipopolysaccharide (LPS) has not been described in detail for strains possessing the virulence-associated epitope recognized by the monoclonal antibody (mAb) 3/1 of the Dresden Panel. About 75 % of cases of community-acquired legionellosis are caused by mAb 3/1-positive strains. In this study, the LPS architecture of the mAb 3/1-positive Corby strain was investigated during its life cycle in broth culture and inside monocytic host cells. During the exponential growth phase in broth, the highly acetylated and therefore strongly hydrophobic mAb 3/1 epitope is expressed continuously, but only 3 % of the bacteria can be detected using mAb 59/1, which recognizes a short-chain variant of the Legionella LPS that is less hydrophobic due to missing acetylations of the O-chain. The percentage of mAb 59/1-positive legionellae increases up to 34 % in the post-exponential growth phase. LPS shed in broth during the exponential phase is mAb 59/1-negative, and mAb 3/1-positive components do not possess short-chain molecules. The LPS pattern expressed and shed inside U937 cells and A/J mouse macrophages points to the same regulatory mechanisms. During the so-called 'pregnant pause', the period for establishment of the replicative phagosomes, the mAb 3/1-positive LPS is shed into the phagosome and seems to pass through the phagosomal membrane, while mAb 59/1-positive LPS is detectable only on the bacterial surface. After egress of the legionellae into the cytoplasm followed by host cell lysis, individual bacteria are mAb 3/1-positive and mAb 59/1-negative. Intracellularly formed Legionella clusters consist of surface-located mAb 3/1-positive bacteria, which are predominantly mAb 59/1-negative. They surround less hydrophobic and therefore closely packed mAb 59/1-positive bacteria. Based on the different degrees of hydrophobicity, bacteria are able to support the expression of two functionally different LPS architectures, namely more hydrophobic LPS for surviving in aerosols and more hydrophilic LPS for close-packing of legionellae inside clusters.

Lipopolysaccharide of Legionella pneumophila shed in a liquid culture as a nonvesicular fraction arrests phagosome maturation in amoeba and monocytic host cells.

After uptake by susceptible host cells, Legionella pneumophila displays the ability to arrest phagolysosome fusion. To elucidate the role of lipopolysaccharide (LPS) in this mechanism, we investigated its influence on Acanthamoeba castellanii, A/J mouse macrophages and human monocytes. For this, legionellae were cultured in broth to the replicative, noninfectious phase or to the infectious phase expressing virulence traits. Shed LPS-enriched outer membrane vesicles (OMV) and LPS species <300 kDa were obtained from L. pneumophila Corby strains possessing the virulence-associated LPS epitope recognized by monoclonal antibody (MAb) 3/1 and its mutant TF 3/1, which has lost this epitope due to a mutation in the lag-1 gene. The shed LPS components were attached by specific antibodies to latex beads and added to the host cells for phagocytosis. We demonstrated for the first time that evasion of lysosomal degradation of phagosomes for up to 5 h can also be set off by LPS that is not tied up in OMV. Moreover, our cell culture models showed that the influence of MAb 3/1-positive and -negative LPS was identical. Our data clearly substantiate that LPS is an independent factor for evading lysosomal degradation, which is independent of the bacterial expression of known virulence traits.

Characterization of Legionella pneumophila isolates from patients in Japan according to serogroups, monoclonal antibody subgroups and sequence types.

We collected 86 unrelated clinical Legionella pneumophila strains that were isolated in Japan during the period 1980-2008. Most (80.2%) belonged to serogroup 1, followed by serogroups 5, 3 and 2. Interestingly, the patients with L. pneumophila serogroup 1 had a significantly higher male-to-female ratio (12.4) than the patients with other L. pneumophila serogroups (2.0) (OR, 10.5; 95% CI, 2.5-44.5). When the serogroup 1 strains were analysed by monoclonal antibody (mAb) typing, the most prevalent subgroup was Benidorm (34.9% of all isolates). Moreover, 79.7% of the serogroup 1 isolates were bound by mAb 3/1, which recognizes the virulence-associated epitope. When all 86 isolates were subjected to sequence-based typing (SBT) using seven loci, they could be divided into 53 sequence types (STs). The ST with the most isolates (seven) was ST1, to which most isolates from patients and environments around the world belong. However, six of the seven ST1 isolates were isolated before 1994. Other major STs were ST306 (n=6), ST120 (n=5) and ST138 (n=5). All ST306 and ST138 isolates, except for one isolate (ST306), were suspected or confirmed to be derived from bath water, which suggests that these strains prefer bath habitats. The sources of all ST1 and ST120 isolates remain unclear. By combining the SBT and mAb data, the 86 isolates could be divided into 59 types (discrimination index, 0.984). This confirms the usefulness of this combination in epidemiological studies.

PCR-based 'serotyping' of Legionella pneumophila.

Currently, several PCR assays based on 16S rRNA and virulence-associated genes are available for detection of Legionella pneumophila. So far, no genotyping method has been published that can discriminate between serogroups and monoclonal subgroups of the most common L. pneumophila serogroup 1. Our first approach was to analyse LPS-associated genes of seven L. pneumophila serogroup 1 strains, and we developed two PCR-based methods specific for serogroup 1. Specific DNA fragments could be amplified from all the serogroup 1 strains (n=43) including the strains from the American Type Culture Collection. In contrast, none of the strains from serogroups 2-15 (n=41) contained these specific gene regions. In a second approach, primers specific for the lag-1 gene, encoding an O-acetyltransferase, which is responsible for the presence of the LPS epitope recognized by mAb 3/1, were designed and tested for their ability to differentiate between mAb 3/1-positive and -negative strains. All mAb 3/1-positive strains (n=30) contained the lag-1 gene, but in turn 4 of 13 tested mAb 3/1-negative strains were also positive in the PCR. Thus, the discrimination between mAb 3/1-positive and mAb 3/1-negative subgroups could not be achieved for all strains. In a third approach, two intergenic regions expected to be specific for monoclonal subgroup Knoxville and closely related subgroups Benidorm/Bellingham were identified and used for selective genotyping. These intergenic regions could not only be amplified in every tested strain belonging to the subgroups Knoxville, Benidorm and Bellingham, but also in some strains of other unrelated subgroups. The two PCR approaches with primers specific for serogroup 1 genes definitely represent a valuable tool in outbreak investigations and for risk assessment. They also might be used for culture-independent diagnosis of legionellosis caused by L. pneumophila serogroup 1.

Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains.

The standard sequence-based method for the typing of Legionella pneumophila serogroup 1 strains was extended by using the gspA and neuA alleles. The use of neuA as a seventh allele for typing significantly increased the index of discrimination calculated for a panel of unrelated strains (from 0.932 to 0.963) and subdivided some known large common complexes (e.g., 1,4,3,1,1,1). This modification to the standard method is proposed as the method of choice in the epidemiological investigation of L. pneumophila infections.

Evaluation of the Duopath Legionella lateral flow assay for identification of Legionella pneumophila and Legionella species culture isolates.

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries' regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.

Incomplete activation of macrophage apoptosis during intracellular replication of Legionella pneumophila.

The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icm-dependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.

Evaluation of different primers for DNA fingerprinting of Legionella pneumophila serogroup 1 strains by polymerase chain reaction.

A DNA fingerprinting method for the characterization of Legionella pneumophila serogroup 1 strains was established. This method was based on the DNA extraction using Chelex 100 and subsequent PCR analysis using primers under conditions of low stringency. Sixteen single primers were tested for the typing of the 10 epidemiologically unrelated reference strains of L. pneumophila serogroup 1 as well as patient isolates and environmental strains isolated from the water system of a hospital where patients with legionellosis were treated. In addition, a combination of two primers (Lpm-1 and Lpm-2) originally established for the specific detection of Legionella strains was tested. The PCR results were compared with two further subtyping methods, i.e. monoclonal antibody analysis and pulsed-field gel electrophoresis. The type strains Philadelphia 1, Knoxville 1, Allentown 1, Benidorm 0303E, Bellingham 1, and France 5811 could be distinguished clearly in experiments using all of the primers. Depending on the primer used, Heysham 1 and Oxford 4032E showed different DNA profiles. The strains Olda and Camperdown 1 were nearly indistinguishable. In contrast, the analysis by PFGE and MAb subtyping revealed distinct types for all 10 reference strains. The discrimination of the patient isolates from two suspected cases of nosocomial legionellosis and environmental isolates was not possible with the 16 single primers used in the study. However, the PCR assay with the combination of Lpm-1 and Lpm-2 as well as the PFGE and MAb analysis were able to differentiate distinct types. The use of the sequence-specific primers under low-stringency annealing conditions allowed both simultaneous gene detection as well as epidemiological typing of Legionella strains.

Rapid detection and enumeration of Legionella pneumophila in hot water systems by solid-phase cytometry.

A new method for the rapid and sensitive detection of Legionella pneumophila in hot water systems has been developed. The method is based on an IF assay combined with detection by solid-phase cytometry. This method allowed the enumeration of L. pneumophila serogroup 1 and L. pneumophila serogroups 2 to 6, 8 to 10, and 12 to 15 in tap water samples within 3 to 4 h. The sensitivity of the method was between 10 and 100 bacteria per liter and was principally limited by the filtration capacity of membranes. The specificity of the antibody was evaluated against 15 non-Legionella strains, and no cross-reactivity was observed. When the method was applied to natural waters, direct counts of L. pneumophila were compared with the number of CFU obtained by the standard culture method. Direct counts were always higher than culturable counts, and the ratio between the two methods ranged from 1.4 to 325. Solid-phase cytometry offers a fast and sensitive alternative to the culture method for L. pneumophila screening in hot water systems.

Comparison of commercially available media for the recovery of Legionella species.

A lack of standardization of environmental monitoring techniques for Legionella spp. complicates the interpretation and comparison of results from different institutions. Since the quality of the culture media has enormous effect on the recovery of Legionella spp. a comparative assessment of commercially available media from four manufactures (Becton Dickenson, BioMerieux, Heipha, and Oxoid) was performed. For this, samples containing infected Acanthamoeba castellanii cells, samples from the External Quality Assurance Scheme (EQA) run by the Health Protection Agency in London and water samples from a hospital in Dresden, Germany, were investigated. The glycine-containing media (GVPC) from four manufactures were equally effective in growing legionellae. However, this medium formulation inhibited some of the non-pneumophila strains tested which was not observed with the selective BMPA and non-selective BCYE-agar.

Phase variation of the multiple banded protein in Ureaplasma urealyticum and Ureaplasma parvum.

Ureaplasma urealyticum and U. parvum are common commensals and, possibly, pathogens of the human urogenital tract. Like other Mycoplasmatales they possess variable surface proteins. The multiple banded (MB) protein shows a striking variability of its molecular weight. This is caused by changes of the number of C-terminal repeating units. In this study, selective pressure was imposed against cytadherence of U. urealyticum and U. parvum. Ureaplasmas were co-incubated with either erythrocytes or HeLa cells and the cell-bound fraction was removed. Additionally, U. urealyticum populations were transferred serially through broth containing specific polyclonal antibodies. Both approaches led to the emergence of escape variants in which no MB protein was detectable. PCR studies with several primers on different parts of the mba gene indicated major differences between wild-type strains and MB-negative escape variants. In experiments with clonal lineages, however, the loss of the MB protein was shown to be reversible. Therefore, it is proposed that the multiple banded proteins of U. urealyticum and U. parvum are subjected to a phase-switching mechanism as it has already been described for several other Mycoplasmatales.

A hospital-associated outbreak of Legionnaires' disease caused by Legionella pneumophila serogroup 1 is characterized by stable genetic fingerprinting but variable monoclonal antibody patterns.

An outbreak of 18 pneumonia cases caused by Legionella pneumophila serogroup 1 occurred at a Swedish university hospital 1996 to 1999. Eight clinical isolates obtained by culture from the respiratory tract were compared to 20 environmental isolates from the hospital and to 21 epidemiologically unrelated isolates in Sweden, mostly from patients, by using pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism analysis (AFLP), and monoclonal antibody (MAb) typing. All patients and most environmental isolates from the outbreak hospital belonged to the same genotypic cluster in both PFGE and AFLP. This genotype was distinctly different from other strains, including a cluster from a second hospital in a different part of the country. The MAb subtype of the outbreak clone was Knoxville except for three isolates that were Oxford. A variation in the MAb reactivity pattern was also found in a second genotypic cluster. These changes in the MAb reactivity pattern were due to the absence or presence of the lag-1 gene coding for an O-acetyltransferase that is responsible for expression of the lipopolysaccharide epitope recognized by MAb 3/1 of the Dresden Panel. In all MAb 3/1-positive strains, the lag-1 gene was present on a genetic element that was bordered by a direct repeat that showed a high degree of sequence homology. Due to this homology, the lag-1 gene region seemed to be an unstable element in the chromosome. MAb patterns are thus a valuable adjunct to genotyping methods in defining subgroups inside a genotypic cluster of L. pneumophila sg 1.

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

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 C-terminal, 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.

Clinical utility of urinary antigen detection for diagnosis of community-acquired, travel-associated, and nosocomial legionnaires' disease.

The clinical utility of Legionella urinary antigen assays for the diagnosis of Legionnaires' disease was assessed by using samples from 317 culture-proven cases. The sensitivities of the Binax enzyme immunoassay (EIA) and Biotest EIA were found to be 93.7 and 94.4% for travel-associated infection and 86.5 and 76.0% for community-acquired infection but only 44.2 and 45.7% for nosocomially acquired infection, respectively.

Detection of Legionella pneumophila antigen in urine samples by the BinaxNOW immunochromatographic assay and comparison with both Binax Legionella Urinary Enzyme Immunoassay (EIA) and Biotest Legionella Urin Antigen EIA.

The new BinaxNOW Immunochromatographic (ICT) Assay for the detection of Legionella pneumophila antigens was used to test 535 urine specimens from patients with and without Legionnaires' disease. The specificity, calculated by testing 112 samples from patients with pneumonia of aetiologies other than Legionella infection, and 167 urine specimens from urinary tract infections, was found to be 97.1% if the manufacturer's guidelines were followed. However, it was determined that the 'false positive' results characterised by very weak bands could be discounted by re-examination of the results at 60 min, yielding a specificity of 100%. With this minor modification of the procedure applied to examination of urine samples from 117 patients with legionellosis confirmed by isolation of L. pneumophila and 70 patients who had seroconverted to L. pneumophila serogroup 1, sensitivity was calculated to be 79.7%. In comparison, the sensitivities of the Binax Urinary Antigen Enzyme Immunoassay (EIA) and Biotest Urin Antigen EIA were estimated to be 79.1 and 83.4%, respectively. Eleven cases (5.9%) were positive by BinaxNOW assay but negative by Binax or Biotest EIA, or both. The sensitivities of all assays increased to c. 94% if only diagnosis of cases confirmed by isolation of serogroup 1 L. pneumophila was considered, although the sensitivity for infections caused by L. pneumophila serogroup 1 monoclonal antibody (MAb) subgroup Bellingham was significantly lower than for other MAb subgroups. The Biotest EIA recognised 10 (45%) of the 22 cases not caused by L. pneumophila serogroup 1, whereas the two Binax kits detected only three each. The ICT assay BinaxNOW can be recommended as a rapid specific test for the diagnosis of Legionnaires' diseases caused by L. pneumophila serogroup 1, although very weak bands should be interpreted cautiously.

A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study.

OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.