221I. de Filippis and M.L. McKee (eds.), Molecular Typing in Bacterial Infections, Infectious Disease, DOI 10.1007/978-1-62703-185-1_15, © Springer Science+Business Media New York 2013

Legionellae are Gram-negative bacteria that can cause sporadic cases and outbreaks of pneumonia when water droplets are inhaled from a variety of natural and man-made sources [ 1 ] . There are more than 50 different species of Legionella and although 20 are documented as human pathogens [ 1 ] , up to 90 % of clinical cases are caused by Legionella pneumophila . Among the 15 serogroups (Sg) character-ized within the species L. pneumophila , Sg1 is responsible for about 85 % of all cases worldwide [ 2, 3 ] .

Typing of L. pneumophila has two principal applications.

Identi fi cation of environmental sources of infections in order to prevent or to –stop an outbreak. Comparison of clinical and environmental isolates is necessary to identify a water reservoir as the source of infections. Studying the dynamics of – Legionella populations.

The fi rst step of Legionella identi fi cation is its auxotroph character for cysteine. Legionellae have proved to be relatively unreactive when traditional biochemical tests are utilized, necessitating more complex identi fi cation methods. Speci fi c anti-bodies are commonly used for rapid discrimination of L. pneumophila serogroup 1 from other L. pneumophila and from other Legionella in latex agglutination assays. They are also used for acute species identi fi cation (e.g., indirect immuno fl uorescence assay), but cross reactions are frequent and molecular techniques such as mip sequencing appear to be the gold standard for the species identi fi cation of Legionella non pneumophila [ 4 ] . Recently matrix-assisted laser desorption ionization time-of- fl ight mass spectrometry (MALDI-TOF-MS) has been described as a useful tool for Legionella species identi fi cation [ 5, 6 ] .

C. Ginevra (*) Laboratoire pathogénie bactérienne et immunité innée, Université Lyon 1, Faculté de médecine Lyon est, INSERM U851, Centre national de référence des légionelles, Hospices civils de Lyon , 7 rue Guillaume Paradin , 69372 Lyon , France e-mail: christophe.ginevra@univ-lyon1.fr

Chapter 15 Legionella pneumophila Typing

Christophe Ginevra

222 C. Ginevra

Usually typing techniques are applied on L. pneumophila serogroup 1 isolates; nevertheless, some of the typing methods described below can be applied on other Legionella isolates.

After genus and species identi fi cation, a fi rst phenotypic screen can be performed by subgrouping L. pneumophila serogroup 1 using panels of monoclonal antibodies as described by Joly et al. and Helbig et al. [ 7, 8 ] . The latter described a standard phenotyping scheme allowing dividing Lp1 into 9 subgroups which is very low for a typing method but which constitutes a interesting fi rst screen which could enhance the discriminatory power of an associated genotyping method [ 9 ] .

The key point of molecular typing is the selection of the molecular marker. The marker has to be variable enough to differentiate two unrelated isolates, but stable enough to remain identical between a same strain isolated on one side from patients’ samples during an outbreak and on the other side from environmental samples dur-ing epidemiological investigations.

For L. pneumophila , most molecular typing methods are based on comparison of DNA banding patterns generated by several methods. More recently, sequence-based methods have been developed [ 9– 13 ] . A MALDI-TOF-MS approach has also been described recently [ 14 ] .

To facilitate the monitoring of travel associated LD, a European group was created in 1986 European working group on Legionella infection (EWGLI). This group has worked on the standardization at an international level of molecular typing methods.

Restriction fragment length polymorphisms (RFLP) is one of the fi rst methods developed for L. pneumophila molecular typing and has been used as the gold stan-dard until recently in some countries such as the UK in which it was used for at least 19 years [ 15 ] . The method is based on probing restriction fragments of chromo-somal DNA with cloned probes composed of randomly selected regions of the L. pneumophila chromosome [ 16 ] . This method has also been use for Legionella longbeachae typing [ 17 ] .

When the probe used for hybridization is derived from rRNA, the method is called ribotyping. Ribotyping was fi rst used for Legionella species identi fi cation by using 16S–23S ITS probe [ 18 ] . It was then used for L. pneumophila subtyping using different probes [ 19 ] .

RFLP typing has a high discriminatory index, but several methods developed later appear to have an equal discriminatory index but are easier to set up (e.g., PCR-based techniques) or have shown to be more discriminative (e.g., Pulse- fi eld gel electrophoresis (PFGE) [ 20 ] ).

PFGE is one of the gold standards for local epidemiology (e.g., outbreak inves-tigation); this method has been used for several years. The method is based on the separation by pulse- fi eld electrophoresis of macrorestriction fragments of the bacte-rial chromosome generated by digestion with an infrequent cutting site restriction endonuclease. PFGE has a high discriminatory index as assessed by several studies; most of these studies described the use of S fi I restriction endonuclease [ 21– 23 ] . Despite its high discriminatory index, this method suffers some drawbacks: it is time consuming (4 days to obtain results), inter-gel reproducibility is poor, electro-phoresis requires speci fi c equipment and computer-aided imaging analysis is

22315 Legionella pneumophila Typing

needed, data are dif fi cult to exchange between laboratories making investigations of travel-associated LD cases harder.

Recently Chang et al. described an improved protocol for L. pneumophila typing reducing to 2 days the total duration of the experiment [ 24 ] . Based on the global genomes sequenced, Zhou et al. evaluated new restriction endonuclease for PFGE typing, they also optimized electrophoretic parameters [ 25 ] . Despite some interna-tional standardization of PFGE typing protocols (restriction endonuclease, plugs preparation, and electrophoretic parameters) data remain dif fi cult to exchange.

PFGE has also been used for L. longbeachae typing [ 26 ] ; moreover, an opti-mized protocol using a double digestion has been described for Legionella anisa typing [ 27 ] .

Several authors have used arbitrary primed PCR (AP-PCR) for L. pneumophila subtyping [ 28– 30 ] . This method is based on the generation of DNA fi nger printing by random ampli fi cation of genome fragments. These techniques allow a good dis-criminatory index, are easy to perform, and give rapid results. The major drawback for these techniques is the lack of reproducibility between laboratories. Several primers have been tested for L. pneumophila subtyping [ 29, 30 ] and some authors recommended combination of the results obtained with two different primers before drawing any conclusion about the relatedness between strains [ 29 ] .

AFLP, one of the methods standardized by the EWGLI, has a high discrimina-tory index. In this method, bacterial DNA is simply digested and speci fi c adapters are ligated to the restriction fragments. These adapters are then use as targets for PCR ampli fi cation. The length polymorphism of ampli fi ed fragments generated is visualized by agarose or acrylamide gel electrophoresis [ 31– 33 ] .

The infrequent-restriction-site PCR (IRS-PCR) assay was developed for L. pneu-mophila molecular typing by Riffard et al. and shows a high discriminatory index in his study, similar to that of PFGE [ 23 ] . IRS-PCR consists of double digestion of genomic DNA with a restriction enzyme that infrequently cuts the chromosome and a second enzyme that frequently cuts it, followed by ampli fi cation of DNA with primers and adapters targeting the extremities of the restricted fragments. This method has the advantage of using low quantities of target DNA, and the separation of ampli fi ed frag-ments can be achieved by conventional agarose gel electrophoresis.

Pourcel et al. fi rst described the use of variable-number tandem-repeat (VNTR) diversity for L. pneumophila typing [ 34 ] . The authors developed this method based on the L. pneumophila Philadelphia genome and then updated it after the sequenc-ing of two others strains (Paris and Lens) [ 11 ] . The method is based on the length polymorphisms of 8 VNTR regions. These VNTR are repetitive sequences and the number of repetitions vary between strains. The length polymorphisms can be eas-ily visualized by PCR ampli fi cation of each locus using fl anking primers and elec-trophoresic migration. This technique is called MLVA for multi-locus VNTR analysis. The MLVA type or pro fi le is composed of a string of allele numbers, cor-responding to the number of repeats at each VNTR locus, separated by commas, in a predetermined order. L. pneumophila MLVA typing has been adapted to an auto-mated multicolored capillary electrophoresis in a multiplex assay by Nederbragt which made the method more accurate and more sensitive than the gel-based method

224 C. Ginevra

[ 35 ] . An MLVA-type database has also been created and is available on the website http://bacterial-genotyping.igmors.u-psud.fr/ .

Conversely to AP-PCR, the ampli fi cation step of AFLP, IRS-PCR, and MLVA is performed in stringent conditions which made inter-laboratories reproducibility higher.

Several methods are based on the comparison of polymorphisms of several DNA fragments.

The method is called multilocus sequence typing (MLST) when targets sequences are parts of housekeeping genes, sequence-based typing (SBT) which targets sequences are part of more variable genes, and multispacer typing (MST) when target sequences are highly variable intergenic region.

Since 2007, SBT is the new gold standard method recommended by EWGLI. This method is based on the sequence comparison of seven genes ( fl aA , pilE , asd , mip , mompS , proA , and neuA ). An allelic pro fi le comprises a string of numbers encompassing the number of individual alleles of the genes separated by commas. Each allelic pro fi le corresponds to a sequence type (ST) (e.g., allelic pro fi le 1,4,3,1,1,1,1 corresponds to ST1) [ 9, 10 ] . The assignment of allelic pro fi le and ST could be done by submitting the raw data sequences on the web interface available on the EWGLI website.

Isolation of clinical or environmental L. pneumophila is not easy. This impairs the epidemiological investigations as both clinical and environmental isolates are required for comparison to fi nd the source of infection. SBT is a PCR-based method which can be applied directly to DNA extracted from clinical or environmental samples without isolates. This direct use of the method on samples as been pub-lished in few cases and gives variable results in some case all genes could be ampli fi ed and sequenced [ 36 ] , in other cases no or few genes could be ampli fi ed and sequenced in environmental or in clinical samples [ 37 ] . To enhance the sensitivity of SBT directly applied on clinical samples, two studies have described the addition of a previous ampli fi cation step leading to nested or semi-nested PCR before sequencing of the target genes [ 38, 39 ] . In their study, Coscollá et al. enhanced the discriminatory index of the method by adding to the six fi rst gene targets of the standard SBT 3 intergenic regions as new targets, but these targets were not included in the EWGLI de fi nition of sequence types.

Fujinami et al. evaluated MALDI-TOF-MS for rapid discrimination of Legionella isolates [ 14 ] . The authors evaluated the use of MALDI-TOF-MS for Legionella species identi fi cation on one hand and L. pneumophila typing on the other hand. In their study, different Legionella species could be differentiated and two set of L. pneumophila iso-lates clustered in the same way when typed by mass spectrometry clusters or by PFGE. Nevertheless, in their study, the authors only tested 23 L. pneumophila isolates and nine other Legionella species. This method should be evaluated on a larger number of iso-lates from more diverse origins. If the high discriminatory index of MALDI-TOF-MS for L. pneumophila typing is demonstrated on a large isolate collection, this method will be a useful tool. MALDI-TOF-MS data can be generated within a few hours after Legionella growth. MALDI-TOF mass spectrometers are increasingly present in microbiology laboratories due to its increasing use for bacterial identi fi cation.

22515 Legionella pneumophila Typing

15.1 Conclusion

Several studies have compared these typing methods for discrimination indexes and also for rapidity, intra- and inter-laboratories reproducibility, and ease to exchange results.

PCR-based methods have the advantage of rapidity and their ease to be per-formed in standard laboratories and can be recommended for initial investigations during outbreaks.

PFGE remains a highly discriminative method, but it can be performed only by specialized laboratories such as reference centers. Results remain still dif fi cult to exchange between laboratories. This method is recommended for local epidemiol-ogy investigation by specialized laboratories.

SBT appears to be the method of choice for the exchange of results and is a pow-erful tool for global epidemiology. The additions of new targets have demonstrated that the discriminative power of this technique can be enhanced easily [ 9, 36 ] . The possible application of SBT method directly on clinical and environmental samples offers new solutions during epidemiological investigations.

Enhanced molecular characterization of Legionellae by SBT coupled with tech-niques like monoclonal antibody testing or PFGE typing allows us to build up a global picture of strain distribution and signi fi cance. The collaborative results obtained by members of EWGLI since 2003 using an SBT scheme show that a minority of strains cause most disease. Several independent studies show that few genotypes (ST1, ST23, ST37, ST40, ST47, ST62, etc.) cause lots of culture-proven LD cases and that these genotypes could be worldwide spread.

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