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1 Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerrannée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France
2 Department of Veterinary Pathology, Faculty of Veterinary Science, University of Liverpool, Leahurst, Neston CH64 7TE, UK
3 Respiratory and Systemic Infections Laboratory, Central Public Health Laboratory, Public Health Laboratory Service, London NW9 5HT, UK
4 Infectious Diseases Laboratories, Institute of Medical and Veterinary Science, Frome Road, Adelaide, SA 5000, Australia
Correspondence
Didier Raoult
raoult{at}medecine.univ-mrs.fr
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of L. drancourtii LLAP12T is X97366 and that for the mip gene sequence is AF407571.
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) and this diversity is matched by that of facultative intra-amoebal parasites, which include numerous established human pathogens (Barker & Brown, 1994; Horn et al., 2001). Following the demonstration that legionellae exploit amoebae as natural hosts (Rowbotham, 1980, 1983), amoebal co-culture methods have been introduced for the recovery of legionellae from environmental samples and have led to the isolation of several organisms that could not be cultured on axenic media (Rowbotham, 1983; Barbaree et al., 1986). As these organisms could not be subjected to conventional methods of bacterial identification, their designation remained Legionella-like amoebal pathogens (LLAPs). The introduction of 16S rRNA gene PCR-based methods finally permitted partial characterization of LLAPs and, on the basis of 16S rRNA gene sequence comparison, many were found to share specific evolutionary homology with members of the Legionellaceae (Fry et al., 1991; Birtles et al., 1996). Although several LLAPs have now been grown axenically (Adeleke et al., 2001), cultivation of others has failed. In this study, an LLAP isolate that had previously been characterized only by 16S rRNA gene sequence analysis (Birtles et al., 1996) was subjected to polyphasic assessment, thereby permitting its accurate classification.
LLAP12T was isolated by T. J. Rowbotham (Public Health Laboratory, Leeds, UK) in co-culture with an Acanthamoeba species from an environmental water source in West Yorkshire, UK (Fallon & Rowbotham, 1990). In our laboratory, LLAP12T was co-cultured routinely with Acanthamoeba polyphaga strain Linc AP-1 in peptone/yeast extract/glucose medium under previously described conditions at 32 °C (Rowbotham, 1983). All attempts to isolate LLAP12T by using buffered charcoal/yeast extract agar failed, despite the use of media from different manufacturers (bioMérieux, Diagnostic Pasteur) and prolonged incubation at 30 or 35 °C, with or without the presence of 5 % CO2.
Phenotypic characterization
The microscopic appearance of LLAP12T was verified by direct observation of infected amoebae using an inverted microscope, and of fixed and stained bacteria by using conventional microscopy. Both approaches revealed large numbers of motile bacteria within amoebal cytoplasmic vacuoles. These organisms stained weakly with Gram stain (negative), but well with Giménez stain. Ultrastructural analysis using transmission electron microscopy (La Scola et al., 2001) indicated that LLAP12T was a polymorphic, Gram-negative organism and confirmed that clusters of bacteria lay within amoebal cytoplasmic vacuoles (Fig. 1).
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), as was the production of polyvalent mouse antibodies (La Scola et al., 2001). This antiserum was adsorbed to the amoebae by mixing the sera with washed amoebae, then shaking the mixture overnight at room temperature. After centrifugation at 10 000 g for 10 min, the supernatant was removed and frozen at –80 °C. The immunofluorescent test (Harrison & Taylor, 1988) was used to assess cross-reactivity between antisera that were raised against LLAP12T and 69 different Legionella antigens, and cross-reactivity between LLAP12T and rabbit antisera that were raised against 80 Legionella antigens. Despite a high (>1/3200) homologous antibody titre to LLAP12T, the mouse antiserum failed to react significantly (>1/100) with any other Legionella antigen preparation. Furthermore, all antisera that were raised against other Legionella taxa failed to react significantly (>1/100) with LLAP12T antigen. Our observations of LLAP12T morphology and its intra-amoebal position are in keeping with those reported for other legionellae. Although serological reactivity among members of the Legionellaceae varies, this approach has long served as a routine method for species identification. It is reasonable to suspect that an isolate that demonstrates no reactivity with a panel of antisera that represents the serological spectrum of the current genus is likely to be a member of a novel taxon.
Sequence analysis
Methods used for amplification and determination of 16S rRNA and macrophage infectivity potentiator (mip) gene sequences and the procedure for determination of DNA G+C content have been detailed elsewhere (Birtles et al., 1996; Ratcliff et al., 1998; La Scola et al., 2001). A partial mip gene sequence for Legionella taurinenesis Turin IT was also determined. A 585 bp fragment of the mip gene of LLAP12T was sequenced and used for further analysis. Sequence comparison revealed that the LLAP12T sequence was most similar (86–88 %) to those of Legionella lytica and Legionella rowbothamii. Phylogenetic reconstruction, derived from an alignment of the LLAP12T mip gene sequence fragment with those of Legionella species with validly published names by using previously described methods (Birtles et al., 1996; Ratcliff et al., 1998), revealed that LLAP12T clustered specifically with, but had diverged from, L. lytica and L. rowbothamii (Fig. 2). This branching order was supported strongly by bootstrap analysis. Interestingly, the LLAP12T mip gene sequence was found to be indistinguishable from that of LLAP4, an organism that was isolated in the same manner as LLAP12T and was characterized partially by 16S rRNA gene sequence analysis (Birtles et al., 1996). Although this earlier study found that among the Legionellaceae, LLAP4 was related specifically to LLAP12T, there was, nonetheless, 1·3 % 16S rRNA gene sequence dissimilarity between them (19 mismatches in an alignment of 1482 bp). This was the first time that interstrain 16S rRNA gene sequence dissimilarity had been found to be greater than that observed between mip gene sequences. Furthermore, the extent of 16S rRNA gene sequence dissimilarity between LLAP4 and LLAP12T was greater than that seen between some different Legionella species (the 16S rRNA genes of L. rowbothamii and L. lytica differ by <1 %). Concern about these observations led us to repeat 16S rRNA gene sequencing for LLAP4 and LLAP12T. Although LLAP4 was no longer extant, DNA preparations remained. The 16S rRNA gene sequences we obtained from LLAP12T and LLAP4 were different from those submitted to GenBank (accession numbers X97366 and X97357, respectively). The new LLAP12T sequence differed at only two positions from that submitted to GenBank (in a 1348 bp alignment), whereas the new LLAP4 sequence differed at 12 positions (in a 1350 bp alignment). Comparison of the new LLAP4 and LLAP12T 16S rRNA gene sequences indicated that they differed at only four sites (<0·3 % of a 1348 bp alignment). Our experiences serve as a warning that sequences held in databases, particularly those deposited in the 1980s and early 1990s when sequencing methodologies were somewhat cruder than those that are currently available, cannot always be relied on. The LLAP4 and LLAP12T 16S rRNA gene sequences held by GenBank have now been revised.
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; Fox et al., 1992; Stackebrandt & Goebel, 1994), this analysis should not be relied on in the justification of species status for LLAP12T. However, the 16S rRNA gene sequences of other Legionella species are more similar to one another than that of LLAP12T is to that of L. lytica. Furthermore, phylogenetic inferral derived from 16S rRNA gene sequence data demonstrates clearly that LLAP12T has diverged significantly from its close relatives. A previous study using these data (Birtles et al., 1996) included LLAP4 and LLAP11, organisms that are no longer viable and thus could not be included in the current study. LLAP11 was found to possess a 16S rRNA gene sequence that was indistinguishable from that of LLAP12T, whereas LLAP4 was shown in this study to possess a sequence that shares >99 % similarity with that of LLAP12T. Taken together, these data provide further evidence that LLAP12T is not unique, but rather represents a distinct clade of legionellae.
Phylogenetic analyses derived from 16S rRNA and mip gene analysis are generally congruent (Ratcliff et al., 1998), although with variation within the genus Legionella of up to 30 %, mip gene sequences potentially offer a more reliable and sensitive basis for comparative analysis. Nonetheless, the combined taxonomic value of 16S rRNA and mip gene sequence analyses is greater than the sum of their individual worth, as congruence in bi- or multilocus analyses provides a good indication that evolutionary relationships inferred from individual genes or gene fragments do indeed represent true phylogenies for the organism. Thus, these data and those derived from the serological assessment provide unequivocal evidence that LLAP12T is a representative of a novel Legionella species. The data also support the inclusion of LLAP4 and LLAP11 in this species, a hypothesis that was first put forward solely on the basis of 16S rRNA gene sequence data (Adeleke et al., 1996).
Effect of temperature on the physiology of intra-amoebal LLAP12T
To assess the effect of temperature on the nature of the association between LLAP12T and its amoebal host, six flasks that each contained 10 ml of a suspension of washed amoebae (5x105 ml–1) in Page's amoeba saline (PAS) solution were seeded with 1 ml supernatant from a 7-day-old infected flask to yield a bacteria : cell ratio of 10 : 1. Infected flasks were incubated at 25, 28, 30, 32, 35 and 37 °C. Every day for 8 days, flasks were shaken, then 100 µl culture was removed, mixed with 300 µl trypan blue (0·13 %) (Sigma-Aldrich) in PAS solution, then transferred to a Nageotte counting chamber. Microscopic examination of amoebae permitted the enumeration of intact cells. Six uninfected amoebal cultures that were prepared concurrently with those described above and incubated and handled in the same way were used as controls.
A. polyphaga grew at all temperatures tested, but higher numbers of cells survived for longer periods at lower incubation temperatures (Fig. 3). Conversely, when infected with LLAP12T, amoebae incubated at these lower temperatures (<35 °C) lysed more rapidly (Fig. 3). After 48 h, LLAP12T infection led to lysis of 53 % of amoebae at 25–32 °C, as opposed to 0 % at 35–37 °C (P<0·05). This observation was confirmed by phase-contrast microscopic observation of infected amoebal cultures in culture flasks. After 48 h, co-cultures that were incubated at 25 and 28 °C already included high numbers of rounded, detached cells that contained a single, large vacuole filled with motile bacilli. This cytopathic effect was not observed in cultures that were incubated at 35–37 °C, even after prolonged incubation, demonstrating that LLAP12T maintained stable infection of amoebae at higher temperatures. This ability may contribute to the propensity of man-made water supplies, which are commonly maintained at higher temperatures than that of natural aquatic environments, to serve as sources of Legionella infection. Previous work has demonstrated that the virulence of legionellae increases when they are associated with amoebae (Cirillo et al., 1999); thus, exposure to conditions that are capable of prolonging this association must serve to enhance this pathogenic potential.
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Description of Legionella drancourtii sp. nov.
Legionella drancourtii (dran.cour'ti.i. N.L. gen. n. drancourtii in honour of Michel Drancourt for his work on intracellular micro-organisms, especially Rickettsiae).
Gram-negative. Does not grow on axenic media. Exists naturally as a strictly intracellular parasite of free-living amoebae. Cytopathic effect on A. polyphaga is restricted to co-cultures that are incubated at lower temperatures (25–32 °C). May be observed as motile rods within vacuoles of amoebae. DNA G+C content is 39·4 mol%.
The type strain is LLAP12T=ATCC 50991T.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Adeleke, A. A., Fields, B. S., Benson, R. F. & 8 other authors (2001). Legionella drozanskii sp. nov., Legionella rowbothamii sp. nov. and Legionella fallonii sp. nov.: three unusual new Legionella species. Int J Syst Evol Microbiol 51, 1151–1160.[Abstract]
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Fry, N. K., Rowbotham, T. J., Saunders, N. A. & Embley, T. M. (1991). Direct amplification and sequencing of the 16S ribosomal DNA of an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia. FEMS Microbiol Lett 67, 165–168.[CrossRef][Medline]
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