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Journal of Clinical Microbiology, July 2003, p. 2932-2939, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.2932-2939.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Sequence-Based Typing of Legionella pneumophila Serogroup 1 Offers the Potential for True Portability in Legionellosis Outbreak Investigation

Valeria Gaia,1* Norman K. Fry,2 Timothy G. Harrison,2 and Raffaele Peduzzi1

Istituto Cantonale di Microbiologia, Lugano, Switzerland,1 Respiratory and Systemic Infection Laboratory, Specialist and Reference Microbiology Division, Health Protection Agency, Colindale, London, United Kingdom2

Received 14 October 2002/ Returned for modification 10 December 2002/ Accepted 31 March 2003


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ABSTRACT
 
Seven gene loci of Legionella pneumophila serogroup 1 were analyzed as potential epidemiological typing markers to aid in the investigation of legionella outbreaks. The genes chosen included four likely to be selectively neutral (acn, groES, groEL, and recA) and three likely to be under selective pressure (flaA, mompS, and proA). Oligonucleotide primers were designed to amplify 279- to 763-bp fragments from each gene. Initial sequence analysis of the seven loci from 10 well-characterized isolates of L. pneumophila serogroup 1 gave excellent reproducibility (R) and epidemiological concordance (E) values (R = 1.00; E = 1.00). The three loci showing greatest discrimination and nucleotide variation, flaA, mompS, and proA, were chosen for further study. Indices of discrimination (D) were calculated using a panel of 79 unrelated isolates. Single loci gave D values ranging from 0.767 to 0.857, and a combination of all three loci resulted in a D value of 0.924. When all three loci were combined with monoclonal antibody subgrouping, the D value was 0.971. Sequence-based typing of L. pneumophila serogroup 1 using only three loci is epidemiologically concordant and highly discriminatory and has the potential to become the new "gold standard" for the epidemiological typing of L. pneumophila.


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INTRODUCTION
 
Legionellosis (infection by members of the genus Legionella) can range from mild respiratory illness to acute life-threatening pneumonia and is invariably acquired from an environmental source. Characterization of clinical and epidemiologically linked environmental isolates of Legionella is invaluable in locating the source and extent of infection, allowing implementation of corrective measures and treatment to prevent further infection. The majority of cases of legionellosis are caused by Legionella pneumophila, particularly serogroup (sg) 1 (20). Previous studies have demonstrated the utility of differentiating isolates belonging to this serogroup of L. pneumophila in order to confirm or refute epidemiological associations in outbreak investigations (5, 19, 22, 28, 29), but only in a local setting.

Isolates of L. pneumophila sg 1 can be rapidly subtyped by using monoclonal antibody (MAb) subgrouping with panels based on the international MAb subgrouping panel (13, 14). However, MAb subgrouping lacks discrimination, dividing L. pneumophila sg 1 into only 8 to 10 phenons, depending on which MAb panels are used (9). Consequently, in addition to this phenotypic typing method, a number of molecular methods have been successfully employed locally for epidemiological purposes. These include ribotyping, amplified fragment length polymorphism (AFLP) analysis, pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) analysis, restriction endonuclease analysis (REA), and arbitrarily primed PCR (9). One of these methods, AFLP analysis (26), has been adopted as an international standard (10) and is now widely used by members of the European Working Group for Legionella Infections (EWGLI). Two recent interlaboratory studies (10, 11) have assessed the reliability and discriminatory power of AFLP analysis for epidemiological typing of L. pneumophila sg 1 isolates. While these studies have demonstrated that the method is robust and rapid and allows the exchange of data between laboratories without the need for exchange of isolates, a program of proficiency testing showed that a significant proportion of laboratories could not correctly identify isolates 100% of the time (10). Incorrect identification was typically a result of data analysis issues (e.g., difficulties in comparing the AFLP banding patterns) rather than of the method itself.

For this reason, we sought to develop a universal typing method, which would be simple, rapid, discriminatory, and truly "portable" and which might in the future be applied by all microbiology laboratories to aid in the investigation of legionellosis.

Multilocus sequence typing (MLST) was first described for Streptococcus pneumoniae by Enright and Spratt (8), who used seven housekeeping genes. Subsequently, several such schemes using nonselective housekeeping genes have been described (http://www.mlst.net). We prefer to reserve the use of the term MLST for schemes such as those described by Enright and Spratt (8) and others that use housekeeping genes (not under selective pressure) and to use "sequence-based typing" (SBT) as a more universal term to describe other schemes not using such targets.

The advantages of sequence data are now well established and include portability between laboratories and the consequent ease with which global databases can be established (21). For this study, the genes chosen included four likely to be selectively neutral (acn, groES, groEL, and recA) and three likely to be under selective pressure (flaA, proA, and mompS). The groESL genes (also known as hsp10/60 and htpA/B) (17, 24) encode the 10-kDa (GroES) and 58-kDa (GroEL) common antigens in L. pneumophila. The 58-kDa common antigen was formerly designated the 60-kDa common antigen (24). The acn gene encodes a major iron-containing protein of L. pneumophila showing aconitase activity (23), and the recA gene encodes the RecA protein of L. pneumophila (30). The flaA gene encodes the flagellum subunit protein of L. pneumophila sg 1 (15), proA encodes a zinc metalloprotease found in L. pneumophila (3), and mompS encodes an outer membrane protein of ca. 29 kDa (6; E. Christoph and W. Ehret, unpublished data).

The aims of this study were (i) to determine the suitability of the various primer pairs and gene targets for amplification and sequencing and (ii) to evaluate an SBT approach, which could be used for the investigation of outbreaks of legionellosis caused by L. pneumophila.


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MATERIALS AND METHODS
 
Bacterial strains. Ninety-five clinical and environmental isolates of L. pneumophila sg 1 from 10 European countries comprising one epidemiologically "unrelated" panel of 79 isolates (panel 1), one epidemiologically "related" panel of 16 isolates (panel 2), and one stability panel of 5 isolates (panel 3) were analyzed (Table 1). Each of these isolates was obtained from the EWGLI Legionella culture collection and has a unique EUL number (http://www.ewgli.org). This collection of isolates was established by members of the EWGLI to facilitate epidemiological typing studies (9, 10, 11). For the unrelated panel (panel 1), 10 laboratories provided their 10 most recent (i.e., consecutive) endemic clinical isolates that conformed to the following criteria: each isolate (i) was obtained from a patient resident within that country during the 2 weeks before onset of symptoms and (ii) was not epidemiologically related (temporally or geographically) to any other isolate included in the panel. Not all laboratories were able to supply 10 isolates, and 11 were obtained from one laboratory, so 79 unrelated isolates were obtained. The epidemiologically related panel (panel 2) of 16 isolates comprised five sets of epidemiologically related isolates and two replicates of the same isolate. The stability panel (panel 3) comprised five variants of the same strain. Each isolate has been extensively characterized previously (1, 2, 9, 10, 11).


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  		TABLE 1. Isolates of Legionella pneumophila serogroup 1 characterized by SBT

Study design. The choice of nonselective and selective gene targets was based on the availability of sequence data for one or more strains of L. pneumophila. The SBT method was evaluated as outlined in the Consensus Guidelines of the European Study Group on Epidemiological Markers (25) and in previous studies (9, 10, 11). Typeability (T) was calculated as the proportion of isolates assigned to a type by sequencing of each target gene. Reproducibility (R) was assessed by analysis of one or more loci from 10 of the isolates in both our laboratories using a number of different conditions (including reagent manufacturers) for DNA extraction, PCR, and DNA sequencing. Initially, sequence data were obtained for the seven genes acn, flaA, groES, groEL, mompS, proA, and recA from 10 of the 16 isolates in the epidemiologically related panel (panel 2, EUL no. 48, 56, 71, 73, 76 to 79, 120, and 121), comprising four sets. The three genes yielding the maximum number of possible sequence types in these four sets, i.e., four sequence types in four sets, were chosen for further analysis of the remaining 6 isolates in panel 2 (EUL no. 40, 47, and 140 to 143) and of the 79 isolates in the epidemiologically unrelated panel (panel 1). Epidemiologic concordance (E) was expressed as the number of epidemiologically related sets of strains found to be indistinguishable by the typing system, divided by the total number of sets in the panel. The index of discriminatory power (D) was determined by calculating Simpson's index of diversity, as described by Hunter and Gaston (18), by the equation

where N is the total number of strains in the sample population, S is the total number of types described, and nj is the number of stains belonging to the jth type (18).

Individual and combined D values were calculated for SBT by using flaA, proA, and mompS, together with MAb subgrouping results from previous studies. The stability of these three genes, targets that are likely to be under selective pressure (flaA, proA, and mompS), was assessed by analysis of the five variants of the same strain (Table 1, panel 3).

DNA extraction and PCR amplification. Genomic DNA was extracted using the Nucleon BACC2 DNA extraction kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom) or the InstaGene Purification Matrix (Bio-Rad, Hercules, Calif.). Oligonucleotide primers targeting regions of each of the genes were designed to amplify 279- to 763-bp products encompassing regions of variation (Table 2). Amplification using PCR was performed in a reaction volume of 50 µl. Each reaction mixture contained 2.5 mM MgCl2, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 µM (each) deoxynucleotide, 10 pmol of each primer (MWG Biotech Ltd., Milton Keynes, United Kingdom, or Microsynth AG, Balgach, Switzerland), and 1.0 to 2.5 U of Taq polymerase (Life Technologies, Paisley, United Kingdom, or Qiagen, Basel, Switzerland). Template DNA (ca. 10 to 100 ng) was added, and reaction mixtures with no added DNA served as negative controls. Amplification was performed in a DNA Engine (MJ Research) or a GeneAmp PCR system (Perkin-Elmer Applied Biosystems, Foster City, Calif.) using the following conditions: 35 cycles of 30 s at 94°C, 30 s at 50°C, and 30 s at 72°C.


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  		TABLE 2. Oligonucleotide primers designed for this study

Purification and DNA sequencing. Amplicons were purified using the Wizard PCR Preps purification system (Promega Corp., Madison, Wis.) or the Qiaquick PCR purification kit (Qiagen). Nucleotide sequences were determined by using the primers used for amplification together with either (i) the Dye Terminator Cycle Sequencing kit (Beckman Coulter), in which case the products were analyzed on a CEQ 2000 XL DNA analysis system or a CEQ 8000 genetic analysis system (Beckman Coulter), or (ii) the ABI PRISM BigDye Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS (Applied Biosystems), in which case the products were analyzed on a model 310 ABI DNA sequencer (Applied Biosystems). Sequencing and analysis were performed according to the instructions supplied by the manufacturers.

Sequence analysis. Sequence analyses were performed using the Editseq and Megalign programs included in the Lasergene package (DNASTAR, Inc., Madison, Wis.); Kodon, version 1.0 (Applied Maths, Kortrijk, Belgium); or BioNumerics, version 3.0 (Applied Maths). For all analyses, data from forward and reverse sequencing primers were combined and aligned manually.

Data analysis. On completion of the sequencing, the complete data set was analyzed in BioNumerics (Applied Maths).

Nomenclature. For each gene of L. pneumophila analyzed, identical sequences were assigned the same allele number [e.g., flaA(1)] and different sequences were assigned distinct allele numbers [e.g., flaA(1), flaA(2), flaA(3), etc.]. For each isolate, the combination of alleles at each of the loci was defined as the sequence type or allelic profile (e.g., 1,2,3) by using a predetermined order, i.e., flaA, proA, mompS. Where sequence data were unavailable, a zero was entered into the sequence string.

Nucleotide sequence accession numbers. The flaA, proA, and mompS sequences from the 95 isolates of L. pneumophila sg 1 are available from the authors or at http://www.ewgli.org.


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RESULTS
 
Typeability. All isolates included in the study, except two, yielded PCR products of the expected sizes and DNA sequences with primers from all genes tested. For two isolates (EUL no. 48 and 56), satisfactory amplification products and DNA sequences were not obtained with primers targeting the aconitase gene (acn).

Sequence variation. The number of nucleotides included in the analysis from each of the gene targets ranged from 182 to 500 bp. All sequences used in the analyses were consensus sequences assembled from the results of the forward and reverse primers. Alleles were numbered consecutively even in the case of a single nucleotide difference.

The initial analysis of 10 of the 16 isolates in the epidemiologically related panel, including the number of polymorphic sites, the percentage of nucleotide substitutions, and numbers of silent and nonsilent mutations, is shown in Table 3, and the analysis of all 95 isolates with the three genes yielding the maximum number of possible sequence types in the initial analysis is shown in Table 4. The highest percentage of polymorphic sites was found with the mompS gene (9.09%), followed by proA (8.76%) and flaA (7.69%); these genes yielded a total of 16, 15, and 12 individual sequence types, respectively. Results of analyses of the 16 isolates in the epidemiologically related panel (panel 1) are shown in Table 5, and the allelic profiles of all 95 isolates with flaA, proA, and mompS are shown in Table 6.


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  		TABLE 3. Sequence variation in seven different loci for 10 isolates of L. pneumophila comprising four sets of epidemiologically related isolates


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  		TABLE 4. Sequence variation in three different loci, for all 95 isolates of L. pneumophila tested


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  		TABLE 5. Allelic profiles of L. pneumophila serogroup 1 from epidemiologically related sets


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  		TABLE 6. Allelic profiles of 95 isolates of L. pneumophila serogroup 1 from analysis of three loci showing MAb subgroup and country of origin

Reproducibility. Data from consensus sequences from loci from all 10 isolates tested by both laboratories using different methods of DNA extraction, PCR cycling conditions, and DNA sequencing platforms were in complete agreement (R = 1.00).

Epidemiologic concordance. All six of the sets of isolates included in the epidemiologically related panel showed compelling evidence of epidemiological relatedness (9, 11). Previous analysis revealed concordant MAb subgrouping, RFLP analysis, REA, and AFLP analysis results (9, 11). Seven genes were sequenced for the 10 isolates representing three sets of epidemiologically related strains and two replicates of the same strain. Epidemiologic concordance (E) was calculated for each of the genes analyzed by using these 10 isolates, and for each locus there was complete concordance (E = 1.00). The aconitase gene (acn), the two common-antigen genes (groES and groEL), and recA could differentiate only two or three sets of strains out of the four sets, whereas proA, mompS, and flaA could distinguish among all four sets of isolates (Tables 3 and 5). The most variable gene was flaA, with 7.14% substitutions over a 182-bp region. Results from the additional two epidemiologically related sets (EUL no. 40, 47, and 140 to 143) comprising clinical and environmental isolates were also epidemiologically concordant (E = 1.00) and gave unique profiles for each set.

Discriminatory power. Individual and combined indices of discrimination (D) were calculated for the three genes flaA, proA, and mompS by using the panel of 79 unrelated isolates (Table 7). Table 7 also shows the increase in the D value when SBT data are combined with MAb subgrouping data.


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  		TABLE 7. Indices of discrimination (D) calculated from the 79 unrelated isolates of L. pneumophila serogroup 1 by using one, two, or three loci with and without MAb subgrouping results

Stability. Analysis of all five variants (EUL 135 to 139) in the stability panel (Table 1, panel 3) resulted in identical sequence profiles: flaA(6), proA(9), mompS(14). This was also a novel sequence profile not previously seen in the other isolates tested (Table 6).


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DISCUSSION
 
Many phenotypic and genotypic methods for the epidemiologic typing of L. pneumophila sg 1 have been described (4, 12, 19, 27). Previous work by members of the EWGLI has sought to identify the best genotypic techniques for standardization (9). By using a well-defined panel of isolates (the same panel of 79 unrelated isolates used in this study), those methods showing good reproducibility (E values of 1.00) and D values of >0.89 were identified. Four methods, AFLP analysis (D = 0.891), RFLP analysis (D = 0.896), PFGE using NotI (D = 0.941), and REA using HhaI and/or HaeIII (D = 0.960 to 0.980), met these criteria; one of these, AFLP analysis, was subsequently adopted as the EWGLI standard method (11). However, the major disadvantage of all of these methods is that they require the interpretation of gel images. Data from these studies show that the comparison of the patterns obtained in different laboratories, even when the highly standardized EWGLI AFLP analysis protocol is followed, is not always satisfactory. The SBT approach exemplified by MLST (21) appears to offer a way to circumvent these problems.

MLST using bacterial housekeeping genes (21) is an adaptation of multilocus enzyme electrophoresis, revealing the slow accumulation of variation within the gene or the protein for which it codes. The choice of genes was based on those that would be likely to be selectively neutral (21). Sequence analysis of a number of loci (6 to 11 loci) can be used to address the population and evolutionary biology of a particular species (7, 21, 31; http://www.mlst.net). It is acknowledged, however, that other techniques may be more useful in studying individual outbreaks, because identification of microvariation is required in order to distinguish between circulating strains within a geographical area (21).

In this study we developed and evaluated an SBT approach for implementation in the investigation of outbreaks of legionellosis caused by Legionella pneumophila sg 1. Of the seven genes investigated, the four selectively neutral genes all showed less variation than the three genes under probable selective pressure (flaA, proA, and mompS). Furthermore, the aconitase primers failed to give satisfactory amplification products and DNA sequences from two epidemiologically related isolates, EUL 48 and EUL 56 (possibly due to sequence variation at the primer binding sites). Given their low overall variability in genetic sequence and the fact that we were seeking to develop a typing method rather than a phylogenetic tool, it was decided not to consider the nonselective genes further as epidemiological typing targets.

None of the three loci flaA, proA, and mompS gave a sufficiently high index of discrimination (range, 0.767 to 0.857) to be used on its own. However, use of a combination of mompS and proA gave a D value of 0.903, and use of all three loci combined gave a D value of 0.924. Although this is below the value of 0.95 considered to be ideal for a typing system used as a single typing method (25), it is still above the value of 0.90 considered to be desirable if the typing results are to be interpreted with confidence (18). Moreover, the D value of 0.903 achieved with three loci is above the value of 0.89 achieved with the AFLP analysis method (9) currently employed in L. pneumophila sg 1 outbreak investigation (9, 10, 11). When use of all three loci was combined with MAb subgrouping, as is usual in legionellosis outbreak investigations, this value rose to 0.971, well in excess of 0.95 (25). The isolates used in this study were chosen because they were well characterized and would allow ready comparison to previous studies using other methods. However, because the unrelated panel consists entirely of clinical isolates, the D values calculated here may well underestimate the true discrimination achieved when the SBT method is applied more widely. For these three genes (flaA, proA, and mompS) with the alleles identified from analysis of the panel 1 isolates, there is the potential to unambiguously resolve ca. 2,500 sequence types. Examination of other strains is likely to identify many more alleles, increasing the number of potential types still further.

While we believe that this study has demonstrated the validity of the SBT approach for L. pneumophila sg 1, there are a number of issues to be resolved before such a method can be used routinely. These include the following. What are the minimum standards required for the generation of suitable data? e.g., for SBT to be sufficiently robust, it is essential that both strands of the amplicon be sequenced. What size of PCR product should be sequenced? Sequencing of smaller (200- to 250-bp) regions showing sufficient variation, possibly by using internal primers, would allow a faster turnaround and be more economical. What are the optimal targets that should be examined? Together with our colleagues and members of the EWGLI, we are investigating additional gene targets (P. C. Lück and J. Etienne, personal communication).

By direct comparison of results from different loci (or distinct areas on the same loci) using the same panel of strains, a consensus should soon be reached, with the aim of using the best combination of gene targets. In an outbreak situation, depending on the numbers of isolates involved, it may still be desirable to screen isolates first by MAb subgrouping and/or AFLP analysis. It is anticipated that following agreement by members of the EWGLI on the most suitable gene targets for epidemiological typing of Legionella pneumophila sg 1, a sequence database, accessed via the EWGLI website, will be established. Following the sequencing of these gene targets from clinical or environmental isolates of L. pneumophila sg 1, users will be able to query the database, identify preexisting allelic profiles, and submit novel profiles.

In conclusion, we have demonstrated the advantages of SBT of L. pneumophila sg 1 over other current methods. By using only three loci, this methodology has the potential to become the new "gold standard" for the epidemiological typing of L. pneumophila.


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ACKNOWLEDGMENTS
 
We thank Barahak Afshar, Nita Doshi, John Duncan, and Oceanis Tzivra of the Respiratory and Systemic Infection Laboratory for expert technical assistance, Jon Green of the PHLS Bioinformatics Unit for assistance with the Worldwide Web-enabled database, and P. Christian Lück and Jürgen Helbig for MAb data for the Corby strain variants.


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FOOTNOTES
 
* Corresponding author. Present address: Istituto Cantonale di Microbiologia, via Mirasole 22a, CH-6500 Bellinzona, Switzerland. Phone: 41 91 814 60 11. Fax: 41 91 814 60 29. E-mail: valeria.gaia{at}ti.ch. Back


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Journal of Clinical Microbiology, July 2003, p. 2932-2939, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.2932-2939.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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